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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de

taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

UNIVERSITATEA DE TIINE AGRICOLE I MEDICIN


VETERINAR CLUJ-NAPOCA
COALA DOCTORAL
FACULTATEA DE ZOOTEHNIE I BIOTEHNOLOGII

Ing. Biolog BLTEANU I. VALENTIN ADRIAN

REZUMAT AL TEZEI DE DOCTORAT

STUDIUL POLIMORFISMELOR GENETICE ALE PROTEINELOR


MAJORE DIN LAPTE LA PRINCIPALELE RASE DE TAURINE,
BUBALINE, OVINE I CAPRINE DIN ROMNIA N SCOPUL
UTILIZRII LOR CA MARKERI GENETICI N AMELIORARE I
TRASABILITATE

CONDUCTOR TIINIFIC
Prof.Univ. Dr. Ing. VLAIC AUGUSTIN

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

CUPRINS
INTRODUCERE.....................................................................................................4
PARTEA I: STUDIU BIBLIOGRAFIC
CAPITOLUL I
COMPOZIIA LAPTELUI DE VAC, BIVOLI, OAIE,
CAPR I MECANISMUL SINTEZEI LUI LA NIVELUL
GLANDEI MAMARE N LACTAIE................................................................5
CAPITOLUL II
TEHNICI MOLECULARE UTILIZATE PENTRU
STUDIUL POLIMORFISMELOR PROTEINELOR DIN LAPTE..................7
CAPITOLUL III
STADIUL ACTUAL AL CERCETRILOR PRIVIND
STUDIUL POLIMORFISMELOR PROTEINELOR DIN
LAPTE LA TAURINE, BUBALINE, OVINE I CAPRINE..............................7
CAPITOLUL IV
IMPORTANA STUDIERII POLIMORFISMELOR
PROTEINELOR MAJORE DIN LAPTE PRIN PRISMA
POSIBILITII UTILIZRII LOR CA MARKERI
GENETICI N AMELIORAREA ANIMALELOR
I TRASABILITATEA PRODUSELOR LACTATE.......................................10
PARTEA II: CERCETRI PROPRII
CAPITOLUL V
SCOPUL I OBIECTIVELE CERCETRII.....................................................13
CAPITOLUL VI
CARTAREA PE CROMOZOMI A GENELOR CARE
CODIFIC PROTEINELE MAJORE DIN LAPTE PRIN
TEHNICA HIBRIDRII N SITU CU FLORESCENA (FISH)...................14

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

CAPITOLUL VII
STUDIUL POLIMORFISMELOR CELOR DOU GENE
CE CODIFIC K-CAZEINA I -LACTOGLOBULINA
LA TAURINE DIN RASA BLAT ROMNEASC
DE TIP SIMMENTAL PRIN TEHNICA PCR-RFLP......................................15
CAPITOLUL VIII
STUDIUL COMPARATIV PRIN TEHNICILE IEF I
PCR-RFLP A POLIMORFISMELOR GENETICE ALE
PROTEINELOR MAJORE DIN LAPTE LA TAURINE
DIN RASA BLAT ROMNEASC
DE TIP SIEMMENTAL.......................................................................................16
CAPITOLUL IX
CARACTERIZAREA POLIMORFISMELOR PROTEINELOR
MAJORE DIN LAPTE LA UNELE RASE DE TAURINE,
BUBALINE, OVINE I CAPRINE DIN ROMNIA FOLOSIND
TEHNICA IEF.......................................................................................................17
CAPITOLUL X
CARACTERIZAREA MOLECULAR A ALELELOR NOI
IDENTIFICATE LA LOCII S1-CAZEINEI I -CAZEINEI
LA RASA SURA DE STEP, VARIETATEA MOLDOVENEASC
I BIVOL ROMNESC......................................................................................21
CAPITOLUL XI
IDENTIFICAREA AUTENTICITII/ORIGINII DECLARATE
A LAPTELUI I PRODUSELOR LACTATE AUTOHTONE
FOLOSIND CA MARKERI GENETICI POLIMORFISMELE
PROTEINELOR MAJORE DIN LAPTE...........................................................23
CAPITOLUL XII
CONCLUZII GENERALE I RECOMANDRI.............................................24
BIBLIOGRAFIE SELECTIV...........................................................................25

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

INTRODUCERE
Lactogeneza poate fi mpartita n doua faze: faza 1 - ncepe n ultima treime a
gestaiei n care au loc procese de difereniere structurale i funcionale ale epiteliului
secretor; faza 2 - ncepe imediat dup natere i implic finalizarea diferenierii celulare,
care coencide cu sinteza i secreia laptelui n cantiti semnificative. Aceste dou faze
sunt eseniale avnd ca efect final declanarea lactaiei.
n laptele rumegtoarelor ntlnim 6 tipuri de proteine majore codificate de 6 gene
nealele, ce se exprim specific n celulele epiteliale ale glandei mamare n lactaie: S1cazeina (S1-CN), -cazeina (-CN), S2-cazeina (S2-CN), K-cazeina (K-CN), lactoglobulina (-CN i -lactoalbumina (-LA).
Variaiile care au aprut n structura acestor gene de-a lungul timpului, au dus la
apariia mai multor alele la aceti loci. Aceste variaii, denumite generic polimorfisme,
sunt cauzate de restructurri ale genelor (substituia unor nucleotide, deleii, inserii etc),
ce pot avea ca efect modificarea nivelului lor de expresie, inactivarea expresiei lor,
modificarea compoziiei n aminoacizi a proteinei etc.
Astfel unele variante genetice de la cei 6 loci au efect pozitiv asupra coninutului
de cazein al laptelui, timpului de coagulare, fermitii coagulului i randamentului de
obinere al brnzeturilor, iar altele au efect negativ asupra acestor parametri; unele
variante genetice ale -CN de la taurine au fost asociate cu declanarea unor boli la
oameni, cum ar fi: diabetul de tip 1, cardiopatia ischemic, sindromul morii subite la nou
nscui, schizofrenia i autismul (variantele A1, B, C), altele nu (variantele A2, A3); unele
variante genetice ale S1-CN au fost asociate cu alergii la copii, n urma consumului de
lapte.
Aplicarea n Romnia a informaiilor furnizate de variantele genetice ale celor 6
proteine majore din lapte, nu poate fi facut fr o caracterizare a raselor/speciilor de
ferm autohtone. Aceast caracterizare realizat n cadrul tezei mele de doctorat, ofer
informaii extrem de valoroase despre frecvena alelelor de la cei ase loci ai proteinelor

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

din lapte de la speciile/rasele autohtone. Ea deschide noi posibiliti de utilizare a acestor


polimorfisme ca markeri genetici n ameliorarea animalelor, n manipularea prin selecie
genetic a componenilor laptelui, n scopul producerii unui lapte alternativ care s
contracareze o serie de maldii, precum i ca markeri genetici de indentificare a
autenticitii, originii i trasabilitii produselor lactate.

PARTEA I: STUDIU BIBLIOGRAFIC

CAPITOLUL I
COMPOZIIA LAPTELUI DE VAC, BIVOLI, OAIE, CAPR I
MECANISMUL SINTEZEI LUI LA NIVELUL GLANDEI MAMARE N
LACTAIE

Cercetrile tiinifice au stabilit c laptele are o compoziie chimic complex, mai


ales n ceea ce privete fraciunile cazeinice, proteinele din zer, enzimele i substanele
antimicrobiene. Laptele reprezint o surs de hran cu valoare biologic ridicat,
coninnd mai mult de 100 de substane dizolvate ntr-o form uor asimilabil, aflate n
suspensie sau sub form de emulsie, necesare funcionrii normale a organismului uman
i animal (Banu i colab., 1998).
Proteinele din laptele sunt mprite n dou mari grupe n funcie de
comportamentul lor la pH acid = 4,6:
1. Fraciunea solubil la acest pH, denumit ,, proteine din zer , este alctuit din lactoglobulin (-LG) i -lactoalbumin (-LA) i alte proteine minore. n procesul de
producere al brnzeturilor prin adaus de cheag, aceast fraciune rmne n zer i nu intr
n constituia lor.
2. Fraciunea insolubil la acest pH, numit ,,cazeina total, este constituit din patru
tipuri de cazeine: S1-cazeina (S1-CN), S2-cazeina (S2-CN), -cazeina (-CN) i Kcazeina (K-CN). Cazeinele se gsesc n laptele proaspt dispersate sub forma unui numr

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

foarte mare de particule solide n suspensie, fiind sudate ntre ele prin molecule de
Ca9(PO4)6. Aceste particule sunt denumite micelii.
Coninutul n protein al laptelui de vaca este n medie de 3,3%, n laptele de
bivoli de 4,1% , n laptele de oaie de 5,3 % , iar n cel de capr de 3,7% (Iurc i
colab., 1998) . Din totalul proteinelor, fractiunea cazeinic reprezint 80%, iar proteinele
serice 20% .
De proporia fraciunii cazeinice i mrimea miceliilor depind n mare masur
proprietile de prelucrare ale laptelui, cantitatea i calitatea brnzei obinute, textura i
gustul diferitelor sortimente de brnzeturi i produse lactate (Buchberger si colab., 2000).
Cele 6 tipuri de proteine majore din lapte sunt codificate de 6 gene nealele, care se
exprim specific n celulele epiteliale ale glandei mamare n lactaie.
Analiza privind segregarea Mendelian a celor 4 gene nealele ce codific cele 4
tipuri de cazeine de la rumegtoare, a scos n evidena faptul c ele sunt situate pe
cromozomii din perechea 6 n urmtoarea ordine: s1-CN, -CN, s2-CN, K-CN
(Threadgill i colab., 1990; Hayes i colab., 1993a). Gena care codific -LG a fost
localizata pe cromozomii din perechea 11 (Hayes i colab., 1993b), iar cea care codific
-LA a fost localizat pe cromozomii din perechea 5 (Soulier i colab., 1989; Vilotte i
colab., 1987).
Sistemul endocrin, n principal prin glanda hipofiz, joac un rol central n toate
aspectele ce implic creterea i dezvoltarea esutului mamar (mamogeneza), declanarea
lactaiei i sinteza laptelui (lactogeneza), meninerea secreiei lactate (galactopoieza) i
apoi intrarea n repaus mamar, prin intermediul a 2 hormoni majori: hormonul de cretere
sau somatotrop (GH sau STH), respectiv prolactina (PRL). Semnalele hormonale
determin activarea transcripional a genelor ce codific proteinele din lapte i sinteza
proteinelor specifice.

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

CAPITOLUL II
TEHNICI MOLECULARE UTILIZATE PENTRU STUDIUL
POLIMORFISMELOR PROTEINELOR DIN LAPTE
Studiile realizate nc din anii 50 folosind electroforeza pe hrtie (PE), au pus n
eviden polimorfismul proteinelor majore din lapte, -LG fiind prima protein al crei
polimorfism a fost identificat de ctre Aschaffenburg i Drewry (1955).
Odat cu apariia unor tehnici de analiz a proteinelor mult mai performante: SGE
(electroforeza n gel de amidon), AE (electroforeza n gel de agaroz), PAGE
(electroforeza n gel de poliacrilamid), IEF (electroforeza prin focalizare izoelectric),
HPLC (cromatografia n faza lichid de nalt performan), Maldi TOF-MS
(spectrometria de

mas),

s-au mbuntit substanial cunotinele legate de

polimorfismul proteinelor majore din lapte la diverse specii de ferm.


Apariia tehniciilor bazate pe analiza ADN: PCR (reacia n lan a polimerazei),
PCR-RFLP (polimorfismul de lungime al fragmentelor de ADN amplificate i
restrictate),

SSCP

(polimorfismul

de

conformaie

al

fragmentelor

de

ADN

monocatenare), RT-PCR (reacia n lan a polimerazei n timp real) i a tehnicilor de


secveniere a ADN-ului, au permis descifrarea polimorfismelor care apar n structura
genelor, care au repercursiuni asupra expresiei genice i compoziiei n aminoacizi a
variantelor genetice ale proteinelor majore din lapte.

CAPITOLUL III
STADIUL ACTUAL AL CERCETRILOR PRIVIND STUDIUL
POLIMORFISMELOR PROTEINELOR DIN LAPTE LA TAURINE,
BUBALINE, OVINE I CAPRINE
Mutaiile care au aprut de-a lungul timpului n structura genelor care codific cele
6 proteine majore din lapte, au dus la apariia mai multor variante genetice la aceti loci.

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

Aceste variaii, denumite generic polimorfisme, indic faptul c fiecare specie proteic
din lapte poate s se prezinte sub dou sau mai multe forme codificate de gene
autozomale (alele) ntre care exist interaciune de co-dominan, ceea ce nseamn c
ambele alele se exprim la indivizii heterozigoi.
Alfa S1-CN este o protein din lapte constituit din 199 de aminoacizi, avnd o
greutate molecular de 23, 614 kDa. La taurine la locusul S1-CN au fost identificate
pn n prezent 10 variante genetice: A, B, C, D, Eyak, Ebali, F, G, H, (Farrell i colab.,
2004) i IRV (Blteanu i colab., 2007a; Blteanu i colab., 2008a,b). Alela IRV a fost
redenumit ISM, deoarece nu a fost evideniat la alte rase de taurine din Romnia, fiind
caracteristic doar rasei Sur de Step, varietatea Moldoveneasc (SM = Sur
Moldoveneasc). La bubaline au fost identificate 3 variante genetice: A, B (Chianese i
colab., 2009) i BRV (Blteanu i colab., 2007c; Blteanu i colab., 2008c). n urma
caracterizrii la locusul S1-CN a populatiilor de Bivol Romnesc, am constatat c
aceast variant genetic este specific rasei Bivol Romnesc, ea fiind redenumit dup
regiunea n care a fost identificat: S1-CN BBT (indicele BT reprezentnd Bivol din
Transilvania). La ovine au fost identificate 9 variante genetice: A, B, C, D, E, F, G, H, I
(Pirisi i colab., 1999; Giambra i colab., 2010). La caprine au fost identificate 17
variante genetice: A, B1, B2, B3, B4, C, D, E, F, G, H, I, L, M, N, 01,02 (Ramunno i
colab., 2004).
Beta-CN este o protein din lapte constituit din 209 aminoacizi, avnd o greutate
molecular de 23,983 kDa. La taurine la locusul -CN au fost identificate pn n
prezent 14 variante genetice: A1, A2, A3, B, C, D, E, A3m, B2, A4, H, F, A5, G (Farrell
i colab., 2004). La bubaline au fost identificate 3 variante genetice: A, B (Ferranti i
colab., 1998) i CRV (Blteanu i colab., 2007c; Blteanu i colab., 2008c). n urma
caracterizrii la locusul -CN a populatiilor de Bivol Romanesc, am constatat c aceast
variant genetic este specific rasei Bivol Romnesc, ea fiind redenumit dupa regiunea
n care a fost identificat: -CN CBT (indicele BT reprezentnd Bivol din Transilvania).
La ovine nu exist dovezi clare privind polimorfismul la nivel proteic (Chianese i

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

colab., 1995). La caprine au fost identificate 8 variante genetice: A, A1, B, C, D, E, 0 i


0 (Cosenza i colab., 2005a; Caroli i colab., 2006)
Alfa S2-CN este o protein din lapte constituit din 207 de aminoacizi, avnd o
greutate molecular de 25,150 kDa. La taurine la locusul S2-CN au fost identificate
pn n prezent 4 variante genetice: A, B, C, D (Farrell i colab., 2004). La bubaline au
fost identificate 3 variante genetice: A, B, C (Ferranti i colab., 1998). La ovine au fost
identificate 2 variante genetice: A, B (Rossi i colab., 1984; Mauriello i colab., 1990).
La caprine au fost identificate 9 variante genetice: A, B, C, E, F, G, D, 0 i 0 (Erhardt i
colab., 2002).
Kappa-CN este o protein din lapte constituit din 169 de aminoacizi, avnd o
greutate molecular de 19,007 kDa. La taurine la locusul K-CN au fost identificate
pn n prezent 11 variante genetice: A, B, C, B2 , E, F, G, Az, H, I, J (Farrell i colab.,
2004). La bubaline au fost identificate 2 variante genetice: A, B (Mitra i colab., 1998).
La ovine au fost identificate o singur variant genetic (Chianese i colab., 1996;
Ceriotti i colab., 2004b). La caprine se cunosc 16 situsuri polimorfice ce corespund
unui numr de 13 variante proteice i 3 mutaii tcute, implicnd un numr de 15 situsuri
polimorfe doar n exonul 4: A, B, B', B", C, C', F, G, H, I, J, L, D, E, K, M (Jann i
colab., 2004b).
Alfa-LA este o protein din lapte constituit din 123 de aminoacizi, avnd o
greutate molecular de 14,175 kDa. La taurine la locusul -LA au fost identificate pn
n prezent 3 variante genetice: A, B, C (Farrell i colab., 2004). La bubaline au fost
identificate 2 variante genetice: A, B (Chianese i colab., 2004). La ovine au fost
identificate 2 variante genetice: A, B (DallOlio i colab., 1989). La caprine au fost
identificate 2 variante genetice: A1, respectiv A2 (Cosenza i colab., 2005b).
Beta-LG este o protein din lapte constituit din 162 aminoacizi, avnd o greutate
molecular de 18,277 kDa. La taurine la locusul -LG au fost identificate pn n
prezent 13 variante genetice: A, B, C, D, Dr, Dyak, E, F, G, W, H, I , J (Farrell i colab.,
2004). La bubaline au fost identificate 2 variante genetice: A, B (Chianese i colab.,
2004). La ovine au fost identificate 3 variante genetice A, B (Kolde i colab., 1983;
9

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

Schlee i colab., 1993) i C (Erhardt i colab., 1989). La caprine au fost identificate 2


variante genetice: A, respectiv B (Yahyaoui i colab., 2000).

CAPITOLUL IV
IMPORTANA STUDIERII POLIMORFISMELOR PROTEINELOR
MAJORE DIN LAPTE PRIN PRISMA POSIBILITII UTILIZRII LOR
CA MARKERI GENETICI N AMELIORAREA ANIMALELOR I
TRASABILITATEA PRODUSELOR LACTATE
Influena variantelor genetice ale proteinelor majore din lapte asupra cantitii
laptelui

La taurine influena variantelor genetice ale proteinelor din lapte asupra cantitii
lui a fost evaluat n numeroase studii. Astfel genotipurile BB de la locusul S1-CN,
A2A2 de la locusul -CN, AA de la locusul -LG i AA (+15) de la locusul -LA, au
fost asociate cu o cantitate mai mare de lapte (Bovenhuis i colab., 1992; Bleck i colab.,
1993b; Ikonen i colab., 1999, Ng-Kwai-Hang i colab., 2006).
La ovine studiile privind influena genotipurilor de la locusul -LG asupra cantitii
laptelui sunt contradictorii. Bolla i colaboratorii (1989), respectiv Caroli i colaboratorii
(1995) au constatat la rasa Sarda c genotipul BB este asociat cu o cantitate mai mare de
lapte. Di Stasio i colaboratorii (1992) au constatat la rasa de ovine Sicilian o
superioritate a indivizilor AB la acest locus n ceea ce privete producia de lapte. La rasa
de ovine Valle del Belice, genotipul AA a fost asociat cu o cantitate mai mare de lapte
(Giaccone i colab., 2000).
La caprine Kumar i colaboratorii (2006) au constatat c genotipurile AA au avut o
producie mai mare n comparaie cu celelalte genotipuri.

10

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

Influena variantelor genetice ale proteinelor majore din lapte asupra calitii
laptelui, proprietilor sale de prelucrare i randamentului de obinere al
brnzeturilor

La taurine genotipul CC de la locusul S1-CN, genotipul BB de la locusul K-CN


i genotipul BB de la locusul -LG au fost corelate cu un coninut mai mare de cazein i
protein total a laptelui (Jakob i colab., 1994; Fitzgerald i colab., 1997; Lunden i
colab., 1997 ). Genotipul BB de la locusul -LG a fost corelat cu un coninut mai mare de
grasime al laptelui (Wedholm i colab., 2006; Heck i colab., 2009). Laptele provenit de
la genotipul CC de la locusul S1-CN, genotipul BB la locusul -CN i genotipul BB la
locusul K-CN, coaguleaz ntr-un timp mult mai scurt n comparaie cu celelalte
genotipuri (Delacroix - Buchet i colab., 1994; Fitzgerald i colab., 1997; Kubarsepp i
colab., 2005). Genotipul BB de la locusul K-CN influeneaz pozitiv randamentul de
transformare al laptelui n brnzeturi n comparaie cu genotipurile AA, diferenele
constatate fiind ntre 2,7-15%, n functie de tipul de brnz fabricat (Van den Berg i
colab., 1992; Fitzgerald i colab.,1997; Walsh i colab., 1998).
La caprine efectele polimorfismului S1-CN asupra calitii laptelui de capra au
fost studiate la rasele Franceze (Mahe i colab., 1994; Delacroix i colab., 1996;
Manfredi i colab., 2000). Rezultatele obinute indica faptul c alela A, n comparaie cu
alelele E i F, are un efect semnificativ (pozitiv) asupra coninutului de protein, grsime
i proprietilor de prelucrare ale laptelui. n experimente de fabricare a brnzeturilor, sau obinut diferene de 7,4% ntre genotipurile AA i EE, de 6,9% ntre genotipurile EE i
FF de14,8% ntre AA i FF. Brnzeturile provenite de la genotipurile AA au avut un gust
mai slab pronunat de capr (datorit lipolizei mai slabe), n comparaie cu genotipurile
FF, care au avut un gust mai pronunat (datorit lipolizei mai accentuate). Rezultate
similare au fost obinute i la rasele Spaniole (Sanchez i colab., 1998), rasele Italiene
(Meggiolaro i colab., 2000), rasele Norvegiene (Vegarud i colab., 1999) i la unele rase
din SUA (Clark i colab., 2000).

11

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

La ovine genotipul CC de la locusul S1-CN are o influena pozitiv asupra


compoziiei laptelui i randamentului de obinere al brnzeturilor. n experimente de
fabricare a brnzeturilor s-au obinut diferene de +3,5%, respectiv +8,6%, ntre genotipul
CC, n comparaie cu genotipurile CD, respectiv DD (Pirisi i colab., 1999). Genotipul
BB de la locusul -LG a fost asociat cu un coninut mai mare de grsime al laptelui i
mai sarac n lactoza, avnd un efect favorabil asupra randamentului de obinere al
brnzeturilor (Celuk i colab., 2006).

Utilizarea polimorfismelor proteinelor majore din lapte ca markeri genetici n


identificarea autenticitii laptelui i produselor lactate

Pe plan mondial un numr mare de metode au fost propuse pentru identificarea


posibilelor falsuri cauzate de amestecuri de lapte interspecifice nedeclarate. Majoritatea
metodologiilor de identificare a autenticitii produselor lactate se bazeaz pe analiza
polimorfismelor proteinelor majore din lapte: a) Electroforeza n gel de poliacrilamid
n condiii native (Kaminarides i colab., 2002); b) Electroforeza n gel de
poliacrilamida n condiii denaturante (Veloso i colab., 2002); c) Electroforeza prin
focalizare izoelectric (Addeo i colab,. 1990; Anonymous, 2001; Blteanu , 2005;
Blteanu i colab., 2007b,c,d; Vlaic i colab., 2008; Blteanu i colab., 2008c); d)
Electroforeza de capilaritate (Lee i colab., 2001); e) Metode imunochimice - ELISA
(Hurley i colab., 2004); f) Cromatografia de nalt performan n faz lichid
inversat (Veloso i colab., 2002); g) Spectrometria de masa (Siciliano i colab.,
2000); h) Tehnici bazate pe analiza ADN (Bottero i colab., 2002).

12

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

PARTEA II: CERCETRI PROPRII

CAPITOLUL V
SCOPUL I OBIECTIVELE CERCETRII

Studiul polimorfismelor proteinelor majore din lapte la speciile / rasele de ferm


autohtone a avut ca scop evaluarea posibilitii folosirii ulterioare a acestor informaii n
mai multe direcii, ca de exemplu:
- Creterea calitii laptelui (n principal al coninutului de cazein), proprietilor sale de
prelucrare i randamentului de obinere al brnzeturilor;
- Obinerea unui lapte hipoalergenic prin selecia unor indivizi ce nu produc S1-CN,
care este principalul alergen din lapte;
- Obinerea unui lapte mai sntos ce conine variante ale CN care nu afectez
santatea umana;
- Identificarea autenticitii/originii laptelui i produselor lactate.
Aplicarea informaiilor furnizate de aceti markeri genetici nu poate fi fcut fr o
cunoatere aprofundat a polimorfismelor genetice ce apar la cei 6 loci la speciile / rasele
autohtone de taurine, bubaline, ovine i caprine. Aceast caracterizare poate oferi
informaii extrem de valoroase n ceea ce privete frecvena alelelor de la cei 4 loci ai
cazeinelor i cei 2 loci ai proteinelor din lactoserum n populaiile autohtone.
n contextul stadiului actual al cunoaterii n domeniu, teza are ca obiective
principale direcii de cercetare cu importan deosebit pe plan mondial, ns mai puin
sau deloc cunoscute pe plan naional:
- Testarea unor protocoale de hibridare FISH (hibridare in situ cu fluorescen), folosind
sonde BAC (cromozom artificial bacterian), care s permit cartarea genelor ce codific
proteinele majore din lapte pe cromozomii metafazici i n nuclei interfazici;

13

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

- Caracterizarea polimorfismelor genetice de la cei 6 loci ce codific cele 6 proteine


majore din lapte, la unele rase de taurine: Blat Romneasc de tip Simmental, Blat
cu Negru Romneasc, Holstein Rou, Brun, Pinzgau de Transilvania i Sura de Step;
- Caracterizarea polimorfismelor genetice de la cei 6 loci ce codific cele 6 proteine
majore din lapte, la bubalinele autohtone din rasa Bivol Romnesc;
- Caracterizarea polimorfismelor genetice de la cei 6 loci ce codific cele 6 proteine
majore din lapte, la caprinele autohtone din rasa Carpatina;
- Caracterizarea polimorfismelor genetice de la cei 6 loci ce codific cele 6 proteine
majore din lapte, la ovinele autohtone din rasele: urcan, Carabaa, igaie, igaie
Ruginie, Merinos de Cluj i Karakul de Botoani;
- Caracterizarea molecular a alelei noi descoperite la Sura de Step, varietatea
Moldoveneasc, redenumit S1-CN ISM, n vederea studierii posibilitii folosirii ei ca
marker genetic de biodiversitate al rasei;
- Caracterizarea molecular a alelelor noi descoperite la rasa

Bivol Romnesc,

redenumite S1-CN BBT i -CN CBT;


- Studierea posibilitii folosirii polimorfismelor genetice ale proteinelor majore din
laptele

speciilor/raselor

de

ferm,

ca

markeri

genetici

de

identificare

autenticitii/originii laptelui, brnzeturilor i altor produse lactate autohtone, lund n


considerare i noile alele identificate la cei doi loci ai cazeinelor la rasa Bivol Romnesc.

CAPITOLUL VI
CARTAREA PE CROMOZOMI A GENELOR CARE CODIFIC
PROTEINELE MAJORE DIN LAPTE PRIN TEHNICA HIBRIDRII N
SITU CU FLORESCEN (FISH)

n cadrul experimentelor realizate am folosit dou sisteme de detecie a sondei


hibridate i anume: un sistem de detecie cu un singur anticorp marcat cu un fluorocrom
rou (TRITC), anticorp care recunoate specific digoxigenina cu care a fost marcat una

14

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

din sonde BAC (sonda ce conine gena WAP) i un sistem dublu, n care un anticorp
primar recunoate specific biotina cu care a fost marcat cealalt sond (sonda ce conine
clusterul de cazeine), care la rndul su este recunoscut de un anticorp secundar marcat
cu un fluorocrom verde (FITC).
Au fost realizate hibridri (cu sondele menionate) pe cromozomi metafazici,
provenii din culturi celulare de fibroblati de iepure i celule mamare tumorale HC11 din
glanda mamar de oarece, precum i pe nuclei interfazici bidimensionali sau
tridimesionali cu structur prezervat preparai din celule HC11.
n toate cazurile au fost obinute semnale de hibridare specifice, care au permis o
cartare corect pe cromozomi, respectiv n teritoriile cromozomiale specifice, a genelor
studiate.

CAPITOLUL VII
STUDIUL POLIMORFISMELOR CELOR DOU GENE CE CODIFIC
K-CAZEINA I -LACTOGLOBULINA LA TAURINE DIN RASA
BLAT ROMNEASC DE TIP SIMMENTAL PRIN TEHNICA PCRRFLP
Studiul polimorfismelor K-CN i -LG a vizat testarea unor protocoale de lucru
PCR-RFLP, care s permit diferenierea celor mai comune alele de la aceti doi loci (A,
respectiv B). Astfel n cazul amplificrii unui fragment de 350 pb din gena K-CN i
digestiei cu enzima Hinf I, s-au observat 3 profile de restricie corespunztoare
genotipurilor AA, AB i BB, de la acest locus. n cazul amplificrii unui fragment de 262
pb din gena -LG i digestiei cu enzima Hae III s-au observat de asemenea 3 profile de
restricie, corespunztoare genotipurilor AA, AB si BB, de la acest locus.
Calcularea frecvenei genotipurilor la locusul K-CN a scos n eviden o frecven
mai mare a genotipului AB n comparaie cu celelalte dou. Calcularea frecvenei

15

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

alelelorde la acest locus, a scos n eviden o frecven mai mare a alelei A, n comparaie
cu alela B.
Calcularea frecvenei genotipurilor de la locusul -LG a scos n eviden o
frecven mai mare a genotipului AB n comparaie cu celelalte 2. Calcularea frecvenei
alelelorde la acest locus, a scos n eviden o frecven mai mare a alelei B n comparaie
cu alela A.
Rezultatele obinute evideniaz c cele 2 teste ADN pot fi folosite pentru
identificarea alelelor comune A i B de la acesti loci, ceea ce permite o genotipizare
precoce a reproductorilor taurini pentru cei 2 markeri genetici.

CAPITOLUL VIII
STUDIUL COMPARATIV PRIN TEHNICILE IEF I PCR-RFLP A
POLIMORFISMELOR GENETICE ALE PROTEINELOR MAJORE DIN
LAPTE LA TAURINE DIN RASA BLAT ROMNEASC DE TIP
SIEMMENTAL

Studiul a fost realizat pe 14 indivizi din rasa Blat Romneasc de tip


Simmental, care au fost genotipizai comparativ la locusul K-CN i -LG att prin PCRRFLP, ct i prin IEF. El a avut ca scop evaluarea eficienei genotipizrii la nivel de
ADN (realizat prin tehnica PCR-RFLP) i la nivelul expresiei genice (realizat prin
tehnica IEF), la locii ce codific proteinele majore din lapte.
n urma analizei comparative a rezultatelor obinute am identificat la locii K-CN,
respectiv -LG aceleai genotipuri, att prin PCR-RFLP, ct i prin IEF. n plus prin
tehnica IEF au fost vizualizate n acelai gel i genotipurile de la ceilali 4 loci ce codific
S1-CN, S2-CN, -CN i -LA, permind o identificare corect a tuturor alelelor, chiar
i a celor mai rare, ce apar cu frecven mai redus.

16

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

Rezultatele obinute scot n eviden faptul c tehnica IEF permite stabilirea mai
corect a genotipurilor la cei 6 loci ce codific proteinele majore din lapte, deoarece pot
fi identificate i alele mai rare, lucru dificil i costisitor cu ajutorul tehnicii PCR.

CAPITOLUL IX
CARACTERIZAREA POLIMORFISMELOR PROTEINELOR MAJORE
DIN LAPTE LA UNELE RASE DE TAURINE, BUBALINE, OVINE I
CAPRINE DIN ROMNIA FOLOSIND TEHNICA IEF
n cazul taurinelor au fost genotipizai prin IEF la cei 6 loci ce codific
proteinele majore din lapte un numr de 693 de indivizi din 7 rase autohtone, dup cum
urmeaz: Blat Romneasc de tip Simmental (236 de indivizi), Blat cu Negru
Romneasc (230 de indivizi), Holstein Rou (13 indivizi), Brun (134 de indivizi),
Pinzgau de Transilvania, varietile neagr (26 de indivizi) i roie (30 de indivizi) i
Sura de Step, varietatea Moldoveneasc (24 de indivizi).
La locusul S1-CN au fost identificate cele 2 variante genetice comune B i C.
Frecvena variantei B este de peste 0,9 la rasele mai ameliorate n direcia unei producii
mai mari de lapte studiate, atingnd frecvena 1 la rasa Holstein Rou. Frecvena cea mai
mic a fost observat la rasa Pintzgau, varietatea negr. Frecvena variantei C, asociat la
multe rase de taurine cu o calitate superioar de procesare n brnzeturi a laptelui, a fost
cea mai mare la rasa Pinzgau Negru (0,308), aceast alel fiind absent la rasa Holstein
Rou. La locusul S1-CN a fost identificat la rasa Sura de Step, varietatea
Moldoveneasc, o nou variant genetic denumit ISM, cu un punct izoelectric ntre cel
al variantelor B i C. Frecvena acestei alele determinat prin IEF este de 0,041 n
populaiile studiate.
La locusul -CN au fost identificate 5 variante genetice A1, A2, A3, B, C.
Frecvena variantei ancestrale A2 este cea mai mare n comparaie cu a celorlalte (peste
0,5), cu excepia rasei Pintzgau, varietatea rosie, la care frecvena variantei A1 este mai

17

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

mare (0,483). Varianta A3 a fost detectat doar un individ din rasa Blat Romneasc de
tip Simmental. Varianta B, asociat la multe rase de taurine cu o calitate superioar de
procesare n brnzeturi a laptelui, are o frecven extrem de redus la toate rasele (ntre
0,039-0,117).Varianta C a fost detectat cu o frecven foarte redus la rasele Blat
Romneasc de tip Simmental, Brun i Pinzgau Negru. La rasa Sura de Step prezena
variantei C a fost evideniat doar la indivizii genotipizai din zona Tazlu i Tupilai, ea
fiind o dovad a infuziei cu rasa Brun. Ea nu a fost detectat la indivizii considerai a fi
rasa curat de la SCDCB Dancu.
La locusul S2-CN au fost identificate o singur variant genetic i anume
varianta A, cu o frecvena egala 1.
La locusul K-CN au fost identificate 3 variante genetice A, B i C. Cea mai mare
frecven a variantei A a fost nregistrat la rasa Blat cu Negru Romneasc (0,835),
iar a variantei B la rasa Brun (0,619). La rasa Blat Romneasc de tip Simmental
frecvena alelei A este foarte mare (0,679). Varianta C a fost detectat cu o frecven
redus la rasele Blat Romneasc de tip Simmental i Brun.
La locusul -LA au fost identificate o singur variant genetic i anume varianta
B, cu o frecven egal 1.
La locusul -LG au fost identificate 3 variante genetice A, B i C. Frecvena
variantei A este cea mai mare la rasa Blat Romneasc de tip Simmental i Sura de
Step (peste 0,5). Frecvena variantei B este mare la toate rasele, la rasa Holstein Rou
fiind de 0,769.
n cazul bubalinelor au fost genotipizai la cei 6 loci ce codific proteinele
majore din lapte un numr de 139 de indivizi din rasa Bivol Romnesc. La locii S1-CN
i -CN au fost identificate cu o frecven de 0,86 cele 2 variante genetice comune: A de
la locusul S1-CN i B de la locusul -CN. Au fost identificate cu o frecven de 0,14
dou variante genetice noi, S1-CN BRV i -CN CRV (redenumite S1-CN BBT i -CN
CBT). Ele au fost observate n linkage pe cromozomul 6. La ceilali 4 loci a fost
identificat doar cte o alel cu o frecven egal cu 1.

18

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

n cazul ovinelor au fost genotipizai la cei 6 loci ce codific proteinele majore


din lapte un numr de 282 de indivizi din 6 rase autohtone, dup cum urmeaz: urcan
(44 de indivizi), Carabaa (40 de indivizi), igaie (45 de indivizi), igaie Ruginie (28 de
indivizi), Merinos de Cluj (35 de indivizi), Karakul de Botoani, varietile neagr (15
indivizi), brumrie (15 indivizi), maro (15 indivizi), sur (15 indivizi), roz (15 indivizi),
alb (15 indivizi).
La toate cele 6 rase de ovine genotipizate polimorfismul proteinelor din lapte este
foarte redus, singurul locus polimorf fiind locusul -LG, unde au fost identificate dou
variante genetice A i B. Varianta A are o frecven mai mare (de peste 0,5) la rasele
urcan, Caraba, Merinos de Cluj, respectiv Karakul, cea mai mare fiind la Karakul,
varietatea alb. Alela B are o frecven mai mare, n comparaie cu A, la rasele igaie i
igaie Ruginie.
La locii S1-CN, -CN, S2-CN, K-CN i -LA a fost identificat cte o singur
variant genetic cu o frecven egal cu 1. Prezena alelei C la locusul S1-CN are o
semnificaie pozitiv, deoarece ea a fost asociat n multe studii cu caliti superioare de
procesare ale laptelui i randamente mai mari de obinere a brnzeturilor.
n cazul caprinelor au fost genotipizai la cei 6 loci ce codific proteinele majore
din lapte un numr de 283 de indivizi din rasa Carpatin.
La locusul S1-CN au fost identificate 4 categorii de alele, asociate cu 4 nivele
diferite de expresie. Rezultatele indic o frecven

de 0,569 a alelelor cu expresie

puternic a S1-CN (A, B, C) i o frecven de 0,431 n cazul alelelor cu expresie medie,


slab i nule. Dac lum n considerare efectivul de 700.000

de capre existent n

Romnia i frecvena calculat a alelelor de tip E, F sau 0 de 43,1%, putem deduce c un


numr de 301.700 de capre produc un lapte cu un coninut mai srac n protein, care
afecteaz substanial calitatea lui i randamentul de obinere al brnzeturilor. Aceste
rezultate pot explica heterogenitatea rasei Carpatin n ceea ce privete unii parametrii
calitativi i de procesare ai laptelui. La locusul S1-CN au fost identificate 2 posibile noi
variante genetice denumite S1-CN 0 i S1-CN X. Prima, identificat cu o frecven de
0,021, se caracterizeaz prin absena S1-CN n lapte, fiind observat ntotdeauna n
19

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

linkage cu un profil necunoscut de la locusul -CN, dovedind transmiterea nlnuit a


celor 2 variante genetice situate pe acelai cromozom. A dou posibil variant genetic,
S1-CN X, a fost observat doar la un individ, n stadiu heterozigot cu alela B. Pe baza
comportamentului electroforetic se pare c prezint n secvena aminoacizilor o
restructurare major, cauzat probabil de acelai fenomen de exon skipping ce
caracterizeaz i alela F. Cu toate acestea, pe baza intensitii de colorarea a benzilor
electroforetice, am concluzionat c face parte probabil din categoria alelelor cu expresie
puternic.
La locusul -CN au fost identificate 3 variante genetice. Varianta genetic comun
A, respectiv varianta C au fost identificate cu o frecven de 0,979. Ele nu au putut fi
difereniate prin IEF deoarece au acelai punct izoelectric. La civa indivizi a fost
identificat cu o frecven de 0,021, o variant genetic necunoscut denumit -CN X
linkat ntotdeauna cu S1-CN 0.
La locusul S2-CN au fost identificate 3 variante genetice. Varianta genetic C a
S2-CN are o frecven mai mare (0,553), n comparaie cu varianta A (0,431), varianta
E avnd o frecvena redus (0,016).
La locusul K-CN au fost identificate 2 variante genetice. Varianta genetic A este
predominant (0,912), varianta B avnd o frecvena redus (0,088). Prezena variantei B
i la rasa Carpatina are o semnificaie pozitiv deoarece ea a fost asociat n diverse studii
cu o calitate superioar de procesare a laptelui n brnzeturi.
La locii -LG i -LA a fost identificat cte o singur variant genetic la fiecare
locus, cu frecvene egale cu 1.

20

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

CAPITOLUL X
CARACTERIZAREA MOLECULAR A ALELELOR NOI
IDENTIFICATE LA LOCII S1-CAZEINEI I -CAZEINEI LA RASA
SURA DE STEP, VARIETATEA MOLDOVENEASC I BIVOL
ROMNESC
La Sura de Step
O nou alel a S1-CN de la taurine a fost descoperit la rasa Sura de Step,
varietatea Moldoveneasc, denumit ISM, fiind complet caracterizat folosind o
metodologie combinat de secveniere a proteinei, cADN-ului i ADN-ului.
Prin analiza Maldi Tof-MS am reuit identificarea unei substituii n poziia 192 a
proteinei mature. n aceast regiune varianta ISM conine glicina ca i variant C, i nu acid
glutamic prezent la varianta B. Celelalte substituii prin care varianta genetic ISM se
difereniaz de B i C, nu au putut fi clar identificate prin analiza Maldi Tof-MS.
Secvenierea comparativ a cADN-rilor din dou probe purttoare ale S1-CN ISM
(BISM, CISM), respectiv a 2 probe de referin (BB, CC), au evideniat mutaiile care
caracterizeaz aceast nou alel: substituia unei adenine din alela C i B (exonul 11,
nucleotid 297, codonul 99 gaA ce codific glutamina), cu o timin n alela ISM (gaT ce
codific acidul aspartic); substituia unei adenine din alela B (exonul 17, nucleotida 620,
codonul 207 gAa ce codific glutamina) cu o guanin n alelele C i ISM (gGa ce codific
glicina). Aceast ultim substituie a fost confirmat i la nivel proteic prin analiza Maldi
Tof-MS. Au fost identificate dou alte substituii comune alelelor B, C i ISM (secveniate
de la rasa Sura de Step, Varietatea Moldoveneasc), care sunt diferite de ale unor
secvene de referin publicate n GenBank, fr efect asupra secvenei aminoacizilor n
protein.
Pe baza substituiei unei adenine din poziia 21 a exonului 11 din alela C (adenina
regsit i n alela B), cu o timin n alela ISM, a fost pus la punct un test PCR-RFLP ,
prin amplificarea i restricia cu enzima BseGI a unui fragment de 422 pb din gena S1-

21

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

CN incluznd exonul 11. Deoarece prezena acestei noi alele a fost observat doar la
indivizi considerai ras curat, apartenena ei la rasa Sura de Step nu poate fi contestat.
Aceast teorie este susinut i de frecvena cu care a fost identificat n populaia de Sura
de Step genotipizat: 0,128; frecvena este destul de mare pentru o nou variant
genetic. Aceasta sugereaz c alela ISM a avut o frecvena i mai mare cnd aceast ras
(i progenitorii ei) era predominant n Romnia.
Pe baza similaritilor i diferenelor observate la nivel molecular ntre alelele S1CN B, C i ISM am concluzionat c aceast nou alel a derivat filogenetic din alela
ancestral C, furniznd prima dovad molecular despre relaiile filogenetice dintre Sura
de Step i reprezentanii genului Bos din Africa i Asia

La Bivolul Romnesc
Secvenierea cADN-urilor S1-CN A (alela comun) i alelei BBT de la Bivolul
Romnesc, a scos n eviden n cazul alelei BBT 2 mutaii punctiforme care o difereniaz
de celelalte alele cunoscute la bivolul Mediteranean i Indian. Deleia ntregului exon 6,
este un tip de restructurare extrem de interesant, care scurteaz proteina matur cu 8
aminoacizi, fa de proteina normal care are 199 aminoacizi. Au mai fost identificate
nc 3 mutaii, care fie sunt asemntoare fie sunt diferite fa de alte alelele comune.
Secvenierea cADN-urilor -CN B (alela comun) i alelei CBT de la Bivolul
Romnesc, a dus la identificarea a 4 mutaii care difereniaz cele 2 variante genetice i
care afecteaz secvena previzionat a proteinei mature. De asemenea au fost identificate
2 mutaii tcute n sensul c, codonul rezultat codific acelai aminoacid n ambele
cazuri.
De vreme ce alelele noi identificate apar cu frecven mare la Bivolului Romnesc,
ele ar putea fi folosite ca markeri genetici de origine/autenticitate a brnzeturile
autohtone de bivoli, cu aplicabilitate imediat.

22

Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

CAPITOLUL XI
IDENTIFICAREA AUTENTICITII/ORIGINII DECLARATE
A LAPTELUI I PRODUSELOR LACTATE AUTOHTONE
FOLOSIND CA MARKERI GENETICI POLIMORFISMELE
PROTEINELOR MAJORE DIN LAPTE

Dac n alte ri UE cazurile de falsificare sunt destul de rare, n Romnia laptele


de vac (produs la un pre mai mic i n cantitate mai mare), este de multe ori adugat
nedeclarat n laptele de bivoli, capr sau oaie, destinat preparrii unor produse lactate
,,autentice, asta mai ales n cazul laptelui prelucrat industrial.
Metodologia European acreditat de identificare a falsurilor din brnzeturi, se
bazeaz pe analiza prin IEF a fraciunilor gama cazeinice, care rezult din hidroliza cu
plasmin a -CN, avnd dezavantajul c nu poate diferenia laptele de capr de cel de
oaie. La ora actual n Romnia nu exist o metodologie de identificare a adausurilor de
lapte interspecifice nedeclarate n produsele lactate.
Cercetrile descrise n acest capitol au avut ca scop studierea posibilitii folosirii
polimorfismelor genetice ale proteinelor din lapte, descrise la toti cei 6 loci la speciile de
ferm studiate, ca markeri genetici de indentificare a autenticitii produselor lactate
autohtone. De asemenea, studierea posibilitii utilizrii noilor alele identificate la rasa
Bivol Romnesc la locii -CN: alela CBT i S1-CN: alela BBT, ca markeri genetici de
origine/autenticitate a brnzeturilor tradiionale romneti de bivoli, a fost un alt
obiectiv al prezentelor cercetri.
Analiza de autenticitate privind depistarea posibilelor amestecuri interspecifice
nedeclarate de lapte, a fost realizat pe numr de 12 sortimente de brnzeturi de pe piaa
din Romnia, dup cum urmeaz: 2 sortimente de vac, 3 de bivoli, 3 de oaie i 4 de
capr. n cazul brnzeturilor de vac s-a constatat c sunt fabricat numai din lapte de
vac. Acest lucru nu este surprinztor deoarece folosirea altor tipuri de lapte la fabricarea
brnzeturilor de vac este puin probabil. n cazul brnzeturilor de bivoli, s-a constatat

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

c majoritatea sunt falsificate cu lapte de vac n proporie de 100%.

n cazul

brnzeturilor de oaie s-a constatat c dou au fost fabricate exclusiv din lapte de oaie, una
fiind falsificat n proporie de aproximativ 50% cu lapte de vac. n cazul brnzeturilor
de capr s-a constatat c trei au fost fabricate exclusiv din lapte de capr, cea de-a patra
fiind falsificat n proporie de 100% din lapte de vac.
Compararea a dou tipuri de brnz de bivoli, una de tip Mozzarella produs din
lapte de la rasa Bivol Italian din regiunea Campania, Italia i una de tip telemea produs
din lapte de la rasa Bivol Romnesc din Transilvania, a scos n eviden n mod clar c
acestea se difereniaz net prin prezena n brnza telemea a variantelor genetice S1-CN
BBT i -CN CBT, variante genetice care sunt absente la Bivolul Italian. Ca urmare
posibilitatea utilizrii celor dou variante genetice (ce nu au mai fost semnalate pn
acum la nici o alt ras de bivoli din Europa sau din Asia), ca markeri genetici de origine,
autenticitate i trasabilitate a brnzeturilor Romneti de bivoli este imediat.

CAPITOLUL XII
CONCLUZII GENERALE I RECOMANDRI

1. Experimentele de citogenetic au permis cartarea corect pe cromozomii metafazici,


respectiv n teritoriile cromozomiale specifice, a celor patru cazeine i a genei WAP. Ca
urmare recomand aceste protocoale de lucru pentru cartarea genelor de interes pe diveri
cromozomi, studiul linkageului sau crossing-overului, studiul cariotipului n vederea
identificrii unor deleii, duplicaii, inversii, translocaii, care nu sunt vizibile prin metode
clasice de bandare a cromozomilor;
2. n urma analizei comparative a rezultatelor obinute am identificat la locii K-CN,
respectiv -LG aceleai genotipuri, att prin PCR-RFLP, ct i prin IEF. In plus prin
tehnica IEF au fost vizualizate n acelai gel i genotipurile de la ceilali 4 loci ce codific
S1-CN, S2-CN, -CN i -LA, permind o identificare corect a tuturor alelelor, chiar
i a celor mai rare, ce apar cu frecven mai redus;

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

3. Au fost genotipizati prin IEF la cei 6 loci ce codific proteinele majore din lapte un
numr de 693 de indivizi din 7 rase de taurine autohtone, 139 de indivizi dintr-o ras
autohton de bubaline, 282 de indivizi din 6 rase autohtone de ovine, 283 de indivizi
dintr-o ras autohton de caprine. n urma genotipizrii au fost stabilite frecvenele
alelelor i genotipurilor la cei ase loci ce codific proteinele majore din lapte;
4. Ca urmare recomand cele tehnica PCR-RFLP pentru genotipizarea precoce a
reproductorilor pentru cei doi markeri, iar tehnica IEF pentru genotipizarea la toi cei 6
loci, n vederea identificrii mai corecte a genotipurilor i evaluarea nivelului de expresie
al acestor alele;
5. Au fost identificate i caracterizate molecular 3 variante genetice noi, una la locusul
S1-CN la Sura de Step i dou la locii S1-CN i -CN la Bivolul Romnesc. La
caprinele din rasa Carpatin au fost identificate 3 posibile noi variante genetice, doua la
locusul S1-CN i una la locusul -CN, care sunt n curs de caracterizare.
6. Folosind o analiz combinat de detecie a polimorfismului fraciunii cazeinice i a
proteinelor din lactoserum de la speciile de ferm autohtone, am reuit o identificare
rapid a speciilor n probele de lapte de amestec i n brnzeturile analizate. Se poate
concluziona c variantele genetice ale celor 6 proteine majore din lapte ntlnite la
speciile de ferm autohtone (majoritatea fiind comune la toate speciile i rasele din
lume), pot fi folosite cu succes ca markeri genetici de identificare a autenticitii/originii
produselor lactate autohtone.

BIBLIOGRAFIE SELECTIV
1. Addeo F., Moio L., Chianese L., Stingo C., Resmini P., Berner I., Krause I., Di Luccia
A., Bocca A., 1990. Use of plasmin to increase the sensitivity of the detection of bovine
milk in ovine and/or caprine cheese by gel isoelectric focusing of 2 -caseins.
Milchwissenschaft, 45:708-711;
2. Aschaffenburg R., Drewry J., 1955. Occurrence of different beta-lactoglobulins in
cow's milk. Nature, 176:218-219;

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

3. Blteanu V.A., Pop F.D., Vlaic A., Carsai T.C., Creang t., Rusu A.R., 2010a.
Characterization of the S1-casein IRV allele provides evidence for phylogeny of the
ancient Romanian Grey Steppe cattle, Moldavian strain. Lucrri tiinifice Seria
Zootehnie Iai, 53: 315-320;
4. Blteanu V.A., Vlaic A., Pop F.D., Carai T.C., Pascal C., Creang t., Zaharia N.,
Pdeanu I., Voia O.S., Sauer M., Sauer I.W., 2010b. The study of S1-casein genetic
marker polymorphism in Carpathian goat breed: synthesis of 2009 research (Project PN II
52104/2008). Lucrri tiinifice Seria Zootehnie Iai, 53: 321-325;
5. Blteanu V.A., Pop F.D., Vlaic A., Rusu A.R., Creang S., 2008b. Molecular
characterization of S1-CN IRV allele discovered in Romanian Grey Steppe cattle and it's
frequency in this breed. Bulletin of USAMV-CN, Anim. Sci. and Biotech., 65: 477;
6. Blteanu V.A., Pop F.D, Vlaic A.Rusu A.R. , 2008c. Multiple mutations events are
characterizing alpha s1 casein BRV and beta casein CRV alleles discovered in Romanian
Buffalo breed. Bulletin of USAMV-CN, Anim. Sci. and Biotech., 65: 477;
7. Blteanu V.A., Vlaic A., Pop F.D., Rusu A. R., Odagiu A., Cighi V., Creanga S.,
2007d. S1-casein alleles frequency in Carpathian goat. Bulletin of USAMV-CN, Anim.
Sci. and Biotech., 63-64/2007: 527;
8. Chianese L., Garro G., Mauriello R., Laezza P., Ferranti P., Addeo F., 1996.
Occurrence of five s1 casein variants in ovine milk. J. Dairy Res., 63: 49-59;
9. Farrell H. M., Jimenez-Flores R., Bleck G. T, Brown. E. M.,. Butler J. E, Creamer L.
K., Hicks C. L., Hollar C. M., Ng-Kwai-Hang K. F., Swaisgood H. E., 2004.
Nomenclature of the Proteins of Cows Milk-Sixth Revision. J. Dairy Sci., 87:16411674;
10. Giaccone P., Di Stasio L., Macciotta N.P.P., Portolano B., Todaro M., Cappio-Borlino
A., 2000. Effect of betalactoglobulin polymorphism on related traits of dairy ewes milk
analysed by repeated measures design. J. Dairy Res., 67: 443-448;
11. Heck J. M. L, Schennink A., Valenberg H. J. F., Bovenhuis H., Visker M. H.,
Arendonk J. A. M, Hooijdonk A. C. M, 2009. Effects of milk protein variants on the
protein composition of bovine milk, J. Dairy Sci., 92:1192-1202;

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

12. Mah M.F., Miranda G., Queval R., Bado A., Zafindrajaona P.S., Grosclaude F.,
1999. Genetic polymorphism of milk proteins in African Bos taurus and Bos indicus
populations. Characterization of variants s1-Cn H and k-Cn J. Genet. Sel. Evol., 31:
239-253;
13. Ng-Kwai-Hang, K. F., 2006. Genetic variants of milk proteins and their effects on the
yield 484 and quality of cheese. Nutrition and Natural Resources, 56: 1-11;
14. Siciliano R.A., Rega B., Amoresano A., Pucci P., 2000. Modern mass spectrometric
methodologies in monitoring milk quality. Analytical Chemistry, 72: 408-415;
15. Threadgill D.W., Womack J.E., 1990. Genomic analysis of the major bovine milk
protein genes. Nucleic Acids Research, 18: 6935-6942;
16. Veloso A.C.A., Teixeira N., Ferreira I.M., 2002. Separation and quantification of the
major casein fractions by reverse-phase high-performance liquid chromatography and
urea-polyacrylamide gel electrophoresis. Detection of milk adulterations. Journal of
Chromatography, 967: 209-218;
17. Vlaic A., Blteanu V.A., F.D. Pop, 2008. Molecular methods used in detection of
cattle milk in buffalo, ewe and goat in dairy products. Lucrri tiinifice Seria Zootehnie,
Iasi, 51: 1016-1021;
18. Vlaic, A., D.C. Pamfil, Ioana Gaboreanu, B. Vlaic, R. Renaville, 2003. Increasing
milk production in cattle using DNA marker assisted selection (Pit-1).

Bulletin of

USAMV-CN, Anim. Sci. and Biotech., 59:188-191;


19. Vlaic A., Ciobanu D.C., Oroian T., Handoca E., 2002.Variantele genetice ale kcazeinei determinate prin tehnica PCR RFLP i asocierea acestora cu nsuirile
produciei de lapte la rasa Brun. Cercetri de genetic vegetal i animal, ASAS
Bucureti i ICCPT Fundulea, vol. VII;
20. Wedholm A., Larsen L B., Lindmark-Mnsson H., Karlsson A. H., Andrn A., 2006.
Effect of Protein Composition on the Cheese-Making Properties of Milk from Individual
Dairy Cows. J. Dairy Sci., 89: 3296-3305;

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

UNIVERSITY OF AGRICULTURAL SCIENCES AND


VETERINARY MEDICINE CLUJ-NAPOCA
DOCTORAL SCHOOL
FACULTY OF ANIMAL HUSBANDRY AND BIOTECHNOLOGY

Eng. Biologist BLTEANU I. VALENTIN ADRIAN

SUMMARY OF Ph.D. THESIS

THE
STUDY
OF
MAJOR
MILK
PROTEINS
GENETIC
POLYMORPHISMS IN THE MAIN CATTLE, BUFFALO, SHEEP AND
GOAT BREEDS FROM ROMANIA WITH THE AIM OF USING THEM
AS GENETIC MARKERS IN BREEDING AND TRACEABILITY

Scientific advisor
Prof.Univ. Eng. VLAIC AUGUSTIN Ph.D.

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

CONTENTS
INTRODUCTION.................................................................................................31
PART I: BIBLIOGRAPHIC STUDY
CHAPTER I
COMPOSITION OF CATTLE, BUFFALO, SHEEP, GOAT
MILK AND MILK SYNTHESIS MECHANISM IN
LACTATING MAMMARY GLAND..................................................................32
CHAPTER II
MOLECULAR TECHNIQUES USED FOR MILK
PROTEINS POLYMORPHISMS STUDY.........................................................33
CHAPTER III
CURRENT STAGE OF THE RESEARCH CONCERNING
THE STUDY OF MILK PROTEINS POLYMORPHISMS
IN CATTLE, BUFFALO, SHEEP AND GOAT.................................................34
CHAPTER IV
THE IMPORTANCE OF MAJOR MILK PROTEINS
POLYMORPHISMS STUDY BY THEIR POSSIBLE
USE IN ANIMAL BREEDING AND DAIRY
PRODUCTS TRACEABILITY...........................................................................37
PART II: PERSONAL RESEARCH
CHAPTER V
THE RESEARCH AIM AND OBJECTIVES........39
CHAPTER VI
CHROMOSOME MAPPING OF THE GENES CODING
FOR MAJOR MILK PROTEINS BY FLUORESCENCE
IN SITU HYBRIDISATION (FISH).......40

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

CHAPTER VII
THE STUDY BY PCR-RFLP OF THE TWO GENES
POLYMORHISM CODING FOR K-CASEIN AND
-LACTOGLOBULIN IN CATTLE BELONGING
TO ROMANIAN SIMMENTAL BREED ......41
CHAPTER VIII
COMPARATIVE STUDY BY IEF AND PCR-RFLP OF
MAJOR MILK PROTEINS GENETIC POLYMORPHISMS
IN CATTLE BELONGING TO ROMANIAN SIMMENTAL
BREED..........................................................................................................42
CHAPTER IX
THE CHARACTERIZATION OF MAJOR MILK
PROTEINS POLYMORPHISMS IN SOME ROMANIAN
CATTLE, BUFFALO, SHEEP AND GOAT BREEDS
BY IEF TECHNIQUE......42
CHAPTER X
MOLECULAR CHARACTERIZATION OF THE NEW
ALLELES IDENTIFIED IN S1-CASEIN AND
-CASEIN LOCI IN ROMANIAN GREY STEPPE
CATTLE, MODAVIAN VARIETY AND ROMANIAN
BUFFALO..............................................................................................................47
CHAPTER XI
IDENTIFICATION OF DECLARED AUTHENTICITY/
ORIGIN OF MILK AND DAIRY PRODUCTS USING
THE MAJOR MILK PROTEINS POLYMORPHISMS AS
GENETIC MARKERS.....48
CHAPTER XII
GENERAL CONCLUSIONS AND RECOMMENDATIONS.........................49
SELECTIVE BIBLIOGRAPHY..........................................................................50

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

INTRODUCTION
Lactogenesis can be divided in two phases: Phase I - begins in the last stage of
gestation and is characterized by cellular, structural and functional differentiation
processes of the secretor epithelium; Phase I - begins immediately after birth and
involves the ending of cellular differentiation, which coincides with milk secretion and
its synthesis in significant quantities. These two phases are essential having as final
effect the beginning of milk production (the start of lactation).
In ruminants milk there are 6 major milk proteins types, which are coded by 6 nonallelic genes specifically expressed in mammary epithelial cells during lactation: S1casein (S1-CN), -casein (-CN), S2-casein (S2-CN), K-casein (K-CN), lactoglobulin (-LG) and -lactoalbumin (-LA).
The mutations occurring in these genes structure during the years, lead to the
appearance of many alleles in these loci. These variations, named generically
polymorphisms, are caused by genes restructuration (substitution of a nucleotide with
another, deletions, insertions etc), which can have as effects: modification of genes
expression levels, inactivation of genes expression and modification in aminoacid
sequence of the proteins codified by these genes.
Some of these genetic variants from the 6 loci have a positive effect on milk casein
content, coagulation time, curd firmness and cheese making efficiency, others having a
negative effect on these parameters; some -CN genetic variants (A1, B, C) were
associated with some illnesses in humans as Diabetes Mellitus type 1, Ischemic Heart
disease, Sudden Infant Death Syndrome, Schizophrenia and Autism; some of S1-CN
genetic variants are incriminated for causing allergies in children, following milk
consumption.
Using of the information provided by the 6 major milk proteins genetic variants,
cannot be done without a characterization of Romanian farm species/breeds.

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

The characterization accomplished in my Ph.D. Thesis, provides extremely valuable


information about the alleles frequencies in the six milk proteins loci in local Romanian
species/breeds. This can open new possibilities of using these polymorphisms as genetic
markers in animal breeding, in manipulation of milk components by genetic selection in
order to create alternative milk formulas in order to overcome some illnesses, or as
genetic markers in authenticity/origin identification of dairy products.

PART I: BIBLIOGRAPHIC STUDY

CHAPTER I
COMPOSITION OF CATTLE, BUFFALO, SHEEP, GOAT MILK AND
MILK SYNTHESIS MECHANISM IN LACTATING MAMMARY GLAND

The scientific research established that milk has a complex composition, mainly
concerning the casein fraction, whey proteins, enzymes and antimicrobial components.
The milk represents a highly valuable food source containing more than 100 components
in suspension or emulsion needed for a normal development of humans and animals
(Banu et al., 1998).
The milk proteins are divided in two main groups according to their behaviour in
acid pH = 4,6:
1. The soluble fraction in acid pH (whey proteins), is composed of -lactoglobulin (LG), -lactoalbumin (-LA) and other minor proteins. In the cheese making process
by adding chimosin, this fraction remains in whey.
2. The insoluble fraction in acid pH (whole casein) is composed of S1-casein (S1CN), S2-casein (S2-CN), -casein (-CN) and K-casein (K-CN). The caseins are found
in fresh milk in a high number of solid particles in suspension, bounded by Ca9(PO4)6
molecules. These particles are named micelles.

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

The protein content of cattle milk is in average 3,3%, in buffalo milk 4,1%, in sheep
milk 5,3 % and in goat milk 3,7% (Iurc et al., 1998). From the total milk proteins the
casein fraction represents 80% and whey proteins 20%.
The proportion of the casein fraction has a significant influence on milk
manufacturing properties, quantity and quality of obtained cheese, texture and flavour of
different cheeses and other dairy products (Buchberger et al., 2000).
The 6 major milk proteins are coded my 6 non allelic genes, which are specifically
expressed in mammary gland during lactation.
The Mendelian segregation analysis of the 4 non-allelic genes coding for the 4
casein types in ruminants, revealed that they are located on the chromosome pair 6 in the
following order: S1-CN, -CN, S2-CN, K-CN (Threadgill et al., 1990; Hayes et al.,
1993a). The gene coding for -LG was located on the chromosome pair 11 (Hayes et al.,
1993b) and that coding for -LA was located on the chromosome pair 5 (Soulier et al.,
1989; Vilotte et al., 1987).
The endocrine system, mainly the pituitary gland, plays a central role in all aspects
involving growth and development of mammary gland (mamogenesis), starting of
lactation and milk synthesis (lactogenesis), maintaining of milk synthesis (galactopoesis)
and then its activity ending, by two major hormones: growth hormone or somatotroph
(GH or STH) and prolactin (PRL). The hormonal signals induce the transcriptional
activation of the genes coding for major milk proteins and specific protein synthesis.

CHAPTER II
MOLECULAR TECHNIQUES USED FOR MILK PROTEINS
POLYMORPHISMS STUDY
The studies accomplished from the early 50 using paper electrophoresis (PE)
evidenced the milk proteins polymorphism, -LG being the first protein in which a
polymorphism was identified by Aschaffenburg and Drewry (1955).

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

Due to the development of more advanced techniques for protein analysis as: SGE
(starch gel electrophoresis), AE (agarose gel electrophoresis), PAGE (polyacrylamide gel
electrophoresis),

IEF

(isoelectric

focusing),

HPLC

(high

performance

liquid

chromatography), Maldi TOF-MS (mass spectrometry), the knowledge concerning the


polymorphisms of major milk proteins in farm species was enriched substantially.
The appearance of the DNA based techniques: PCR (polymerase chain reaction),
PCR-RFLP (restriction fragments length polymorphism), SSCP (single strand
conformation polymorphism), RT-PCR (real time polymerase chain reaction) and DNA
sequencing techniques, allowed the identification of many polymorphisms in the genes
structure, having repercussions on gene expression and aminoacid composition of milk
proteins.

CHAPTER III
CURRENT STAGE OF THE RESEARCH CONCERNING THE STUDY OF
MILK PROTEINS POLYMORPHISMS IN CATTLE, BUFFALO, SHEEP
AND GOAT
The mutations which appeared over the years in the structure of these genes coding
for the six major milk proteins, lead to the appearance of many genetic variants in these
loci.
These variations, named polymorphisms, indicate the fact that each milk protein is
found in several forms codified by autosomal codominant genes named alleles, meaning
that both alleles are expressed in heterozygous individuals.
Alpha S1-CN is a composed of 199 aminoacids, having a molecular weight of 23,
614 kDa. In cattle in S1-CN locus 10 genetic variants were identified so far: A, B, C,
D, Eyak, Ebali, F, G, H, (Farrell et al., 2004) and IRV (Blteanu et al., 2007a; Blteanu et
al., 2008a, b). The IRV allele was renamed ISM because it was not evidenced in other
Romanian breeds, being specific to Romanian Grey Steppe cattle, Moldavian variety (SM

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

= Moldavian variety). In buffalo, 3 genetic variants were identified so far: A, B


(Chianese et al., 2009) and BRV (Blteanu et al., 2007c; Blteanu et al., 2008c).
Following the characterization in S1-CN locus of Romanian Buffalo populations it was
established that this genetic variant is specific to this breed, being renamed according to
the region in which it was identified S1-CN BBT (BT = Transylvanian Buffalo). In
sheep, 9 genetic variants were identified so far: A, B, C, D, E, F, G, H, I (Pirisi et al.,
1999; Giambra et al., 2010). In goat, 17 genetic variants were identified so far: A, B1, B2,
B3, B4, C, D, E, F, G, H, I, L, M, N, 01 and 02 (Ramunno et al., 2004).
Beta-CN is a protein composed of 209 aminoacids, having a molecular weight of
23,983 kDa. In cattle in -CN locus 14 genetic variants were identified so far: A1, A2,
A3, B, C, D, E, A3m, B2, A4, H, F, A5, G (Farrell et al., 2004). In buffalo, 3 genetic
variants were identified so far: A, B (Ferranti et al., 1998) and CRV (Blteanu et al.,
2007c; Blteanu et al., 2008c). Following the characterization of -CN locus of
Romanian Buffalo populations it was established that this genetic variant is specific to
this breed, being renamed according to the region in which it was identified -CN CBT
(BT = Transylvanian Buffalo). In sheep there is no clear evidence of polymorphism at
the protein level (Chianese et al., 1995). In goat 8 genetic variants were identified so far:
A, A1, B, C, D, E, 0 and 0 (Cosenza et al., 2005a; Caroli et al., 2006).
Alpha S2-CN is a protein composed of 207 aminoacids, having a molecular weight
of 25,150 kDa. In cattle in S2-CN locus 4 genetic variants were identified so far: A, B,
C, D (Farrell et al., 2004). In buffalo, 3 genetic variants were identified so far: A, B, C
(Ferranti et al., 1998). In sheep, 2 genetic variants were identified so far: A, B (Rossi et
al., 1984; Mauriello et al., 1990). In goat, 9 genetic variants were identified so far:: A, B,
C, E, F, G, D, 0 and 0 (Erhardt et al., 2002).
Kappa-CN is a protein composed of 169 aminoacids, having a molecular weight of
19,007 kDa. In cattle in K-CN locus 11 genetic variants were identified so far: A, B, C,
B2, E, F, G, Az, H, I, J (Farrell et al., 2004). In buffalo, 2 genetic variants were identified
so far: A, B (Mitra et al., 1998). In sheep, 1 genetic variant was identified so far
(Chianese et al., 1996; Ceriotti et al., 2004b). In goat, 16 polymorphic sites were
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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

identified, corresponding to 13 protein variants and 3 silent mutations, involving 15


polymorphic sites just in exon 4: A, B, B', B", C, C', F, G, H, I, J, L, D, E, K, M (Jann et
al., 2004b).
Alpha-LA is a protein composed of 123 aminoacids, having a molecular weight of
14,175 kDa. In cattle in -LA locus, 3 genetic variants were identified so far: A, B, C
(Farrell et al., 2004). In buffalo, 2 genetic variants were identified so far: A, B (Chianese
et al., 2004). In sheep, 2 genetic variants were identified so far: A, B (DallOlio et al.,
1989). In goat, 2 genetic variants were identified so far: A1 and A2 (Cosenza et al.,
2005b).
Beta-LG is a protein composed of 162 aminoacids, having a molecular weight of
18,277 kDa. In cattle in -LG locus 13 genetic variants were identified so far: A, B, C,
D, Dr, Dyak, E, F, G, W, H, I, J (Farrell et al., 2004). In buffalo, 2 genetic variants were
identified so far: A, B (Chianese et al., 2004). In sheep, 3 genetic variants were identified
so far: A, B (Kolde et al., 1983; Schlee et al., 1993) and C (Erhardt et al., 1989). In goat
2 genetic variants were identified so far: A and B (Yahyaoui et al., 2000).

CHAPTER IV
THE IMPORTANCE OF MAJOR MILK PROTEINS POLYMORPHISMS
STUDY BY THEIR POSSIBLE USE IN ANIMAL BREEDING AND DAIRY
PRODUCTS TRACEABILITY
The influence of major milk proteins genetic variants on milk quantity

In cattle the influence of major milk proteins genetic variants on milk quantity was
evaluated in several studies. The BB genotypes form S1-CN locus, A2A2 from -CN
locus, AA from -LG locus and AA (+15) genotype from -LA locus, were associated
with a higher milk quantity (Bovenhuis et al., 1992; Bleck et al., 1993b; Ikonen et al.,
1999, Ng-Kwai-Hang et al., 2006).

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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In sheep the studies concerning the influence of -LG genotypes on milk quantity
are contradictory. Bolla et al. (1989) and Caroli et al. (1995) observed in Sarda sheep that
BB genotype is associated with a higher milk quantity. In another study made on a
Sicilian sheep breed, Di Stasio et al. (1992) observed that AB genotypes had the highest
milk production. In Valle del Belice breed, Giaccone et al. (2000) observed that AA
genotype is associated with a higher milk quantity.
In goat Kumar et al. (2006) observed that individuals with AA genotypes at the LG locus had a higher milk production when compared to other genotypes.

The influence of major milk protein genetic variants on milk quality, milk
manufacturing properties and cheese making efficiency

In cattle the CC genotype from S1-CN locus, the BB genotype from K-CN locus
and the BB genotype from -LG locus were associated with a higher casein and whole
protein content in milk (Jakob et al., 1994; Fitzgerald et al., 1997; Lunden et al. 1997).
The BB genotype from -LG locus was correlated with a higher fat content in milk
(Wedholm et al., 2006; Heck et al., 2009). The milk from CC genotypes in S1-CN, the
BB genotypes from -CN locus and BB genotypes from K-CN has a shorter coagulation
time (Delacroix - Buchet et al., 1994; Fitzgerald et al., 1997; Kubarsepp et al., 2005). The
BB genotype from K-CN locus has a positive influence on cheese yield, in comparison
with AA genotypes, the observed differences being situated between 2,7-15% ,
depending on the cheese type produced (Van den Berg et al., 1992; Fitzgerald et al.,
1997; Walsh et al., 1998).
The effects of S1-CN polymorphism on goat milk quality were studied in French
breeds (Mahe et al., 1993; Delacroix et al. 1996, Manfredi et al., 2000). The results
obtained indicated that A allele in comparison with E and F has a significant effect
(positive) on protein and fat content and milk manufacturing properties. In cheese making
experiments the following differences were obtained concerning the cheese quantity:
+7,4% between AA and EE genotypes, +6,9% between EE and FF and +14,8% between
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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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AA and FF genotypes. The cheese obtained from AA genotypes had a weak specific goat
flavour (because of a weaker lipolysis), in comparison with FF genotypes which had a
more pronounced goat flavour (because of a higher lipolysis). Similar results were
obtained in Spanish breeds (Sanchez et al., 1998), Italian breeds (Meggiolaro et al.,
2000), Norwegian breeds (Vegarud et al., 1999) and SUA (Clark et al., 2000).
In sheep the CC genotype from S1-CN locus has a positive influence on milk
composition and cheese yield. In cheese making experiments the following differences
were obtained concerning the cheese yield, +3,5% and +8,6% respectivelly, between the
CC genotype, in comparison with CD and DD genotypes (Pirisi et al., 1999). The BB
genotype from -LG locus was associated with a higher fat content in milk and lower in
lactose, having a favorable effect on cheese yield (Celuk et al., 2006).

The use of major milk protein polymorphism as genetic markers in


authenticity identification of milk and dairy products

At international level a high number of methods were proposed for the identification
of possible adulteration caused by undeclared inter-specific milk mixtures. In most cases
dairy products authenticity identification is based on major milk protein polymorphism
analysis: a) Polyacrylamide gel electrophoresis in native conditions (Kaminarides et
al., 2002); b) Polyacrylamide gel electrophoresis in denaturant conditions (Veloso i
colab., 2002); c) Isoelectric focusing electrophoresis (Addeo et al., 1990; Anonymous,
2001; Blteanu, 2005; Blteanu et al., 2007b,c,d; Vlaic et al., 2008; Blteanu et al.,
2008c); d) Capilarity electrophoresis (Lee et al., 2001); e) Immunochemical methods
- ELISA (Hurley et al., 2004); f) Reverse phase high performance liquid
chromatography (Veloso et al., 2002); g) Mass spectrometry (Siciliano et al., 2000); h)
DNA based techniques (Bottero et al., 2002).

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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PART II: PERSONAL RESEARCH

CHAPTER V
THE RESEARCH AIM AND OBJECTIVES

The study of major milk protein polymorphisms in local species / breeds had as a
goal the evaluation of the possibility of using this information in several directions as:
- improving milk quality (especially of casein content), its manufacturing properties and
cheese making efficiency;
- obtaining of a hypoallergenic milk by selection of individuals with no S1-CN in milk,
which is the main milk allergen;
- obtaining of a healthier milk which is containing -CN genetic variants which dont
have a negative effect on human health;
- authenticity / origin identification of milk and dairy products.
The using of information provided by these genetic markers cannot be done without
the knowledge of genetic polymorphisms occurring in the six loci in cattle, buffalo, sheep
and goat local breeds. This characterization can provide extremely valuable information
about the allele frequencies in the four casein loci and the two whey protein loci in local
breeds.
In the context of actual research knowledge in this research field, the proposed
project has as major objectives research directions with importance on international level,
but little or not known at the national level:
- Setting up of a FISH protocol (fluorescence in situ hybridisation) using BAC probes
(bacterial artificial chromosome), in order to map on metaphase chromosomes and
interphase nuclei the genes coding for major milk proteins;
- The characterization of genetic polymorphisms in the 6 loci coding for the 6 major milk
proteins in some cattle breeds: Romanian Simmental, Romanian Black and White,
Brown, Red Holstein, Transylvanian Pinzagau, Romanian Grey Steppe cattle;

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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- The characterization of genetic polymorphisms in the 6 loci coding for the 6 major milk
proteins in Romanian Buffalo;
- The characterization of genetic polymorphisms in the 6 loci coding for the 6 major milk
proteins in Carpathian goat;
- The characterization of genetic polymorphisms in the 6 loci coding for the 6 major
milk proteins in some local ovine breeds: Turcana, Carabasa, Tigaie, Rusty Tigaie, Cluj
Merinos and Botosani Karakul;
- The molecular characterization of new discovered allele (renamed S1-casein ISM) in
Romanian Grey Steppe Cattle, Moldavian variety, in order to study the possibility of
using it as biodiversity genetic marker;
- The molecular characterization of new discovered alleles in Romanian Buffalo breed
renamed S1-casein BBT and -casein CBT;
- The study of the possibility to use milk proteins polymorphisms from native farm
species/ breeds milk, to identify authenticity and origin of milk, cheeses and other dairy
products, considering also the 2 new casein allele identified in Romanian Buffalo.

CHAPTER VI
CHROMOSOME MAPPING OF THE GENES CODING FOR MAJOR
MILK PROTEINS BY FLUORESCENCE IN SITU HYBRIDISATION
(FISH)

In these experiments I used two detection systems of the hybridised probe: a


system with a single antibody labelled with a red fluorophore (TRITC), antibody
recognising specifically the digoxigenin, which was used to label one of the BAC probes
(the probe containing WAP gene), and a double system, in which a primary antibody is
specifically recognising the biotine (used to label the second probe containing the casein
cluster), which is further recognised by a secondary antibody labelled with a green
fluorophore (FITC).

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
taurine, bubaline, ovine i caprine din Romnia n scopul utilizrii lor ca markeri
genetici n ameliorare i trasabilitate

The hybridising with mentioned probes was done on metaphase chromosomes,


obtained from rabbit fibroblasts cell cultures and mouse mammary tumour cells HC11.
The hybridisation was also done on bi-dimensional and three-dimensional interphase
nuclei with a preserved structure, prepared from HC11 cells. In all cases were obtained
specific hybridisation signals, which allowed a correct mapping on the chromosomes and
specific chromosomes territories of studied genes.

CHAPTER VII
THE STUDY BY PCR-RFLP OF THE TWO GENES POLYMORHISM
CODING FOR K-CASEIN AND -LACTOGLOBULIN IN CATTLE
BELONGING TO ROMANIAN SIMMENTAL BREED
The study of K-CN i -LG genes polymorphism had a goal testing some PCRRFLP protocols, which two allow the identification of the two common A and B allele
from these loci. In the case of K-CN a 350 bp fragment was amplified and digested with
Hinf I, three restriction profiles being observed corresponding to AA, AB and BB, from
this locus. In the case of -LG, a 262bp fragment was amplified and digested with Hae
III, three restriction profiles being observed corresponding to AA, AB and BB, from this
locus. The calculation of genotypes frequencies in K-CN locus, revealed a higher
frequency of AB genotype, in comparison with the other two. The calculation of genes
frequencies this locus revealed a higher frequency of A allele, in comparison with B
allele. The calculation of genotypes frequencies in -LG locus revealed a higher
frequency of AB genotype, in comparison with the other two. The calculation of genes
frequencies this locus revealed a higher frequency of B allele, in comparison with A
allele.
The obtained results are indicating that the two DNA tests can be used in
identification of the common A and B allele from the two loci, allowing a precocious
genotyping of cattle for the two genetic markers.

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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CHAPTER VIII
COMPARATIVE STUDY BY IEF AND PCR-RFLP OF MAJOR MILK
PROTEINS GENETIC POLYMORPHISMS IN CATTLE BELONGING TO
ROMANIAN SIMMENTAL BREED

The study was done on 14 individuals belonging to Romanian Simmental breed,


which were comparatively genotyped in K-CN and -LG loci by PCR-RFLP and IEF.
This experiment had as a goal the evaluation of genotyping efficiency at the DNA level
(by PCR-RFLP) and at the expression level directly from milk (by IEF), in the loci
coding form major milk proteins.
Following the comparative analysis were identified in K-CN and -LG loci the
same genotypes by PCR-RFLP and IEF. Moreover by IEF were identified in the same gel
the genotypes in the other 4 loci coding for S1-CN, S2-CN, -CN and -LA, allowing
a correct identification of all allele, even of those which are very rare.
The obtained results are evidencing the fact that IEF technique allows to establish
more precisely the genotypes in the 6 loci, even of the rare allele, difficult and costly
identifiable by PCR.

CHAPTER IX
THE CHARACTERIZATION OF MAJOR MILK PROTEINS
POLYMORPHISMS IN SOME ROMANIAN CATTLE, BUFFALO, SHEEP
AND GOAT BREEDS BY IEF TECHNIQUE
n cattle were genotyped by IEF in the 6 loci coding for major milk proteins a
number of 693 individuals belonging to 7 breeds, as follows: Romanian Simmental (236
individuals), Romanian Black and White (230 individuals), Red Holstein (13
individuals), Brown (134 individuals), Transylvanian Pinzgau, black variety (26

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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individuals) and red variety (30 individuals) and Romanian Grey Steppe cattle,
Moldavian variety (24 individuals).
In S1-CN locus two common genetic variants were identified, namely B and C.
The frequency of B allele was 0,9 in improved breeds in direction of higher milk
production, the highest frequency being observed in Red Holstein breed. The lowest
frequency was observed in Black Pintzgau. The frequency of C variant, associated in
many cattle breeds with a higher milk processing quality, was the biggest in Black
Pintzgau (0,308), this allele being absent in Red Holstein breed. In S1-CN casein locus
a new allele, renamed ISM, was identified in Romanian Grey Steppe cattle, Moldavian
variety, with an isoelectric point between those of B and C genetic variants. The
frequency of this new allele, identified by IEF, was 0,041 in analysed populations.
In -CN locus five genetic variants were identified: A1, A2, A3, B, C. The
frequency of ancestral allele A2 was the highest in almost all breeds (over 0,5) in
comparison with the others allele. The exception was observed in Red Pintzgau, in which
the frequency of A1 variant was higher (0,483), in comparison with the other alleles. The
A3 variant was detected only in one individual belonging to Romanian Simmental breed.
The B variant, associated in many cattle breeds with a higher milk processing quality, has
an extremely reduced frequency in all breeds (between 0,039-0,117). The C variant was
detected with a very low frequency in Romanian Simmental, Brown and Pinzgau, black
variety, breeds. In Grey Cattle the presence of C variant was detected only in individuals
from Tazlau i Tupilati, this being a proof of infusion with Brown breeds. It was not
detected in individuals, considered pure breed, from SCDCB Dancu.
In S2-CN locus only one genetic variant was identified, namely A variant, with
the frequency 1.
In K-CN locus three genetic variants, named A, B and C, were identified. The
highest frequency of A variant was observed in Black and White breed (0,835) and of B
variant was observed in Brown (0,619). In Romanian Simmental breed the frequency of
A allele is very high (0,679). The C variant was detected with a low frequency in
Romanian Simmental and Brown breeds.
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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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In -LA locus only one genetic variant was identified, namely B, with the
frequency 1.
In -LG locus three genetic variants were identified: A, B and C. The frequency of
A variant was the highest in Romanian Simmental and Grey Steppe cattle (over 0,5). The
frequency of B allele is high in all breeds, in Red Holstein breed being 0,769.
In buffalo were genotyped by IEF in the 6 loci coding for major milk proteins a
number of 139 individuals belonging to Romanian Buffalo. In S1-CN and -CN loci
were identified with a 0,86 frequency two common allele: A in S1-CN and B in -CN
locus. Two new genetic variants, named S1-CN BSM and -CN CSM, were identified
with a frequency of 0,14. They were observed in linkage on chromosome 6. In the other
four loci just one allele was identified with the frequency 1.
In sheep were genotyped by IEF in the 6 loci coding for major milk proteins a
number of 282 individuals belonging to six breeds, as follows: Turcana (44 individuals),
Carabasa (40 individuals), Tigaie (45 individuals), Rusty Tigaie (28 individuals), Cluj
Merinos (35 individuals), Botosani Karakul, black variety (15 individuals), dark grey
variety (15 individuals), brown variety (15 individuals), light grey variety (15
individuals), pink variety (15 individuals), white variety (15 individuals).
In all six ovine breeds genotyped, the milk proteins polymorphisms was very
reduced, the only polymorphic locus being -LG, in which two genetic variants were
identified A and B. The A variant had the highest frequency (over 0,5) in Turcana,
Carabasa, Cluj Merinos, and Karakul respectively, having the highest frequency in
Karakul, white variety. The B allele had a higher frequency in comparison with A allele
in Tigaie and Rusty Tigaie breeds.
In S1-CN, -CN, S2-CN, K-CN and -LA just one allele was identified in each
locus with the frequency 1. The presence of C allele in S1-CN locus has a positive
meaning, because this allele was associated in many studies with higher milk processing
parameters and higher cheese yield.
In goat were genotyped by IEF in the 6 loci coding for major milk proteins a
number of 283 individuals belonging to Carpathian goat.
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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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In S1-CN four allele categories, associated with four expression levels were
identified. The results indicate a 0,569 frequency of high expression allele in S1-CN (A,
B, C) and a frequency of 0,431 in the case of medium, low expression and null alleles. If
we consider that in Romania there are 700.000 goats and the calculated frequency of E, F
and 0 allele is 43,1%, we can deduce that a number of 301.700 of Carpathian goat are
producing a milk with a low protein content, which are significantly affecting milk
quality and cheese making efficiency. These results can explain the heterogeneity of
Carpathian goat concerning some milk quality parameters. In S1-CN locus, two new
possible alleles were identified, named S1-CN 0 and S1-CN X. The first was identified
with a 0,021 frequency and is characterised by the absence of S1-CN in milk. It was
observed always in linkage with an unknown profile in -CN locus, proving the linkage
of the two alleles on the same chromosome. The second genetic variant, S1-CN X, was
observed just in one individual in a heterozygous condition with B allele. Based on the
electrophoresis profile, it seems to present in the sequence a major restructuration,
probably caused by an exon skipping phenomenon as that found in F allele. Based on the
electrophoresis bands colour intensity, was concluded that this variant belongs to high
expression alleles category.
In -CN locus three genetic variants were identified. The common A and C
variants were identified with a 0,979 frequency. They were not differentiated by IEF due
to the same isoelectric point. In some individuals was identified, with a frequency of
0,021, an unknown genetic variant named -CN X, always in linkage with S1-CN 0.
In S2-CN locus three genetic variants were identified. The variant C of S2-CN
had the highest frequency (0,553), in comparison A variant (0,431), the E allele having a
low frequency (0,016).
In K-CN locus two genetic variants were identified. The A variant was
predominant (0,912), the B variant having a low frequency (0,088). The presence of B
variant in Carpathian goat has a positive significance, as long as this allele was associated
in different studies with a high milk processing quality.
In -LG i -LA, just one allele was identified in each locus with the frequency 1.
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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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genetici n ameliorare i trasabilitate

CHAPTER X
MOLECULAR CHARACTERIZATION OF THE NEW ALLELES
IDENTIFIED IN S1-CASEIN AND -CASEIN LOCI IN ROMANIAN
GREY STEPPE CATTLE, MODAVIAN VARIETY AND ROMANIAN
BUFFALO
In Romanian Grey Steppe cattle
A new genetic variant in cattle S1-CN locus, renamed ISM, was identified in
Romanian Grey Steppe cattle, Moldavian variety, being complete characterized using a
combined methodology of protein, cADN and DNA sequencing.
By Maldi Tof-MS analysis, a substitution in the position 192 of mature protein
was identified. In this region ISM variant is containing Gly as C variant and not glutamic
acid, present in B variant. The other substitutions which are making the difference
between ISM variant and B or C were not clearly identified by Maldi Tof-MS analysis.
Comparative analysis of chromatograms obtained following sequencing (with the
forward and reverse primer, respectively) of the 2 samples carriers of S1-CN ISM (BISM,
CISM) and 2 reference samples (BB, CC), revealed the mutations characterizing this new
genetic variant: substitution of an adenine from C and B allele (exon 11, nucleotide 297,
codon 99 gaA coding for Glu) with a thymine in the ISM allele (gaT coding for Asp);
substitution of an adenine from the B allele (exon 17, nucleotide 620, codon 207 gAa
coding for Glu) with guanine in C and ISM alleles (gGa coding for Gly). This last
substitution was confirmed at the protein level by Maldi Tof-MS analysis.
There are 2 additional substitutions observed in the B, C and ISM alleles (sequenced
from Romanian Grey Steppe cattle, Moldavian variety), that differ from some sequences
published in GenBank, with no effect on aminoacid sequence of protein.
Based of the substitution of an adenine from position 21 of exon 11 from C allele
(adenine found in B allele too), with a timine in ISM allele, a PCR-RFLP test was
developed based on the amplification and restriction with BseGI enzyme of a 422 base

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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pairs (bp) fragment from S1-CN gene, including entire exon 11. Because the presence
of this new S1-CN allele was noticed only in pure breed individuals, its origin in this
breed is unquestionable. This theory is also sustained by the high frequency of this allele
in the genotyped Grey Steppe cattle population: 0,128, frequency relatively high for a
new genetic variant. This suggests that ISM variant had an even higher frequency at the
time when this breed and its progenitors were the only ones existing in Romania.
Based on similarities and differences observed at the molecular level, between S1CN B, C and ISM allele, was concluded that this new allele was issued directly from the
ancestral C allele, bringing the first molecular clue about phylogenetic relationships
among the Romanian Grey Steppe cattle and Bos genus representative breeds from Africa
and Asia.

In Romanian Buffalo
The cADN sequencing of S1-CN A (common allele) and BSM alleles from
Romanian Buffalo, revealed two point mutations characterizing BSM allele, different from
the other known allele in Mediterranean and Indian Buffalo. The deletion of entire exon 6
is a very interesting type of restructuration, shortening the sequence of mature protein
with 8 aminoacids, in comparison with the normal protein which has 199 aminoacids.
Three further mutations were identified, which are similar or different from those found
in common allele.
The cADN sequencing of -CN B (common allele) and CBT alleles, leaded to
identification of four further mutations differentiating the two variants and affecting the
predictable protein sequence. Furthermore two additional silent substitutions were
identified with no effect on protein sequence, as long the resulting codons are coding for
the same aminoacids.
As long as these new identified allele are specific to Romanian Buffalo they could
be easily used as genetic markers for Romanian Buffalo cheeses authenticity/origin
identification.

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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CHAPTER XI
IDENTIFICATION OF DECLARED AUTHENTICITY/ORIGIN OF MILK
AND DAIRY PRODUCTS USING THE MAJOR MILK PROTEINS
POLYMORPHISMS AS GENETIC MARKERS

If in other EU countries, products adulteration cases are quite rare, in Romania


undeclared cow milk (produced at a lower price and in higher quantities), is often added
into buffalo, goat or ewe milk, normally fated to produce ,,authentic dairy products,
especially in the case of industrial processed milk.
The official European methodology for cheeses authenticity identification is based
on the IEF analysis of gamma casein fractions, resulting from the hydrolysis of -CN by
plasmin, having the disadvantage that it cannot be differentiated goat and sheep milk.
Up-to-date in Romania there is no identification methodology of undeclared interspecific milk addition in dairy products.
The researches described in this Chapter had as a main goal the study of
possibility to use milk proteins polymorphisms, found in whole six loci from native farm
species studied, as genetic markers to identify authenticity and origin of dairy products.
Also, the study of possibility of using the two alleles identified in Romanian Buffalo CN CSM and S1-CN BSM, as genetic markers for origin/authenticity identification of
Romanian Buffalo cheeses, was another goal of this research.
The authenticity analysis concerning the possible identification of undeclared
inter-specific milk mixtures was done on 12 cheese types form Romanian market, as
follows: 2 types of cattle cheeses, 3 types of buffalo cheeses, 3 types of sheep cheeses
and 4 types of goat cheeses.
In the case of cattle cheeses it was found that these were produced only form cattle
milk. This is not surprising because the using of other milk types in cattle cheese
production in less probable. In the case of buffalo cheeses, it was observed that the
majority of them were produced exclusively from cattle milk. In the case of sheep

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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cheeses it was observed that two of them were produced exclusively from sheep milk,
and one had a 50% cattle milk. In the case of goat cheeses it was observed that three of
them were exclusively produced from goat milk, the forth one being falsified 100% with
cow milk.
The comparison between two buffalo cheeses types, a Mozzarella type produced
form Italian buffalo milk from Campania region, Italy and a salty cheese produced from
Romanian Buffalo from Transylvania, clearly revealed that they can be differentiated by
the presence in salty cheese of the S1-CN BSM and -CN CSM genetic variants, which are
absent in Italian Buffalo. Therefore the possibility of using these genetic variants (which
were not observed so far in other European or Asian buffalo breeds), as genetic markers
for authenticity origin and traceability identification of Romanian buffalo cheeses is
possible immediately.

CHAPTER XII
GENERAL CONCLUSIONS AND RECOMMENDATIONS
1. The cytogenetic experiments allowed a correct mapping on metaphase chromosomes
and specific chromosome territories of the four caseins and WAP gene. As a consequence
I recommend these protocols for mapping of genes on specific chromosomes territories,
the study of linkage, crossing over and of the karyotype, in order to identify deletions,
duplications, inversions, translocations, which are not visible by classical cytogenetic
methods.
2. Following the comparative analysis were identified in K-CN and -LG loci the same
genotypes by PCR-RFLP and IEF. Moreover by IEF were identified in the same gel the
genotypes in other 4 loci coding for S1-CN, S2-CN, -CN and -LA, allowing a
correct identification of all alleles, even of those which are very rare.
3. Were genotyped by IEF in the 6 loci coding for major milk proteins a number of 693
individuals belonging to 7 cattle breeds, 139 individuals belonging to a local buffalo
breed, 282 individuals belonging to six local sheep breeds and 283 individuals belonging
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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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to a local goat breed. Following genotyping alleles and genotypes frequencies, in the 6
loci coding for major milk proteins, were calculated.
4. As a consequence I recommend the PCR-RFLP technique for precocious genotyping
of cattle for the two markers and the IEF technique for genotyping in the 6 milk protein
loci, to a more precisely genotyping and for the evaluation of alleles expression levels.
5. There were identified and molecular characterized 3 new genetic variants, one in
Romanian Grey Steppe cattle in S1-CN locus and two in S1-CN and -CN loci in
Romanian Buffalo. In Carpathian goat breed were identified 3 new possible genetic
variants, two in S1-CN locus and one in -CN, which are in progress of
characterization.
6. Using a combined analysis for detection of casein fraction and whey proteins
polymorphisms found in Romanian farm species, were rapidly identified the species in
the bulk milk samples and analysed cheeses. It can be concluded that the genetic variants
of major milk proteins found in Romanian farm species (the majority of them being
found in all world breeds), can be successfully used as genetic markers in identification
of authenticity/ origin dairy products.

SELECTIVE BIBLIOGRAPHY
1. Addeo F., Moio L., Chianese L., Stingo C., Resmini P., Berner I., Krause I., Di Luccia
A., Bocca A., 1990. Use of plasmin to increase the sensitivity of the detection of bovine
milk in ovine and/or caprine cheese by gel isoelectric focusing of 2 -caseins.
Milchwissenschaft, 45:708711;
2. Aschaffenburg R., Drewry J., 1955. Occurrence of different beta-lactoglobulins in
cow's milk. Nature, 176:218-219;
3. Blteanu V.A., Pop F.D., Vlaic A., Carsai T.C., Creang t., Rusu A.R., 2010a.
Characterization of the S1-casein IRV allele provides evidence for phylogeny of the
ancient Romanian Grey Steppe cattle, Moldavian strain. Lucrri tiinifice Seria
Zootehnie Iai, 53: 315-320;

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Studiul polimorfismelor genetice ale proteinelor majore din lapte la principalele rase de
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4. Blteanu V.A., Vlaic A., Pop F.D., Carai T.C., Pascal C., Creang t., Zaharia N.,
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