导　　师： 陈宏;袁志发

学科专业： 090501

授予学位： 博士

作　　者： ;

机构地区： 西北农林科技大学

摘　　要： 本研究采用了RAPD、PCR-RFLP、微卫星、PCR-SSCP、序列分析共5种方法对2个牛品种/(秦川牛和黑白花牛共60头/)、6个中国固有绵羊品种/(多胎品种小尾寒羊，双胎品种大尾寒羊，单胎品种兰州大尾羊、蒙古羊和哈萨克羊共190余只/)进行了标记研究，旨在找到牛羊多胎性状的合适的分子标记，为进一步对牛双胎基因、绵羊多胎基因的探索和高繁殖率牛羊的选育提供科学依据。本研究主要得到了以下几个方面的结果： 1牛双胎性状的标记研究： 1．1微卫星标记 选择位于在不同染色体上的12个微卫星位点，对单胎母牛混合样、随机双胎母牛混合样以及极端个体/(连续产双胎的母牛/)混合样进行扩增，结果发现有4个微卫星位点HEL13、BM1824、ETH152及INRA005可以作为双胎的标记位点。即：①在EHL13位点极端个体混合样缺一条230bp的等位基因；②在BM1824位点极端个体混合样缺一条263bp的等位基因；③在ETH152位点极端个体混合样多出222bp和206bp两条等位基因；④在INRA005位点极端个体混合样缺一条217bp的等位基因。这种现象在两个牛种中均存在。 1．2PCR-RFLP标记 FSHR基因5’端/(包括侧翼序列和第一外显子/)的限制性内切酶TaqⅠ的PCR-RFLP标记研究发现，在秦川牛中双胎母牛的A基因频率/(0.55/)和AA基因型频率/(0.5/)分别高于单胎母牛的A基因频率/(0.35/)和AA基因型频率/(0.3/)：在黑白花奶牛中双胎母牛的B基因频率/(0.5937/)和BB基因型频率/(0.6875/)均高于单胎母牛的B基因频率/(0.5416/)和BB基因型频率/(0.4167/)，也就是FSHR基因的5’端的PCR-RFLP标记在一个品种内单胎母牛和双胎母牛之间有一定趋势，但两个品种的趋势不同。 1．3 PCR-SSCP柝记 牛的FSHR基因第10外显子的PCR-SSCP标记研究发现，秦川牛的双胎牛突变基因型的频率为60％，远远高于单胎牛的突变基因型频率/(20％/)；奶牛中双胎牛的突变型频率为31.25％，单胎牛的突变型频率为6.67％。因此用FSHR基因第10外显子的多态性对牛的双胎性状标记是可靠的。 1．4 序列分析牛的FSHR基因第10外显子的序列分析发现秦川牛和奶牛中突变型的DNA序列在FSHR基因的第1506个碱基发生了突变，即T→C，所编码的氨基酸没有变。 2绵羊多胎性状的标记研究 2．1 RAPD标记 从30条RAPD引物中筛选出的5条随机引物对小尾寒羊的产羔性状进行标记，扩增结果发现引物0424-198的第6条带与小尾寒羊的产羔数成显著正相关/(P＜0.05/)，第7条带与产羔数之间成显著负相关/(P＜0.05/)；引物OPW19的第1条带与产羔数之间成极显著的正相关/(P＜0.01/)；CYO184引物的第7、第8条带接近正相关。 2．2 微卫星标记选用位于绵羊第6号染色体上与Fec~8基因紧密连锁的微卫星OarAE101，BM1329，BM143和OarHH55以及位于第4号染色体上的OarHH35对小尾寒羊等6个绵羊品种的产羔性状标记研究发现： /(1/)5个微卫星位点在6个绵羊品种的PIC都达到了高度多态/(＞0.7/)水平，说明中国固有绵羊品种具有丰富的遗传多样性。 （2）5个微卫星位点标记小尾寒羊的高繁殖率性状，以oarAE101效果最好，找到了6个有 效标记；其次为 BM1329，找到了 4个有效标记：BM143和 oarHH35一般，分别找到了 2个和 3个 有效标记；在oarHH55位点上没有找到有效标记。 （3）5个微卫星位点对小尾寒羊产羔性状标记研究产生的15个有效标记，表现为： ①与产羔数有极显著正相关（尸＜0刀1）的标记有5个，其相关大小顺序为OarAE10lE（113hp， r。O．5295）＞＊M143－＊J（118加门06加，r一0．4432）＞Ors＊N35－N（139加，t一0．4355）＞Otf＊日ic卜J （103hp，r—0．3792）＝OarAE10lEJ（ll3b／103hp，r—0。3792）： ②与产羔数呈显著正相关（P＜005）的标记有 2个，其相关大小顺序为 OarHH35NT （门9加门27…，r＝0．31 52）＞＊M门29－M（门帅，r＝0．31刀）； ③与产羔数呈极显著负相关（P＜0刀1）的标记有4个，其负相关强弱顺序为OarAE10fK （门比p／l hp，r——0．5295）＞OarAE10lF（11比p，r——0．5202）＞BM1329－N（168hp，r——4091） ＞OarAEI川－K（IO比p，r＝－o．3792）； ④与产羔数呈显著负相关（P叩．05）的标记有4个，其负相关强弱］l匝序为BM143上门， r＝－0．爿刀）＞SMI 329＊（旧6加，r一0．3053）＞＊M1329－FN（1％加门68加，r——0．33％） ”OarHH35－OU（137hP／125bP，f——0．1920）。如果按夹角大小衡量更为明确，即标记与产羔数间的 夹角凡是在 72度以下的为正有效标记，夹角大于约！of度的标记为负有效标记，与相关关系的 结果一致。 （4）品种间的基因频率比较发现，小尾寒羊的频率显著高于其它品种且与其产羔数呈正相 关的基因位点有：OarHH35－N（139bp，OarAE川IE（113hp）和OarAE10！－J（103hp）。而与产羔数呈 极显著负相关的且与单胎品种的频率相近（兰州大尾羊除外）的基因位点有：＊。rAEIO卜厂1门印） 和OafAE10lK（］olbp），这两个标记可以作为单胎性状标记；在BM143－E（116hp）和BM143－J（106hp） 小尾寒羊除低于哈萨克羊的基因频率外，高于其余的4个品种，而E与产羔 60 cows from two bovine breeds /(Qinchuan cattle and Holstein cattle/) and 197 sheep from six ovine breeds /(Small Tail Han sheep with prolific traits , Big Tail Han sheep with twinning trait, and Mongolia sheep and Kazakh sheep and Lanzhou Big Tail sheep with 1 lamb per lambing /) were analysed by RAPD, PCR-RFLP, microsatellite, PCR-SSCP and squencing markers , The object was to obtain effective markers for t ovine and ovine fecundity traits , as well as provide some scientific basis for searching for twining gene in bovine and prolific gene in ovine and for selecting and breeding prolific bovine and ovine . The study results were as follows: 1 The marker results for bovine twining traits: 1.1 Micosatellite markers 12 microsatellites from Bos tauras autosome were amplified effectively in DNA pools of monovular cows , twinning cows and special twinning cows /(often bear twin calves/), of which 4 microsatellite loci could marker bovine twining traits. As follows: Special twining cows have no 230bp allele compared with monovular cows at HEL13 locus. Special twining cows have no 263bp allele compared with monovular cows at BM1824 locus. /(3/) Special twining cows have 222bp and 206bp alleles, but monovular cows have not that at ETH152 locus. Special twining cows have no 217bp allele compared with monovular cows at INRA005. These results appeared in two cattle breeds 1. 1.2 PCR-RFLP markers PCR-RFLP in FSHR gene 5'-flanking region which includes exon 1 digested with restriction endonuclease Taq I demonstrated that the frequency of allele A /(0.5500/) and genotype AA /(0.5000/) in twinning cows were higher than that of allele A /(0.3500/) and genotype AA /(0.300/) in monovular cows for Qinchuan cattle, while The frequency of allele B /(0.5937/) and genotype BB/(0.6875/) in twinning cows were higher than that of allele B /(0.5416/) and genotype BB /(0.4167/) in monovular cows for Holstein cattle. 1.3 PCR-SSCPmarkers Results of bovine FSHR gene exon 10 analysed with PCR-SSCP showed that the mutation frequency with 60/% in twinning cows was higher than in monovular cows /(20/%/) in Qinchuan cattle; and the mutation frequency was 31.25/% in twinning cows and 6.67/% in monovular cows for Holstein cattle respectively. The results provided a reliable evidence for bovine prolific reproductive traits. 1.4 Sequencing Sequencing for FSHR gene exon 10 demostrated that there was a mutation locus at 1506bp, i.e. T-C, but amino acid which coded had no change. These results appeared in two cattle breeds. 2 Molecular markers for ovine fecundity traits: 2.1 RAPD markers 5 RAPD primers selected from 30 primers were used to estimate the correlation between the markers and litter size of Small Tail Han sheep. The results showed that the sixth band of primer 0424-198 had a significant positive correlation with litter size /(P<0.05/), and the seventh band of this primer had a significant negative correlation with litter size /(<0.01/); The seventh and eighth bands of primer CY0184 almost reached significant positive correlation with litter size. 2.2 Microsattelites markers Four microsatellite loci mapped at chromosome 6 and lingked to FecB gene /(OarHH35, OarAElOl, BM1329, BM143, OarHH55/) and another microsatellite locus mapped at chromosome 4 /(OarHH55/) were selected to detect the correlation with litter size traits in six sheep breeds. The results were followes: /(1/) The PIC of 5 microsatelite loci all reached high polymorphism /(PIO0.7/), it identified Chinese sheep have abundant genetic diversity. /(2/) 5 mcrosatellite loci makers for the litter size of Small Tail Han sheep indicated that the OarAElOl was best one , BM1329 was better than BM143 and OarHH35, there were no any effective markers at OarHH55. /(3/) 15 effective markers from above five microsatellites were discovered to marker the litter size of Small Han Sheep. Realts as follows: 5 markers from five microsatellites had been detected the significant positive correlation with the litter size /(.PO.01/), i.e. OarAElOl-E /(113bp, r = 0.5295/) > BM1

关 键 词： 牛 双胎性状 绵羊 多胎性状 微卫星 序列分析 分子标记

分 类 号： [S813]

领　　域： [农业科学] [农业科学]