杨属派间5个种特异性InDel引物及在派间杂交子代鉴别中的应用  

Species-Specific Indel Markers in Five Species from Different Poplar Sects and Their Application in Identification of Inter-Specific Hybrids

作　　者：戴晓港 韩峭子 尹佟明 Dai Xiaogang;Han Qiaozi;Yin Tongming(State Key Laboratory of Tree Genetics and Breeding Co-Innovation Center for Sustainable Forestry in Southern China Key Laboratory of Tree Genetics and Biotechnology of Educational Department of China Key Laboratory of Tree Genetics and Silvicultural Sciences of Jiangsu Province College of Forestry,Nanjing Forestry University,Nanjing 210037)

机构地区：[1]林木遗传育种全国重点实验室、南方现代林业协同创新中心、林木遗传与生物技术教育部重点实验室、江苏省林木遗传和高效培育重点实验室、南京林业大学林学院,南京210037

出　　处：《林业科学》2024年第2期78-86,共9页Scientia Silvae Sinicae

基　　金：江苏省(现代农业)重点研发项目(BE2021366);十四五国家重点研发计划项目(2021YFD2200202)。

摘　　要：【目的】远缘杂交可以实现有利基因的聚合,是种质创新的重要手段。杨属有些派间杂交困难,且杂交子代从形态学难以区分,本研究开发杨属不同派间的特异性引物,为派间杂交创制新种质的真实性鉴定提供技术支撑。【方法】利用在美洲黑杨和小叶杨中种特异性InDel位点设计引物,并利用SeqHunter2依次比对胡杨、山杨、小叶杨和大叶杨基因组,再用杨属5个派的自然群体材料进行种内保守及种间多态引物的试验验证,筛选出杨属不同派的特异性引物序列,对杨属不同派间杂交子代进行真实性鉴别。【结果】将从美洲黑杨基因组设计的5000对InDel引物依次分别比对胡杨、山杨、小叶杨和大叶杨基因组,最后筛选出341对引物在上述5个种中是通用的。从不同染色体上共选取48对引物并合成,利用5个树种各1个DNA检测得到43对引物是通用的,其中19对引物在不同种中扩增条带单一且存在差异。利用自然群体混合DNA对试验验证的19对通用引物进行种间多态性和种内保守性检测,初步获得4对多态性引物。最后再用自然群体DNA对这4对引物进行扩增检测,发现引物Chr12_4341229和Chr13_10582756在美洲黑杨、小叶杨、山杨、大叶杨和胡杨中均只能扩增出一个位点,引物Chr12_4341229扩增条带分别为148、152、113、152和159 bp,引物Chr13_10582756扩增条带分别为140、144、116、140和159 bp。利用上述2对引物对小胡杨2号、黑胡杨和黑小杨进行鉴定,发现所有杂交子代个体均能检测它们2个亲本的特征带。引物Chr12_4341229和Chr13_10582756一起使用,可以用于美洲黑杨、小叶杨、山杨、大叶杨和胡杨任意组合杂交子代的真实性鉴别。【结论】本研究获得了杨属5个派间的5个种的特异性InDel引物2对,可以有效地进行杨属不同派5个种之间任意组合种间杂交子代的真实性鉴别,为进一步开展杨树派间杂交创制新种质�【Objective】Distant hybridization is an important way for germplasm innovation which can achieve the aggregation of favorable genes.Cross-incompatibility often occurs in some of Populus sects,and interspecific hybrids are difficult to distinguish from its morphology.In this study,species-specific InDel markers were developed to identify the interspecific hybrids from different sects of Populus,so as to lay a foundation for the new germplasm innovation in poplar.【Method】The primers were developed from species-specific InDel sites in P.deltoides and P.simonii and were used to blast sequentially the genomes of P.euphratica,P.davidiana,P.simonii and P.lasiocarpa using SeqHunter2.Natural individuals from different sects were used to verify the species-specific primers in intra species conservation and inter species polymorphism by PCR amplification test,and the identified species-specific primer sequences for different sects of Populus were screened,and the authenticity of interspecies hybrid progenies among any combinations from the five species in different Populus sects was identified.【Result】Five thousands of InDel primers were designed from the genome of P.deltoides and used to blast sequentially the genomes of P.euphratica,P.davidiana,P.simonii and P.lasiocarpa.A total of 341 primer pairs were detected as universal markers among the five species.A total of 48 primer pairs from different chromosome were selected and synthesized,and 43 were identified as universal in five species from different sects,of which 19 primer pairs were conservative within individual species and produced length polymorphism of amplified bands in different species.The mixed DNA from natural population was used to test the 19 pairs of universal primers for interspecific polymorphism and intraspecific conservation by PCR amplification,and 4 pairs of polymorphic primers were preliminarily obtained.Finally,natural population DNA was used to amplify and detect these 4 pairs of primers,and it was found that primer pairs of Chr12_4341

关 键 词：杨属 远缘杂交 物种特异性引物 种间杂交分子鉴别 

分 类 号：S722.3[农业科学—林木遗传育种] S718.46[农业科学—林学]