【作者】 决肯·阿尼瓦什；

【导师】 刘守仁； 谢庄；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 我国是世界养羊大国,新疆是我国主要绵羊生产基地,巴什拜羊是新疆乃至国内外不可多见的地方优良品种,具有特殊生物学特征和特性。近年来,虽然对巴什拜羊进行了本品种选育为主的多方面常规育种工作,但对巴什拜羊的生物学特性和遗传多样性方面分子水平的研究很少,未见从形态特征到分子遗传标记的系统研究报告。本文采用从形态标记、细胞遗传标记、生化标记到分子遗传标记的各项研究方法,比较系统的研究了巴什拜羊的生物学特性和遗传多样性,为今后肉羊生产中常规育种与分子育种相结合的研究、传统畜牧业的提升、区域经济的发展提供一些实践和理论依据。1.巴什拜羊生物学特性研究发现(1)巴什拜羊种质特性研究显示：巴什拜羊的外形呈方圆形,躯体各部位结构匀称,肉用型明显,遗传性稳定、属于结实性体质。毛属粗毛,毛色以宗红色毛为主,有黑色和白色毛。角形以公母羊都无角为主,有两角和多角形。巴什拜断奶公、母羔羊生长指标之间没有明显差异,生长指标的差异随着年龄的增加而加大,到成年差异最显著(P<0.01)。巴什拜羊从断奶到成年一直保持屠宰率、净肉率和骨肉比等主要产肉指标平均56%、45%和1：4kg以上的水平,放牧条件下春季到秋季抓膘性能达20—25%1巴什拜羊受胎率97%,繁殖力110%、羔羊成活率98%以上。巴什拜羊的基础生理指标在正常范围之内,血液生理生化指标中血清胆固醇含量低其它地方绵羊品种,其它成分属于正常范围之内。巴什拜公、母羊胆小,容易受惊,野性大,但合群性好,在正常情况下很难将牧群分开,具有良好的放牧特性,表现为采食快、爬山能力强,采食过程中的选择性不强,采食前进速度和采食速度较快。(2)巴什拜羊产肉性能研究显示：巴什拜羊在没有任何补给草料的自然放牧条件下,4.5月龄断奶羔羊平均屠宰率为56.00%、胴体重为19.0kg、净肉率为45.7%、骨肉比为1：4.00kg,周岁公、母羊和成年公、母羊同样的保持这个水平。以上四个主要产肉指标居全国首位,基本接近欧洲6月龄育肥羔羊的中等水平巴什拜羔羊眼肌面积15.60cm2,腰部肌厚和大腿肌厚各4 cm2,臀脂占活重的5%,总脂肪占活重的19.39%。肉色鲜红、肉嫩多汁,营养成分全面,蛋白质含量19.38%、脂肪含量10.1%,胆固醇含量42mg/100g,脂肪酸种类齐全,必需脂肪酸含量44.25%,必需氨基酸和鲜味氨基酸含量占总氨基酸含量的88.27%,微量元素含量丰富。(3)生长规律研究显示：巴什拜羊一出生就进入最高体重增长速度和强度,也是从初生到0-60日龄绝对增长速度最高,公羔393g-295g/d,母羔372g-275g/d,相对增长强度强,公羔80.38%-19.22%、母羔78.37%一18.92%,4月龄羔羊体重35.5kg,达到成年羊体重的一半以上,典型的早熟绵羊品种。(4)巴什拜羊与野生盘羊杂交结果分析显示：野生盘羊杂交三代(回交二代)羔羊的瘦肉重显著的高于纯巴什拜羊(P<0.05),而肌内脂肪和背部脂肪层厚度显著的低于纯巴什拜羔羊(P<0.05),脂臀重、总脂肪重、脂臀占活重率、总脂肪占活重率、腰部脂肪厚度等指标及显著的低于纯巴什拜羊(P<0.01)。回交二代羊其屠宰率、胴体重、净肉率和骨肉比与纯巴什拜羊的成绩相似,但总脂肪比纯巴什拜羊减少3910g,净肉重增多了15.0%,脂臀重减少了85.3%,腰部及大腿肌层各增厚0.5cm,腰部和背部脂肪层各减薄1.5cm和1.Ocm,总脂肪含量减少53.36%,尾脂肪、肌内脂肪、肾脂肪和大网膜(脂肪)都有减少的趋势。纯巴什拜羊和盘羊杂交后代羊肉的常规营养成分中,水分、蛋白质含量、钙、磷和胆固醇之间有一定的数值差异,但差异不显著(P>0.05),脂肪含量之间存在显著差异(P<0.05)。所有脂肪酸含量之间不存在显著差异(P>0.05)。纯巴什拜羊肉成分中钾的含量显著(P<0.05)的高于杂交后代羊,杂交后代羊肉中锌的含量级显著(P<0.01)的高于纯巴什拜羊,其它成分差异不显著(P>0.05)。纯巴什拜羊和盘羊杂交后代羊肉的每个氨基酸含量之间差异不显著,但必需氨基酸和鲜味氨基酸总合值之间存在显著差异(P<0.05)。巴什拜羊改良效果明显,有保留本身的优点,也吸收野生盘羊的优点,杂交后代出现双重效益的杂种优势。2.巴什拜羊多态性研究发现(1)形态标记研究证实：巴什拜羊的毛色有红、黑、白三种颜色,受三对有显性等级的复等位基因的控制,显性等级为红>黑>白,处于Hardy-Weinberg不平衡状态(P<0.05)。红毛基因的基因、基因型频率和产肉性能比其它毛色羊占优势,巴什拜羊的不同毛色可以作产肉性能的形态遗传标记参考。巴什拜羊角与其它的绵羊一样受三对复等位基因的控制和从性遗传的支配。但角形遗传比较特殊,有多角型,多角羊在公、母羊中都有,是特有的品种标志,有角巴什拜羊的屠宰率低于无角羊。巴什拜羊群体中角形基因处于Hardy-Weinberg不平衡状态(P<0.05)。这与实际育种工作方向相符。巴什拜羊的白脸性状受一对基因的控制,而且绝对显性的常染色体遗传。(2)细胞遗传标记研究显示：巴什拜羊正常体细胞的染色体数为2n=54,性染色体构成为,雄性XY；雌性XX。正常体细胞(2n=54)占总观察细胞数的比率为90.42%,而2n≠54的细胞频率为9.58%。发现部分染色体的结构和数量变异,可能属于正常范围内,也可能是试验失误。(3)生化遗传标记证实：巴什拜羊LDH同工酶含量顺序为LDH1> LDH3> LDH2 > LDH5> LDH4;与其它品种之间存在多态性。LDH同工酶中LDH1和LDH3与巴什拜羊的体重有着级显著(P<0.001)的正相关,LDH2、LDH4和LDH5与巴什拜羊的体重呈负相关,而且LDH2和LDH4呈级显著(P<0.001)的负相关。LDH1和LDH3可以作巴什拜羊产肉性状早期选种的辅助遗传标记参考。Es同工酶中除了Es5与巴什拜羊的体重呈极显著(P<0.001)的正相关外,其余的全部呈负相关,而且Es1、Es3、Es4呈级显著(P<0.01)的负相关,表明,Es同工酶中只有Es5可以作巴什拜羊产肉性状的早期选中辅助遗传标记参考。Tf基因座的七个等位基因中Tfa和Tfp为优势基因,基因频率分别为0.3750和0.2292。Hb基因座具有A、B两个等位基因,各基因频率为0.5208和0.4792。发现巴什拜羊的HbAB介于高原型与平原型之间,海拔适应范围比较广,可作为早期选择的遗传标记参考。HbAB基因型各类群的各座位均处于Hardy-Weinderg平衡状态(P>0.05),比较适合于公羊的早期种选择。(4)微卫星遗传标记研究证实：①在10个微卫星位点中,共检测到110个等位基因,平均每个座位等位基因数11个。②巴什拜羊红毛品系、黑毛品系、白毛品系和瘦肉型新品系的平均多态信息含量(PIC)分别为0.7926、0.7690、0.7721和0.8320,平均杂合度(H)分别为0.8174、0.7985、0.7921和0.8468,遗传多样性丰富。③巴什拜羊群体的总近交系数为-0.1782,群体内近交系数为-0.2112,群体间基因分化系数为0.0237,巴什拜羊2.37%的遗传变异来自群体间,而97.63%的遗传变异是由个体间的差异引起的；基因流Nm平均值为8.9167,没有遗传分化现象。聚类分析发现,红毛品系与黑毛品系亲缘关系较近,之后与白毛品系相聚,最后与瘦肉型新品系聚在一起,聚类结果与品系育成史基本一致。④微卫星座位BM143、OARJMP8、BL1038、OARHH35、ILSTS018、BMS648 BM143、BM4311八个标记对巴什拜羊红毛、黑毛、白毛三个品系的体重体尺都有不同程度的显著性相关,但与野生盘羊杂交的瘦肉型品系没有任何显著性相关。其中BMS648对红毛品系的所有生长指标都呈级显著的相关(P<0.01),BL1038对体长、胸围、管围有极显著相关(P<0.01),体高和体重有显著相关(P<0.05),BM143对胸围、管围体重有极显著相关(P<0.01),对体长有显著相关(P<0.05), ILSTS018对体长、管围、体重有极显著相关(P<0.01),对胸围有显著相关(P<0.05),其他座位没有相关或相关性不大。OARHH35对黑毛品系的体重有极显著相关(P<0.01),对体长和胸围有极显著相关(P<0.05),其他座位没有相关或相关性不大。BMS648座位对白毛品系的体高和体长有极显著相关(P<0.01),对胸围和体重有显著相关(P<0.05),其他座位没有相关或相关性不大。对差异显著的座位不同基因型进行多重比较显示：同一座位不同基因型对不同品系生长指标产生正、负效应。(5)巴什拜羊Callipyge基因和Leptin检测发现采用PCR-SSCP技术分析巴什拜羊双肌臀(CLPG)基因和瘦素(LEPTIN)基因SNPs,发现CLPG基因和LEPTIN基因的SNPs在巴什拜羊群体中存在多态性,在CLPGD和CLPGE引物扩增区域四个群体都发现了AA、AB和BB三种基因型,但没有发现A→G碱基突变。LA、LB引物扩增区域发现AA、AB基因型,LC引物扩增区域发现、BB两个纯合基因型。更多还原

【Abstract】 China is one of the largest sheep producing country in the world. Xinjiang Provinces is its major production base for sheep. Bashibai sheep is a best breed in found in mainly in Xinjiang its other provinces and also in few other countries. Bashibai sheep possess special biological features and characteristics. Recently, conventional breeding of Bashibai sheep was performed, with little research about biological characteristics and genetic diversity at the molecular level and no research has been carried out from morphological characteristics to the systematic study with molecular genetic markers.In this study, we have used various research methods as morphological marker, cell genetic markers, biochemical markers of molecular genetic markers, the biological characteristics and genetic diversity of Bashibai sheep was more systematic studied, sheep production in the future by the conventional and molecular breeding of systematic breeding methods, the improvement of modern animal husbandry, development of regional economy to provide some practical and theoretical basis.1. Biological characteristics studied are:(1) Bashibai sheep germplasm characteristicsBashibai sheep showed a square-round shape, the body symmetry of all parts of the structure, apparently the meat-type with genetic stability and strong body. Coarse hair, mainly coat with red hair, also black and white hair, absences/presences of horn and polygons.There was no significant difference at weaning between male and female lambs by growth parameters, growth index differences increase with age, the most significant difference for the adult sheep was (P<0.01).Dressing percentage, meat percentage and meat-bone ratio from weaning to adult was found with an average of 56%,45% and 4:1. Performance from spring to autumn fatten up to 20-25% wtih grazing conditions.Pregnancy rate, fertility rate and lamb survival rate was 97%,110% and 98% respectively.Bashibai sheep was common in the normal physiological range, physiological and biochemical indicators of blood serum cholesterol were recorded low in comparison of other indigenous sheep breeds while other components are within the normal range.Bashibai sheep was timid, easily frightened, good gregarious, difficult to separate the herd in normal circumstances, with good grazing characteristics, showed faster feeding, climbing ability, the choice of feeding process is not strong, feeding rate and feed forward faster.(2) Meat production performance of Bashibai sheepBashibai sheep in the natural grazing conditions with absence of any forage supply, the average dressing percentage of 4.5 months old weaned lambs was 56.00%,19.0kg carcass weight,45.7% meat percentage, meat-bone ratio of 4:1, aged and adult male, ewes also maintain this level. These four major meat production index ranks first in China, which is close to the middle level of 6 months old lamb in Europe.Bashibai sheep loin eye area 15.60 cm2, waist thick and big thigh muscle thick 4 cm, hip fat and total fat each accounted for 5% and 19.39% of live weight.Bashibai sheep with flesh red, tender and juicy, full nutrition, trace elements abundant, protein content 19.38%, fat content 10.1%, cholesterol content 42mg/100g, essential fatty acid content 44.25%, essential amino acids and delicious amino acids accounting for 88.27% ototal amino acid.(3) Growth pattern of Bashibai sheepBashibai sheep was typically premature sheep breeds, the highest absolute growth rate and intensity of weight gain is 0-60 days from birth, male lambs 393g-295g/d, female lambs 372g-275g/d.High relative growth intensity,80.38%-19.22% for male lambs, 78.37%-18.92% for female lambs, four-month old lamb weight 35.5kg, more than half of adult sheep weight.(4) Bashibai sheep hybrid with Ovis Argali (wild sheep) analysisHybridization of three generations (backcross generation) lean meat weight was significantly higher than pure Bashibai sheep (P<0.05), and intramuscular fat and back fat thickness was significantly lower than pure Bashibai sheep (P<0.05), hip fat weight and total fat weight, fat hip and live weight ratio, total fat and live weight ratio, waist fat thickness were measured and found significantly lower than pure Bashibai sheep (P<0.01).The rate of the dressing percentage, lean meat weight, meat percentage and meat-bone ratio of backcross generation were similar with pure Bashibai sheep, but total fat 3910g less than pure Bashibai sheep, lean meat weight increased 15.0%, lipid hip decreased 85.3%, waist and thigh muscle thick added 0.5 cm, waist and back fat layer thinning of 1.5cm and 1.0cm, total fat content decreased 53.36%. Tail fat, intramuscular fat, kidney fat and large networks membrane fat were decreased.Conventional meat nutrients, as moisture, protein, calcium, phosphorus and cholesterol level showed some differences, but not significant (P> 0.05), fat content is significant (P<0.05).Hybrid progeny and pure Bashibai sheep, no significant difference (P>0.05) was recorded between the fatty acidsPure Bashibai sheep components of meat potassium were significantly (P<0.05) higher than hybrid progeny, zinc level was significantly (P<0.01) lower, other components are not significant difference (P>0.05).Hybrid progeny and pure Bashibai sheep between each amino acid is not significantly different, but the essential amino acids and delicious amino acids combined significant difference (P<0.05).Hybrid progeny improved significantly, have retained Bashibai sheep’s own advantages, also absorb the advantages of Ovis Argali, double-efficient hybrids heterosis. 2. Polymorphism study of Bashibai sheep(1) Morphological markersBashibai sheep hair with red, black, white, is affected by three levels dominant control of multiple alleles, rating of the red> black> white, in the Hardy-Weinberg disequilibrium (P>0.05). Red-gene’s gene, genotype frequency and meat productivity advantage over others hair color of sheep, the different hair color can be used for meat production performance of morphological genetic markers.Bashibai sheep angular genetic rather special, with polygonal, polygonal in the male and female sheep, a unique species mark. The angular affected by three pairs of alleles from the genetic control and domination. The dressing percentage of horn sheep was lower than non-horn sheep, which features in the same group, same age, same sex and live weight sheep showed more prominent. The angular gene in Hardy-Weinberg genetic imbalance is (P> 0.05). This is consistent with the direction of actual breeding. Bashibai sheep white face traits controlled by a pair of genes, and certainly autosomal dominant genetic.(2) Cell genetic markers Bashibai sheep normal body cells of chromosome number 2n=54, sex chromosome constitute the male XY,female XX. Normal somatic cells (2n=54) of the total observed number of cells was 90.42%, while the 2n≠54 cell frequency of 9.58%. Some chromosome structure and number of variations may fall within the normal range.(3) Biochemical genetic markersBashibai sheep LDH isoenzyme levels LDH1> LDH3> LDH2> LDH5> LDH4; with polymorphisms among other species.LDH1 and LDH3 has a significant (P<0.01) positive correlation to the sheep’s weight. LDH2, LDH4 and LDH5 was negatively correlated with body weight, LDH2 and LDH4 significantly (P<0.01) negative correlation. LDH1 and LDH3 could be the early selection of sheep meat production traits subsidies genetic markers.Es isozymes in addition to Es5 was significantly (P<0.01) positive correlation with body weight, the rest of all was negative correlation, and Esl, Es3, Es4 was significantly (P<0.01) negative. So only Es5 can be used for Bashibai sheep meat production characteristics to the early genetic markers.Tfa and Tfp was the dominant gene of the seven alleles of Tf loci, gene frequencies were 0.3750 and 0.2292. Hb locus A, B alleles, the gene frequency of 0.5208 and 0.4792. We found that HbAB between plateau and the plain, with wide range of altitude adaptation, can be used as early selection of genetic markers. HbAB genotype of each group of the seats are in Hardy-Weinderg equilibrium (P> 0.05), more appropriate for male sheep’s early selection.(4)Microsatellite markersa. In 10 microsatellite loci, a total of 110 alleles were detected, the average number of alleles per locus 11.b. The average of Bashibai sheep red hair strain, black hair strain, white hair strain and new lean strain polymorphism information content (PIC) was 0.7926,0.7690,0.7721 and 0.8320, heterozygosity (H) were 0.8174,0.7985,0.7921 and 0.8468, genetic diversity.c. The global heterozygote deficit across all populations was-0.1782, inbreeding as-0.2112, coefficient of genetic differentiation was 0.0237, It was indicated that 2.37% of the total genetic variation could be explained by breed differences and remaining 97.63% by differences among individuals for each population. The average gene flow was 8.9167. The cluster analysis showed that the genetic relationship between red hair strain and black hair strain was nearer, and then got together with white hair strain, and at last got together with new lean sheep. The cluster analysis was generally in accordance with their breeding history.d. Microsatellite loci of BM143, OARJMP8, BL1038, OARHH35, ILSTS018, BMS648 BM143, BM4311 eight markers have different significantly correlated with body size and weight on three strains of red hair, black hair, and white hair. However, the lean strains have no significant correlation. BMS648 for all growth parameters were significantly correlated (P<0.01) on red hair strait. BL1038 has significant correlation (P<0.01) with body length, chest and tibia circumference, body height and weight were significantly correlated (P<0.05). BM143 has significant correlation (P<0.01) with chest and tibia circumference, body weight and length was significantly correlated (P<0.05). ILSTS018 has significant correlation (P<0.01) with body length, tibia circumference and body weight, chest circumference was significantly correlated (P<0.05). Others loci were not relevant. OARHH35 has significant correlation (P<0.01) with body weight on black hair strait, and body length and chest circumference were significantly correlated (P<0.05). Others loci were not relevant. BMS648 has significant correlation (P<0.01) with body height and weight on white hair strait, chest circumference and body weight were significantly correlated (P<0.05). Others loci were not relevant.Significant difference between the loci of the different genotypes of multiple comparisons showed that: the same loci on different strains of different genotypes have positive and negative effects on growth index.(5) Callipyge and Leptin geneThe double muscled gene (CLPG) and leptin gene (LEPTIN) SNPs of Bashibai sheep detected by PCR-SSCP, found CLPG and LEPTIN SNPs polymorphism.CLPG1 and CLPG2 primer amplification regions found AA, AB and BB genotypes in the four groups but did not find mutation A→G. L1, L2 regions found AA, AB genotype, L3 regions found AA, BB homozygous genotype.更多还原