大菱鲆(Turbot)微卫星标记的筛选与应用
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摘要
本研究利用微卫星标记,以亲缘关系较远的大菱鲆父母本杂交构建的一个F1家系为作图群体,构建了大菱鲆遗传连锁图谱并对相关生长性状如:体长,体重,体厚进行了QTL分析。本研究的具体内容如下:
1、采用富集文库-菌落原位杂交的方法构建大菱鲆微卫星富集文库,共筛选得到952个克隆,通过序列比对和重复序列筛查,得到含有重复序列的独立克隆753个,占全部克隆数的79.10%。通过tandem repeats finder (TRF)软件的进一步筛查,得到1004个含串联重复序列的位点,其中微卫星位点831个(82.77%),小卫星序列位点173个(17.23%)。利用实验室设计的软件对序列进行分析,得到结论如下:1)微卫星重复序列中,重复序列数目所占比例与累计长度的所占比例情况基本一致,两碱基重复类型累计长度所占的比例最大,其次是四碱基、三碱基、六碱基和五碱基类型,而单碱基重复类型在本研究中鲜有发现;2)重复单位拷贝数变异能力分析表明,变异系数最大的是三碱基;3)重复序列重复单位长度与其拷贝数的相关分析表明,二者呈负相关(r = -0.5, P= 0.072),即随着重复单位的增加,其拷贝数在减少。以上结果为物种之间微卫星分布频率和丰度的比较、微卫星标记的开发及微卫星进化和功能的研究等工作提供基础。
2、本研究利用了富集文库-菌落原位杂交的方法开发的653个微卫星标记,利用含有F1代亲本在内的多个个体对位点进行多态性评价,共得到284个多态性的位点,其中158个标记在所构建图谱家系亲本间具有多态性,分别有125个和118个标记可用于母本和父本遗传连锁图谱的构建。卡方检验显示,母本分离标记中有偏分离标记(P<0.01)10个(8.0%),其它115个标记符合孟德尔1:1分离规律可用于连锁分析;父本分离标记中有偏分离标记(P<0.01)13个(11.0%),其它105个标记符合孟德尔1:1分离规律可用于连锁分析。利用父母本的分离标记,分别对大菱鲆母本和父本作图。所构建的母本连锁图谱共30个连锁群,110个标记连锁群总长度为919.1 cM,平均标记间隔为12.9 cM。所构建的父本连锁图谱共21个连锁群,110个标记连锁群总长度为721.7 cM,平均标记间隔为9.9 cM。根据计算,母本连锁图谱的预期长度为1695.0 cM,覆盖率为35.5%;父本连锁图谱的预期长度为1137.3 cM,覆盖率为48.2%。连锁分析结果显示,大菱鲆母本遗传连锁图谱比父本遗传连锁图谱长49.0%,这可能说明大菱鲆不同性别存在不同的重组率。
利用所构建的图谱对大菱鲆体长,体重和体厚进行QTL定位。在父本连锁图谱上仅检测得到一个可能与体长相关的QTL。QTL定位,为我们从事今后的分子标记辅助育种工作提供了参考。
Genetic linkage maps were constructed for turbot (Scophthalmus maximus L.) using microsatellite markers in an F1 family and quantitative trait loci(QTL) associated with the growth of turbot were detected and located in the maps.
1. In present study, by construction the microsatellite enrichment libraries and colony hybridization, microsatellites were isolated for the turbot (Scophthalmus maximus L.) covering the whole genome in a large scale. By 14 different probes detection, 952 clones were sequenced and after cluster analysis, 753 individual positive clones were finally obtained. Using Vector NTI Suite 8.0 (Invitrogen) and tandem repeats finder (TRF) software, a total of 1004 tandem repeat sequences were found , including 831 microsatellites and 173 minisatellites, which accounted for 82.77% and 17.23% of total repeat sequences respectively. The results of analyzing the different nucleotide unit crossing the whole genome of turbot were also represented. It showed that: the di-nucleotide unit was the dominant repeat unit among the six repeat unit types, whose number was the most abundant, and the repeat unit cumulative length was the largest as well, while the penta-nucleotide type was disadvantage in the whole repeat unit types. The CV (Coefficient of Variation) was introduced to evaluate the ability of the repeated variation of microsatellites. The AGG (126.11) motif was the most variable repeat unit type in this research with a CV value 126.11, but the average CV of di-nucleotides was the most common type among the six repeat unit types as the basis of origin and evolution of repeat sequences. In this study, a negative correlation between the length of repeat units and the average copy number was detected (r = -0.5, P= 0.072).
2. Six hundred and fifty-three primer pairs were designed and 284 primer pairs with high polymorphisms were screened, which were developed from the SSR-enriched libraries. The genetic linkage map of turbot was performed using an F1 family, and the map was constructed using 158 microsatellite markers among all the polymorphic markers. Of these segregating markers, 125 were mapped to the female framework map and 10 (8.0%) markers showed a significant distortion from the expected 1:1 ratio (P<0.01). final female linkage map consisted of 30 linkage groups spanning 919.1 cM with an average distance of 12.9 cM. And 118 were assigned to 22 linkage groups in male map, and 13 (11.0%) markers showed a significant distortion from the expected 1:1 ratio (P<0.01). The total length of the male map was 721.7 cM with an average distance of 9.9 cM. By using two kinds of methods to estimate the genome length, the average estimate length was 1695.0 cM for female and 1137.3 cM for male with coverage of 35.5%and 48.2% respectively. In Turbot, the female map was 49.0% larger than the male map, which may reflect sex-specific recombination rates in Turbot.
In addition, only one possible QTL for the turbot standard length associated with the growth of Turbot was detected and located in the female and male maps. The QTL in related to the growth clustered on linkage group would be very useful for improving this trait by molecular marker-assisted selection.
1、采用富集文库-菌落原位杂交的方法构建大菱鲆微卫星富集文库,共筛选得到952个克隆,通过序列比对和重复序列筛查,得到含有重复序列的独立克隆753个,占全部克隆数的79.10%。通过tandem repeats finder (TRF)软件的进一步筛查,得到1004个含串联重复序列的位点,其中微卫星位点831个(82.77%),小卫星序列位点173个(17.23%)。利用实验室设计的软件对序列进行分析,得到结论如下:1)微卫星重复序列中,重复序列数目所占比例与累计长度的所占比例情况基本一致,两碱基重复类型累计长度所占的比例最大,其次是四碱基、三碱基、六碱基和五碱基类型,而单碱基重复类型在本研究中鲜有发现;2)重复单位拷贝数变异能力分析表明,变异系数最大的是三碱基;3)重复序列重复单位长度与其拷贝数的相关分析表明,二者呈负相关(r = -0.5, P= 0.072),即随着重复单位的增加,其拷贝数在减少。以上结果为物种之间微卫星分布频率和丰度的比较、微卫星标记的开发及微卫星进化和功能的研究等工作提供基础。
2、本研究利用了富集文库-菌落原位杂交的方法开发的653个微卫星标记,利用含有F1代亲本在内的多个个体对位点进行多态性评价,共得到284个多态性的位点,其中158个标记在所构建图谱家系亲本间具有多态性,分别有125个和118个标记可用于母本和父本遗传连锁图谱的构建。卡方检验显示,母本分离标记中有偏分离标记(P<0.01)10个(8.0%),其它115个标记符合孟德尔1:1分离规律可用于连锁分析;父本分离标记中有偏分离标记(P<0.01)13个(11.0%),其它105个标记符合孟德尔1:1分离规律可用于连锁分析。利用父母本的分离标记,分别对大菱鲆母本和父本作图。所构建的母本连锁图谱共30个连锁群,110个标记连锁群总长度为919.1 cM,平均标记间隔为12.9 cM。所构建的父本连锁图谱共21个连锁群,110个标记连锁群总长度为721.7 cM,平均标记间隔为9.9 cM。根据计算,母本连锁图谱的预期长度为1695.0 cM,覆盖率为35.5%;父本连锁图谱的预期长度为1137.3 cM,覆盖率为48.2%。连锁分析结果显示,大菱鲆母本遗传连锁图谱比父本遗传连锁图谱长49.0%,这可能说明大菱鲆不同性别存在不同的重组率。
利用所构建的图谱对大菱鲆体长,体重和体厚进行QTL定位。在父本连锁图谱上仅检测得到一个可能与体长相关的QTL。QTL定位,为我们从事今后的分子标记辅助育种工作提供了参考。
Genetic linkage maps were constructed for turbot (Scophthalmus maximus L.) using microsatellite markers in an F1 family and quantitative trait loci(QTL) associated with the growth of turbot were detected and located in the maps.
1. In present study, by construction the microsatellite enrichment libraries and colony hybridization, microsatellites were isolated for the turbot (Scophthalmus maximus L.) covering the whole genome in a large scale. By 14 different probes detection, 952 clones were sequenced and after cluster analysis, 753 individual positive clones were finally obtained. Using Vector NTI Suite 8.0 (Invitrogen) and tandem repeats finder (TRF) software, a total of 1004 tandem repeat sequences were found , including 831 microsatellites and 173 minisatellites, which accounted for 82.77% and 17.23% of total repeat sequences respectively. The results of analyzing the different nucleotide unit crossing the whole genome of turbot were also represented. It showed that: the di-nucleotide unit was the dominant repeat unit among the six repeat unit types, whose number was the most abundant, and the repeat unit cumulative length was the largest as well, while the penta-nucleotide type was disadvantage in the whole repeat unit types. The CV (Coefficient of Variation) was introduced to evaluate the ability of the repeated variation of microsatellites. The AGG (126.11) motif was the most variable repeat unit type in this research with a CV value 126.11, but the average CV of di-nucleotides was the most common type among the six repeat unit types as the basis of origin and evolution of repeat sequences. In this study, a negative correlation between the length of repeat units and the average copy number was detected (r = -0.5, P= 0.072).
2. Six hundred and fifty-three primer pairs were designed and 284 primer pairs with high polymorphisms were screened, which were developed from the SSR-enriched libraries. The genetic linkage map of turbot was performed using an F1 family, and the map was constructed using 158 microsatellite markers among all the polymorphic markers. Of these segregating markers, 125 were mapped to the female framework map and 10 (8.0%) markers showed a significant distortion from the expected 1:1 ratio (P<0.01). final female linkage map consisted of 30 linkage groups spanning 919.1 cM with an average distance of 12.9 cM. And 118 were assigned to 22 linkage groups in male map, and 13 (11.0%) markers showed a significant distortion from the expected 1:1 ratio (P<0.01). The total length of the male map was 721.7 cM with an average distance of 9.9 cM. By using two kinds of methods to estimate the genome length, the average estimate length was 1695.0 cM for female and 1137.3 cM for male with coverage of 35.5%and 48.2% respectively. In Turbot, the female map was 49.0% larger than the male map, which may reflect sex-specific recombination rates in Turbot.
In addition, only one possible QTL for the turbot standard length associated with the growth of Turbot was detected and located in the female and male maps. The QTL in related to the growth clustered on linkage group would be very useful for improving this trait by molecular marker-assisted selection.
引文
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