[研究意义]采用SLAF-seq测序技术开发乔木柳SNP位点对乔木柳育种发展有重大意义.[方法]本研究根据两亲本和杂交的F1代遗传群体经亲本鉴定195株为研究对象,利用SLAF-seq测序技术进行测序.选择同科基因组大小相似的三角叶杨的基因组作为参考基因组,通过电子酶切预测,最终选择Hae Ⅲ和Hpy166 Ⅱ两种酶进行酶切试验,长度在314 ～364 bp之间的酶切片段序列定义为SLAF标签,预测可得SLAF标签126008个,位于重复序列区的SLAF标签比例为1.60％.为进一步评估酶切方案的有效性与酶切效率,以水稻的测序数据为对照,通过SOAP软件比对水稻基因组(大小为382M)的测序reads与三角叶杨的基因组.[结果]通过研究得到双端比对效率为96.67％,酶切效率为92.06％,SLAF建库正常.采用本次研究中开发得到的SLAF标签277 333个,按照等位基因数和基因序列之间的差异,共获得99 526个多态性SLAF标签,比例达到35.89％.按照遗传学通用等位编码规则,成功编码了58 763个多态性SLAF标签.为构建遗传图谱进一步对SLAF标签进行过滤,最终获得6744个SLAF标签,进一步进行SNP位点的开发,在各连锁群上共开发得到9488个SNP位点.[结论]SLAF-seq技术可以很好地应用于乔木柳SNP位点的开发,可利用所开发的SNP标记,联系群体中不同个体的表型,关联定位与某性状紧密连锁的分子标记,进而可以实现优良基因的精细定位.%[Objective] The use of SLAF-seq sequencing technology to develop the SNP site of arbor willow is of great significance to the development of arbor willow seed.[Method] In this study,two parents and 195 F1 hybrid generation were used as tested objects and sequenced by SLAF-seq technology.Taken the similar sibs cottonwood genome as reference genome,electronic enzyme projections for reference species genome sequence and Hae Ⅲ and Hpy166 Ⅱ enzymes as combination,the sequence of enzyme fragment length of 314-364 bp was defined as SLAFs,predicted 126 008 SLAFs,SLAF labels in repetitive sequences was 1.60 ％.In order to further evaluate the effectiveness of the proposed scheme enzyme and enzyme digestion efficiency,taking rice sequencing data as comparison,comparison between rice genome sequencing reads and reference genome by SOAP software with 382 M rice genome.[Result] The double blast efficiency was 96.67 ％,enzyme digestion efficiency was 92.06 ％,which indicated that SLAF database was normal.In the end,277 333 high quality SLAFs were produced,99526 polymorphism SLAFs were acquired including 35.89 ％ polymorphism according to the difference between the number of alleles and gene sequence.99526 polymorphism SLAFs were coded by genetics of common alleles of encoding rules,having 58763 labels coding success.SLAFs were further filtered to get 6744 SLAFs to construct genetic map and then developed SNP sits,finally,9488 SNP sits on each chain group were developed.[Conclusion] SLAF-seq technology could be well applied in SNP sites of arbor willow,using the developed SNP marker with different individual phenotype in group to position some traits closely chain of molecular markers,the precise positioning of good genes would be realized.
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