簸箕柳×三蕊柳种特异性KASP标记开发及种间杂交子代鉴别  

Development of Species-Specific KASP Markers and Identification of Inter-Specific Hybrids from Salix suchowensis×S.triandra

作　　者：戴晓港 魏铭辰 Dai Xiaogang;Wei Mingchen(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4期119-126,共8页Scientia Silvae Sinicae

基　　金：国家自然基金面上项目(32371905);江苏省(现代农业)重点研发项目(BE2021366);南京林业大学2023年大学生创新训练计划项目。

摘　　要：【目的】开发簸箕柳和三蕊柳种特异性KASP引物,为开展簸箕柳和三蕊柳杂交选育速生、抗虫新种质的真实性鉴定提供参考。【方法】利用簸箕柳和三蕊柳重测序数据,开展全基因组SNP位点分析,筛选出种特异性的SNP位点并设计KASP引物,利用SeqHunter2检测设计引物在簸箕柳中的通用性,并利用簸箕柳和三蕊柳自然群体材料对合成的引物进行实验验证,筛选出簸箕柳和三蕊柳种特异性KASP引物,并对它们的杂交子代进行真实性鉴别。【结果】将三蕊柳和簸箕柳重测序的数据比对到三蕊柳基因组,共获得个6598144个SNPs。经过筛选,最终在簸箕柳中检测到674144个和三蕊柳相比为纯合突变的位点。从每条染色体上随机选取100个位点用于KASP引物设计,在1900个SNPs位点中,750个SNP位点可以成功设计出KASP引物。从每条染色体上选取2组,共选取38组引物,通过SeqHunter2检测得到11组引物在簸箕柳中是通用的。利用簸箕柳和三蕊柳自然群体DNA对合成的10组通用引物进行种内保守和种间差异性检测,获得4组在簸箕柳、三蕊柳和种间杂交子代中聚类明显的引物。引物Stri08_82809、Stri14_11602、Stri14_12274和Stri17_10731在簸箕柳自然群体中分别扩增出纯合G/G、T/T、A/A和T/T位点,在三蕊柳自然群体分别扩增出纯合A/A、C/C、G/G和C/C位点,而在真实杂交子代中分别扩增出G/A、T/C、A/G和T/C杂合位点。利用上述4组引物对簸箕柳×三蕊柳的杂交子代进行鉴定,发现在31个杂交子代中,29个个体扩增出杂合位点,说明同时遗传了2个亲本的特征位点,为真实的种间杂交子代。有2个个体扩增的位点为纯合且和母本相同,说明这2个子代为簸箕柳花粉污染的子代。【结论】本研究获得了簸箕柳和三蕊柳种间特异性KASP标记4组,可以准确快速的开展簸箕柳和三蕊柳种间杂交子代的真实性鉴别,为进一步簸箕柳和三蕊柳种间杂�【Objective】In this study,species-specific KASP primers were developed to identify the interspecific hybrids from Salix suchowensis and S.triandra,which would lay a foundation for the authenticity identification of breeding new germplasm of fast growth and insect resistance in willow.【Methods】Whole genome SNPs were analyzed for selecting species-specific SNPs,and designing KASP primers based on the resequencing data of S.suchowensis and S.triandra.SeqHunter2 was used to detect the versatility of the designed primers in S.suchowensis,and natural population materials from S.suchowensis and S.triandra were used to test validation of the primers for species-specific and versatility identification by PCR amplification test.The selected species-specific KASP primers were used for the authentication identify of their hybrids.【Results】The all resequencing data of S.suchowensis and S.triandra were aligned to the S.triandra genome,and a total of 6598144 SNPs were detected over the whole genome.After filtering by a house perl script,674144 homozygous SNP sites were identified in S.suchowensis,which were homozygous mutation sites with S.triandra.One hundred SNPs were randomly selected from each chromosome for KASP primer design based on the genome sequence of S.triandra and 750 primer pairs were successful designed among the 1900 randomly selected SNP sites.SeqHunter2 detection revealed that 11 primers were versatile in S.suchowensis and S.triandra from the 38 primer pairs randomly selected from different chromosomes.Ten synthesized versatile primer pairs were used for for intra species conservation and inter species difference detection,and four of them could get an obvious clustering in S.suchowensis,S.triandra and its hybrids.Primer pairs of Stri08_82809,Stri14_11602,Stri14_12274 and Stri17_10731 amplified homozygous site of G/G,T/T,A/A and T/T respectively in the population of S.suchowensis,and homozygous site of A/A,C/C,G/G and C/C respectively in the population of S.triandra.For the hybrids identification,th

关 键 词：簸箕柳 三蕊柳 种间杂交 物种特异性KASP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]