基于物种特异性InDel标记鉴别美洲黑杨与小叶杨的杂交子代  

Identification of Interspecific Hybrids between Populus deltoides and P.simonii by Using Species-Specific InDel Marker

作　　者：戴晓港[1] 陈晨[1] 薛良交 吴怀通 尹佟明[1] Dai Xiaogang;Chen Chen;Xue Liangjiao;Wu Huaitong;Yin Tongming(Jiangsu Key Laboratory for Poplar Germplasm Enhancement and Variety Improvement,Nanjing Forestry University,Nanjing 210037)

机构地区：[1]江苏省杨树种质创新与品种改良重点实验室南京林业大学,南京210037

出　　处：《林业科学》2021年第11期79-84,共6页Scientia Silvae Sinicae

基　　金：江苏省(现代农业)重点研发项目(BE2021366);国家自然科学基金项目(31500533)。

摘　　要：【目的】针对杨树种间杂交子代苗期形态不易区分的问题,利用美洲黑杨和小叶杨物种特异性InDel位点开发标记对种间杂交子代进行快速鉴别,为黑杨派和青杨派种间杂交子代鉴定、分子标记辅助育种等研究提供重要的参考技术。【方法】以美洲黑杨、小叶杨及其杂交子代为试验材料,根据美洲黑杨和小叶杨重测序结果分析物种特异性InDel位点并设计引物,采用非变性聚丙烯酰胺凝胶电泳对引物进行通用性和物种特异性检测,用筛选出的物种特异的引物,对种间杂交子代的真实性进行鉴别。【结果】分别将10个个体的美洲黑杨和小叶杨重测序序列比对到美洲黑杨基因组,共获得长度差异大于5 bp的InDel位点359733个,其中种内特异、种间存在差异的InDel位点18418个。采用Primer3在美洲黑杨基因组中的17956个位点成功设计引物。在不同染色体共选取10对引物,采用SeqHunter2将引物序列比对到小叶杨基因组,共有6对引物在小叶杨中是通用的。采用非变性聚丙烯酰胺凝胶电泳检测,发现引物Psi2-1和Psi13-14在美洲黑杨和小叶杨中存在差异。利用美洲黑杨和小叶杨自然群体各50个个体对上述2个引物进行检测,发现引物Psi2-1在美洲黑杨中扩增出128 bp的特异性产物,在小叶杨中扩增出139 bp的特异性产物,在真实的种间杂交子代中扩增出128 bp和139 bp 2条产物;引物Psi13-14在美洲黑杨中扩增出132 bp的特异性产物,在小叶杨中扩增出144 bp的特异性产物,在真实的种间杂交子代中扩增出132 bp和144 bp 2条产物。2对引物对美洲黑杨与小叶杨种间杂交子代鉴别准确率达100%。【结论】获得了美洲黑杨和小叶杨物种特异性InDel标记2对,上述标记可以有效地用于美洲黑杨与小叶杨种间杂交子代的真实性鉴别。【Objective】The identification of interspecific poplar hybrids at seedling stage is challenging because of the morphological similarity.Here,we developed species-specific InDel markers to identify the interspecific hybrids between Populus deltoides and P.simonii,in order to lay a foundation for the identification of hybrids and molecular marker-assisted breeding in poplar.【Method】Ten individuals of each P.deltoides and P.simonii were resequenced to screen species-specific InDels,which were further applied for primer designing.The resulted primers were tested for their versatility and specificity using 8%non-denature polyacrylamide gel electrophoresis.The selected species-specific polymorphic primers were used to identify interspecific hybrid progeny between P.deltoides and P.simonii.【Result】Resequencing reads from each ten individuals of P.deltoides and P.simonii were mapped onto the genome of P.deltoides for comparisons,and a total of 359733 InDel sites with a length difference larger than 5 bp were obtained,of which 18418 sites are conserved within species but different between species.Based on the genome sequence of P.deltoides,17956 primer pairs were successfully designed using Primer3.Ten primer pairs from different chromosomes were selected and tested for their versatile in P.simonii by SeqHunter2 software.A total of six primer pairs were versatile between P.deltoides and P.simonii.The electrophoresis with 8%non-denature polyacrylamide gel showed that primers Psi2-1 and Psi13-14 were polymorphic between P.deltoides and P.simonii.Both primer pairs were then screened in natural population of P.deltoides and P.simonii each with fifty clones separately.The primers Psi2-1 amplified specific bands of 128 bp and 139 bp respectively in P.deltoides and P.simonii.And both of these bands were amplified in interspecific hybrids.Primers Psi13-14 amplified specific bands of 132 bp and 144 bp respectively in P.deltoides and P.simonii.And both of these two bands were successfully amplified in the interspecific h

关 键 词：美洲黑杨 小叶杨 种间杂交 物种特异性引物 分子鉴别 

分 类 号：S722.34[农业科学—林木遗传育种] S718.46[农业科学—林学]