目标区域扩增多态性(TRAP):一种新的植物基因型标记技术

TRAP是一种新的基于PCR的植物基因型标记技术,具有简单、稳定、效率高的特点。借助日益增长的庞大的生物序列信息,TRAP利用生物信息工具和EST数据库信息,产生目标候选基因区多态性标记。TRAP技术采用两个18核苷酸引物产生标记。一个为固定引物,依据EST序列设计;另一个为随机引物,针对外显子和内含子的特点,设计为分别富含GC或AT核心区的任意序列。PCR扩增前5个循环采用35℃的退火温度,后35个循环采用50℃的退火温度。对不同的植物种类,每一个PCR反应可产生多达50个可统计DNA片段。本文在阐述了TRAP的原理与流程后,对该技术的优势和应用情况进行了总结,并对其前景做了展望。     

虾源副溶血弧菌致病基因检测与ERIC-PCR分型

为了监测上海市养殖对虾中副溶血弧菌致病基因的携带情况及潜在致病株流行情况,采用PCR方法对上海地区不同对虾养殖单位中分离获得的19株副溶血弧菌分离株及1株标准菌株进行6种致病基因的检测筛查,并采用ERIC-PCR方法进行基因分型分析。结果发现:19株副溶血弧菌中未检测出携带tdh、ORF8基因,只有1株携带trh基因,而tox RS/new基因携带率较高,19株中有14株检出阳性,基因携带率为73.7%。对能够引起对虾急性肝胰腺坏死病(AHPND)的副溶血弧菌特异性质粒基因的检测中发现,AP1和AP2基因均有3株检测出阳性,基因携带率均为15.8%。通过ERIC-PCR分型技术将20株副溶血弧菌分成7个不同的类群,其分辨力指数DI值为0.811,表明ERIC-PCR具有较好的基因分型能力,分型结果发现该方法能够有效区分不同时间段获得的菌株,而对不同地理位置获得的菌株区分并不明显。以上结果表明从养殖对虾中分离得到的副溶血弧菌致病基因携带率总体偏低,但存在一定的流行风险,需要防范一些新的致病株的流行,这些可以为对虾养殖单位中副溶血弧菌致病流行株的预防控制提供相关参考。     

短枝木麻黄无性系鉴定及其指纹图谱构建

[目的]构建木麻黄无性系的DNA指纹图谱鉴定体系,为今后短枝木麻黄的品种鉴定、品种权保护、新品种的选育提供依据。[方法]从71对EST-SSR引物中筛选出扩增条带清晰、多态性好、扩增稳定的12对引物,采用降落PCR和毛细管电泳的基因分型方法,以采集的9个短枝木麻黄标准无性系的基因分型结果为参照,对华南沿海地区采集的109个短枝木麻黄无性系单株进行鉴定,并结合引物组合法构建基于EST-SSR分子标记的短枝木麻黄无性系鉴定体系。[结果]12对EST-SSR引物对109个短枝木麻黄无性系单株进行PCR扩增,共检测到50个等位基因,每个位点的等位基因数为3～6个,平均为4.2个,每对引物可区分2～5个短枝木麻黄无性系。根据鉴定结果,最终所有样品被划分为22个无性系,除已知的9个标准无性系外,另有13个无性系为不知名无性系,说明不同地区的无性系存在重复命名现象;利用引物组合法进一步优化后,发现只需7对引物就可将22个短枝木麻黄无性系完全区分,并依此建立了基于7对EST-SSR引物的22个短枝木麻黄无性系的指纹图谱,每个无性系都获得了一个7位数的指纹图谱编码。[结论]利用7对EST-SSR引物构建的22个短枝木麻黄无性系的DNA指纹图谱可用于短枝木麻黄无性系的鉴别,研究表明,华南沿海地区部分短枝木麻黄无性系存在命名混乱、同物异名、无性系数量少的现象,新品种选育工作亟待开展和加强。     

绵羊MKRN3基因多态性与产羔数的关联分析

本实验旨在探究绵羊MKRN3基因g.275981C>T与g.276999C>T位点多态性与绵羊产羔数之间的关系,以期为绵羊高繁殖力分子育种提供新的遗传标记。利用全基因组重测序结合Sequenom MassARRAY~?SNP技术对常年发情绵羊品种(小尾寒羊、策勒黑羊和湖羊)和季节性发情绵羊品种(滩羊、苏尼特羊和草原型藏羊)MKRN3基因2个多态位点多态性进行检测,并与小尾寒羊产羔数进行关联分析。结果表明:MKRN3基因g.275981C>T位点与g.276999C>T位点均存在3种基因型;g.276999C>T位点基因型频率和等位基因频率在2种发情模式绵羊品种间差异均达到极显著水平(P<0.01);g.275981C>T位点在6个绵羊品种中均表现为低度多态(PIC<0.25),g.276999C>T位点在6个绵羊品种中均表现为中度多态(0.25T位点在苏尼特羊和策勒黑羊中均处于哈代温伯格平衡状态(P>0.05),g.276999C>T位点在6个绵羊品种中均处于哈代温伯格平衡状态;关联分析表明,g.275981C>T和g.276999C>T位点不同基因型与小尾寒羊第1、2、3胎产羔数均无显著关联。可见,MKRN3基因2个多态位点均不适合用于小尾寒羊产羔数选育。     

绵羊GTF2A1基因多态性及其与产羔数的关联分析

本研究旨在探究绵羊GTF2A1基因g.89505005G>A位点多态性与产羔数之间的关系,以期寻找与绵羊产羔数有关的分子标记。基于前期利用全基因组选择信号分析获得的候选基因GTF2A1,采用Sequenom MassARRAY~?SNP技术对多羔和单羔的绵羊群体及产羔数存在差异的小尾寒羊群体进行g.89505005G>A位点多态性检测,并与产羔数进行关联分析。结果表明:GTF2A1基因g.89505005G>A位点存在3种基因型:AA、AG和GG,且基因型频率和等位基因频率在单、多羔品种间差异均达到显著水平;该位点在小尾寒羊、滩羊、苏尼特羊、萨福克羊、杜泊羊以及草原藏羊6个品种中均处于哈代温伯格平衡状态;关联分析表明:g.89505005G>A位点多态性与小尾寒羊第1、第2以及第3胎产羔数均存在显著关联,AA型各胎产羔数均高于GG型(P<0.05)。综上,A等位基因可能是提高绵羊产羔数的一个潜在有效的DNA标记。     

Effect of selection and selective genotyping for creation of reference on bias and accuracy of genomic prediction

Reference populations for genomic selection usually involve selected individuals, which may result in biased prediction of estimated genomic breeding values (GEBV). In a simulation study, bias and accuracy of GEBV were explored for various genetic models with individuals selectively genotyped in a typical nucleus breeding program. We compared the performance of three existing methods, that is, Best Linear Unbiased Prediction of breeding values using pedigree‐based relationships (PBLUP), genomic relationships for genotyped animals only (GBLUP) and a Single‐Step approach (SSGBLUP) using both. For a scenario with no‐selection and random mating (RR), prediction was unbiased. However, lower accuracy and bias were observed for scenarios with selection and random mating (SR) or selection and positive assortative mating (SA). As expected, bias disappeared when all individuals were genotyped and used in GBLUP. SSGBLUP showed higher accuracy compared to GBLUP, and bias of prediction was negligible with SR. However, PBLUP and SSGBLUP still showed bias in SA due to high inbreeding. SSGBLUP and PBLUP were unbiased provided that inbreeding was accounted for in the relationship matrices. Selective genotyping based on extreme phenotypic contrasts increased the prediction accuracy, but prediction was biased when using GBLUP. SSGBLUP could correct the biasedness while gaining higher accuracy than GBLUP. In a typical animal breeding program, where it is too expensive to genotype all animals, it would be appropriate to genotype phenotypically contrasting selection candidates and use a Single‐Step approach to obtain accurate and unbiased prediction of GEBV.     

Genotyping of groups of cows to improve genomic breeding values of new traits

Genotyping females and including them into the reference set for genomic predictions in dairy cattle is considered to provide gains in reliabilities of estimated breeding values for selection candidates. This should especially be true for low heritability traits. By the use of simulation, we extended a genomic reference set for an existing trait by including a fixed number of genotyped first‐crop daughters for one or two generations of reference sires. Moreover, we calculated results for the effects of a similar strategy in a situation where for a new trait the recording of phenotypes has recently started. For this case, we compared the effect of two different genotyping strategies: first, to phenotype cows but to genotype their sires only, and second, to collect phenotypes and genotypes on the same cows. We studied the effects on validation reliabilities and unbiasedness of predicted values for selection candidates. We found that by extending the reference set with genotyped daughters it is possible to increase the validation reliability of genomic breeding values. In the case of a new trait, it is always better to collect and use genotypes and phenotypes on the same animals instead of using only sire genotypes. We found that the benefits that can be achieved are sensitive to the sampling strategy used when selecting females for genotyping.     

绵羊Tacr3基因多态性与产羔数的关联分析

本研究旨在探究绵羊速激肽受体3(tachykinin receptor 3,Tacr3)基因g.21484478A>C、g.21560640C>T和g.21560688T>C位点多态性与绵羊产羔数之间的关系,为绵羊高繁殖力分子育种提供新的遗传标记。利用全基因组重测序结合Sequenom MassARRAY?SNP技术对常年发情绵羊品种(小尾寒羊、湖羊和策勒黑羊)和季节性发情绵羊品种(滩羊、苏尼特羊和草原型藏羊)Tacr3基因3个多态位点进行检测,并与小尾寒羊产羔数进行关联分析。结果表明,3个位点均存在3种基因型,其中g.21484478A>C位点存在AA、CC和CA基因型,g.21560640C>T位点存在TT、CC和TC基因型,g.21560688T>C位点存在TT、CC和CT基因型,3个多态位点基因型频率和等位基因频率在两种发情模式绵羊品种间均达到差异极显著水平(P<0.01)。群体遗传学分析表明,g.21484478A>C位点在滩羊和策勒黑羊中均表现为中度多态(0.25T位点在6个绵羊品种中均表现为中度多态;g.21560688T>C位点在小尾寒羊、苏尼特羊、湖羊和草原型藏羊中均表现为低度多态。卡方适合性检验表明,g.21560640C>T位点在6个绵羊品种中均处于哈代-温伯格平衡状态(P>0.05);g.21484478A>C位点在草原型藏羊中处于哈代-温伯格不平衡状态(P<0.05);g.21560688T>C位点在滩羊、策勒黑羊中处于哈代-温伯格不平衡状态(P<0.05)。关联分析表明,3个多态位点与小尾寒羊第一、二、三胎产羔数均无显著关联(P>0.05)。推测Tacr3基因3个多态位点均不适用于小尾寒羊产羔数选育。     

鲁中肉羊BMPR1B、BMP15和GDF9基因多态性与产羔数关联分析

为分析绵羊已知多羔主效基因BMPR1B、BMP15和GDF9的多态性与鲁中肉羊产羔数之间的关系,采用Sequenom MassARRAY■SNP技术检测鲁中肉羊BMPR1B、BMP15和GDF9基因中已知主效位点多态性,并与产羔数进行关联分析。结果表明:BMPR1B基因在鲁中肉羊中存在FecB突变,GDF9基因6个分型位点仅4个位点(G1、G3、G4、G5)在鲁中肉羊中存在多态,BMP15基因中5个分型位点在鲁中肉羊中均不存在多态性。FecB基因3种基因型(CC、CT、TT)突变频率分别为0.16、0.58和0.26,此位点多态性较高(0.25相似文献