薄壳山核桃SCAR标记开发及其在品种间的多态性

Development of SCAR marker and its polymorphism in Carya illinoensis

【目的】获得品种间具有多态性且稳定可靠的序列特异性标记,用于薄壳山核桃杂交育种中的分子鉴定。【方法】选取5个性状差异较大的品种Van Deman、Moore、Nacono、Davis和Pawnee,提取高质量基因组DNA,经合适的限制性内切酶酶切后分别构建文库,利用Illumina HiSeq高通量测序仪获得每种样品的简化基因组数据。再进行序列与基因库的比对和各品种间的差异片段比对,获得差异片段和位点,设计适合特异性片段扩增的PCR引物。然后通过试验筛选这些引物,并进行多个品种的多态性分析。【结果】5个品种的基因组DNA经简化基因组测序后,共获得25963442个reads,合计3816.62 Mb碱基,其(A+T)/(C+G)的值约为1.63,长度质量指标Q20%大于95%,Q30%大于90%。经初步组装、精确组装和品种序列差异比对后,共获得特异序列3916条,在差异位点上设计3893对引物,然后进一步分析引物扩增可靠性,包括二级结构、碱基比例等,挑选其中1133对引物,再根据理论扩增产物与核桃基因组的同源性,挑选出扩增产物同源性最高的候选引物473对,占可用引物数量的40%。利用PCR技术,测试这473对引物在23个山核桃品种中的多态性差异,最终获得能产生多态性位点的86对引物,占被筛选引物对1/5左右。将其中80对单一条带用于品种鉴定和亲缘关系分析。利用这些引物对的组合,可以将其中17个品种鉴定出来。基于这些多态性片段进行的亲缘关系分析结果表明,聚类结果与实际的亲缘关系有部分一致性,如揭示了Choctaw与Dependable、Moore与barton之间的亲缘关系。【结论】利用简化基因组测序方法可以挖掘出薄壳山核桃品种的序列特异性分子标记,运用这些标记引物的组合可以进行大量品种的鉴定。所开发的80对引物可用于山核桃杂交育种中亲本组合的选择。

【Objective】Obtain stable and reliable sequence-specific markers of cultivars for hybrid breeding of Carya illinoinensis.【Method】Five cultivars including Van Deman,Moore,Nacono,Davis and Pawnee were selected to extract high-quality genomic DNA.The libraries were constructed by restriction endonuclease digestion.Simplified genomic data of each sample were obtained by Illumina HiSeq high-throughput sequencer.After sequence alignment and BLAST search,the PCR primers suitable for specific fragment amplification were designed.Then these primers were screened through experiments and the polymorphism of several varieties was analyzed.【Result】After simplified genome sequencing,genomic DNA of five cultivars with different traits obtained a total of 25963442 reads,totaling 3816.62 Mb.The value of(A+T)/(C+G)was about 1.63,the length quality index Q20%was more than 95%,and Q30%was more than 90%.After preliminary assembly,precise assembly and sequence alignment,a total of 3916 specific sequences were obtained.3893 pairs of primers were designed on the different sites.Then,the reliability of primer amplification was further analyzed,considering secondary structure,base ratio and so on.Totally,1133 pairs of primers were selected,and then the homology between the amplified products and walnut genome was determined in theory.Finally,473 pairs of candidate primers with the highest homology were selected,accounting for 40%of the available primers.The polymorphism of 473 pairs of primers in 23 pecan cultivars was tested by PCR,and 86 pairs of primers which could produce polymorphic loci were obtained,accounting for about 1/5 of the selected primers.Among them,80 pairs of single bands were used for identification and genetic analysis.Seventeen of these primer pairs could be identified.The analysis of genetic relationship based on these polymorphic fragments showed that the clustering results were partly consistent with the actual genetic relationship,such as revealing the genetic relationship between Choctaw and Dependable,Moore and Barton.【Conclusion】Using simplified genome sequencing to mine specific molecular markers of C.illinoinensis is a simple and rapid method to find genetic differences.On this basis,it is feasible to use multiple primer combinations for cultivar identification.In addition,80 pairs of primers were used to assist the analysis of genetic relationship,which was also beneficial to the selection of parent combinations in hybrid breeding of pecan.