安徽白山羊多胎性能相关分子标记的AFLP筛选
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摘要
为了提高山羊繁殖性能,以增加山羊规模化生产的经济效益,为高繁殖力山羊标记辅助育种提供基础材料,以黄淮山羊的安徽地方类群(安徽白山羊)不同繁殖率群体为试验对象,进行了扩增片断长度多态性(AFLP,amplified fragment length polymorphism)分析.
试验设计了8条EcoRⅠ和8条TaqⅠ引物,共计64对引物组合,采集了10只高繁殖性能(连续3胎,每胎产4羔以上)的多胎安徽白山羊和10只低繁殖性能(连续3胎,每胎只产1羔)的安徽白山羊的耳组织样,构建了代表安徽白山羊高繁殖力和低繁殖力的两个池DNA,利用AFLP技术分析了安徽白山羊两个极端群体DNA水平的差异,以期获得与安徽白山羊多胎性能相关的分子标记,并通过这些分子遗传标记加快多胎安徽白山羊的选育进展,为安徽白山羊的进一步选育提供一定的理论依据.
研究结果表明,AFLP技术适用于山羊基因组DNA的差异分析。实验AFLP图谱条带清晰、丰富,共获得32条差异片段,其中阳性差异片段24条,阴性差异片段8条。选择其中差异条带多、清晰的引物组合12对进行了重复实验,所获得的差异条带经过回收、TA克隆测序获得两条差异条带的序列信息,一条序列长374bp,另一条序列长254bp。所获得的两条差异条带序列经比对后发现374bp的序列存在同源序列,而254bp的序列没有同源序列.
为验证AFLP分析所获得的序列能否作为安徽白山羊多胎性能的分子标记,实验根据比对结果针对374bp的序列,重新设计引物,并进行了PCR-RFLP分析验正。实验采集了104只安徽白山羊耳组织样,其中高产群12只,低产群14只,普通群78只。结果在安徽白山羊群体中发现两种基因型,其中AA型12只,AB型92只,没有发现BB型.高产群、低产群和普通群的AB基因型频率、AA基因型频率分别为0.9167,0.7123,0.9103和0.0833,0.2857,0.0897。高产群和普通群的AA基因型频率都比相应的低产群要低,可推测等位基因B对安徽白山羊高繁殖性能有正向作用,而A基因对安徽白山羊的繁殖性能有负向效应。
To improve reproductive performance of goats can improve economic benefit.In this study technology of amplified fragment length polymorphism(AFLP) was used in different prolificacy white goats of Anhui province,which can offer basic matetial of Marker Assistant Breeding in prolific Anhui white goats.
This experiment designed 8 random primers of EcoRⅠand TaqⅠ,respectively,namely 64 pairs of primer combinations.Twenty individual ear tissue samples were collected from Anhui white goats,ten from high prolificacy,and other from low prolificacy.DNA was extracted from ear tissues and constructed two DNA pools.Technology of AFLP was used to analyze the differences between high and low prolificacy goats,aimed to obtain molecular marker for fecundity of Anhui white goats.The purpose was to improve the Anhui white goats breeding.
The result of this study showed the technology of AFLP was suitable for DNA analysis of Anhui white goats.Bands of the AFLP linkage map were clear and abundant,and 32 differential bands were obtained,24 of them were positive bands,8 of them were negative bands.Twelve pairs primers,which amplified clear and abundant bands,were screened from 64 pairs primers that were used in repeated experiment,and obtained two clear differential bands.These two differential bands were extracted through gel extraction kit and purified and cloned and sequenced,and then two sequenceinformations were obtained,one of them was 374bp and the other was 254bp.These two sequences were blasted in NCBI database,the sequence length 374bp had homologous sequences,but the sequence length 254bp had no homologous sequences.
If the 374bp sequence was a molecular marker for fecundity of Anhui white goats,a pair primers was designed based on the blast result,and then verified by polymerase chaine reaction restriction fragment length polymorphisms(PCR-RFLP).The result showed two genotypes were found in 104 Anhui white goats,AA genotype were 12,AB genotype were 92 and BB were not found.The frequency of genotype AA in prolific group,low prolificacy group and normal group was 0.9167,0.7123 and 0.9103,respectively.The frequency of genotype AB in three groups was 0.0833,0.2857 and 0.0897,respectively.The frequency of genotype AA in prolific group and normal group were lower than low prolificacy group.It can be inferred that gene B made positive effect in prolificacy in Anhui white goat,and gene A made negative effect in prolificacy.
试验设计了8条EcoRⅠ和8条TaqⅠ引物,共计64对引物组合,采集了10只高繁殖性能(连续3胎,每胎产4羔以上)的多胎安徽白山羊和10只低繁殖性能(连续3胎,每胎只产1羔)的安徽白山羊的耳组织样,构建了代表安徽白山羊高繁殖力和低繁殖力的两个池DNA,利用AFLP技术分析了安徽白山羊两个极端群体DNA水平的差异,以期获得与安徽白山羊多胎性能相关的分子标记,并通过这些分子遗传标记加快多胎安徽白山羊的选育进展,为安徽白山羊的进一步选育提供一定的理论依据.
研究结果表明,AFLP技术适用于山羊基因组DNA的差异分析。实验AFLP图谱条带清晰、丰富,共获得32条差异片段,其中阳性差异片段24条,阴性差异片段8条。选择其中差异条带多、清晰的引物组合12对进行了重复实验,所获得的差异条带经过回收、TA克隆测序获得两条差异条带的序列信息,一条序列长374bp,另一条序列长254bp。所获得的两条差异条带序列经比对后发现374bp的序列存在同源序列,而254bp的序列没有同源序列.
为验证AFLP分析所获得的序列能否作为安徽白山羊多胎性能的分子标记,实验根据比对结果针对374bp的序列,重新设计引物,并进行了PCR-RFLP分析验正。实验采集了104只安徽白山羊耳组织样,其中高产群12只,低产群14只,普通群78只。结果在安徽白山羊群体中发现两种基因型,其中AA型12只,AB型92只,没有发现BB型.高产群、低产群和普通群的AB基因型频率、AA基因型频率分别为0.9167,0.7123,0.9103和0.0833,0.2857,0.0897。高产群和普通群的AA基因型频率都比相应的低产群要低,可推测等位基因B对安徽白山羊高繁殖性能有正向作用,而A基因对安徽白山羊的繁殖性能有负向效应。
To improve reproductive performance of goats can improve economic benefit.In this study technology of amplified fragment length polymorphism(AFLP) was used in different prolificacy white goats of Anhui province,which can offer basic matetial of Marker Assistant Breeding in prolific Anhui white goats.
This experiment designed 8 random primers of EcoRⅠand TaqⅠ,respectively,namely 64 pairs of primer combinations.Twenty individual ear tissue samples were collected from Anhui white goats,ten from high prolificacy,and other from low prolificacy.DNA was extracted from ear tissues and constructed two DNA pools.Technology of AFLP was used to analyze the differences between high and low prolificacy goats,aimed to obtain molecular marker for fecundity of Anhui white goats.The purpose was to improve the Anhui white goats breeding.
The result of this study showed the technology of AFLP was suitable for DNA analysis of Anhui white goats.Bands of the AFLP linkage map were clear and abundant,and 32 differential bands were obtained,24 of them were positive bands,8 of them were negative bands.Twelve pairs primers,which amplified clear and abundant bands,were screened from 64 pairs primers that were used in repeated experiment,and obtained two clear differential bands.These two differential bands were extracted through gel extraction kit and purified and cloned and sequenced,and then two sequenceinformations were obtained,one of them was 374bp and the other was 254bp.These two sequences were blasted in NCBI database,the sequence length 374bp had homologous sequences,but the sequence length 254bp had no homologous sequences.
If the 374bp sequence was a molecular marker for fecundity of Anhui white goats,a pair primers was designed based on the blast result,and then verified by polymerase chaine reaction restriction fragment length polymorphisms(PCR-RFLP).The result showed two genotypes were found in 104 Anhui white goats,AA genotype were 12,AB genotype were 92 and BB were not found.The frequency of genotype AA in prolific group,low prolificacy group and normal group was 0.9167,0.7123 and 0.9103,respectively.The frequency of genotype AB in three groups was 0.0833,0.2857 and 0.0897,respectively.The frequency of genotype AA in prolific group and normal group were lower than low prolificacy group.It can be inferred that gene B made positive effect in prolificacy in Anhui white goat,and gene A made negative effect in prolificacy.
引文
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