35份北方地区杨树种质资源的遗传多样性及种质指纹图谱的构建

Construction of Genetic Diversity and Germplasm Fingerprint Profiles of 35 Populus Germplasm Resources from Northern Region

以辽宁省杨树研究所杨树(Populus L.)种质资源保存圃于2005年秋季采用1年生根桩苗方式栽植的(株行距4 m×8 m)35份北方地区常见的杨树种质为研究对象(黑杨派种质14份、黑杨派杂交种种质11份、青杨派杂交种种质9份、白杨派种质1份),2022年6月份采集大树嫩叶,采用十六烷基三甲基溴化铵法(CTAB)提取DNA;从已报道的杨树简单重复序列(SSR)引物序列中选取42对简单重复序列引物用于分析;选取7份不同类型种质材料进行引物筛选,选出18对扩增条带清晰、多态性丰富、稳定性强的简单重复序列引物,进行供试材料的聚合酶链式反应(PCR)扩增(95℃预变性5 min;95℃变性30 s,50℃退火30 s,72℃延伸30 s,35个循环;最后72℃延伸10 min;4℃保存),采用ABI3730型基因分析仪对扩增片段进行毛细管电泳检测。采用DataFormater、POPGENE32软件进行数据转换并分析电泳结果,计算总位点数、多态位点数、期望杂合度、观测杂合度等遗传多样性指标;采用NTSYS 2.1软件中的简单匹配(SM)相似系数程序,计算任意两个品种间的相似系数;采用非加权组平均法(UPGMA)进行聚类分析;采用毛细管电泳荧光简单重复序列分子标记技术,分析35份杨树种质资源遗传多样性和遗传关系、构建35份杨树种质的简单重复序列指纹图谱,补充全国杨属分子基因库。结果表明:筛选出的18对简单重复序列引物,共检测到总位点数235个、多态位点比例为100%、每对引物检测到多态位点为6~18个(平均13.06个)、平均期望杂合度为0.799、平均观测杂合度为0.689、平均多态信息含量为0.769。18对引物,共扩增特异性位点70个、每对引物检测到特异性位点为1~9个(平均3.89个)、特异性比率为31.63%。非加权组平均法聚类分析表明,35份杨树种质资源遗传相似系数在0.753~0.979之间(平均0.814),具有较高的遗传相似性;当遗传相似系数在0.815时,参试样品可分为4个大类,第Ⅰ大类又可分为3个亚类,聚类分析结果与种质的系谱关系和表型特征分类基本吻合,能够较好地反映种质间的亲缘关系。通过引物GCPM-1255、PMGC-2525、ORPM-103、WPMS14构建35份杨树种质资源的简单重复序列指纹图谱,可将全部种质区分开。

The study was conducted using 35 common Populus L.germplasm accessions from northern China,which were planted in the germplasm preservation nursery of Poplar Research Institute of Liaoning Province in the fall of 2005 using 1-year-old root cutting seedlings(plant spacing:4 m×8 m).The germplasm accessions included 14 Aigeiros Germplasm,11 Aigeiros hybrid germplasm,9 Tacamahaca hybrid germplasm and 1 Leuce germplasm.In June 2022,tender leaves were collected from the mature trees,and DNA was extracted using CTAB method.From the reported Populus SSR primer sequences,42 pairs of SSR primers were selected for analysis.Seven diverse germplasm accessions were used for primer screening,and 18 primer pairs with clear amplification bands,rich polymorphism,and high stability were selected for polymerase chain reaction(PCR)amplification of the test materials(95℃pre-denaturation for 5 min;95℃denaturation for 30 s,50℃annealing for 30 s,72℃extension for 30 s,35 cycles;final extension at 72℃for 10 min;storage at 4℃).The amplified fragments were detected using capillary electrophoresis on an ABI3730 genetic analyzer.Data Formatter and POPGENE32 software were used for data conversion and analysis of the electrophoresis results,calculating genetic diversity indices such as total loci,polymorphic loci,expected heterozygosity,and observed heterozygosity.The simple matching(SM)similarity coefficient program in NTSYS 2.1 software was used to calculate the similarity coefficients between any two varieties.Unweighted group average method(UPGMA)clustering analysis was performed.Capillary electrophoresis fluorescence-based SSR molecular marker technology was used to analyze the genetic diversity and relationships of the 35 Populus germplasm accessions,and to construct the SSR fingerprint profiles of the 35 Populus accessions,supplementing the national Populus molecular genetics database.The results showed that the 18 selected SSR primer pairs detected a total of 235 loci,with a 100%polymorphism rate.Each primer pair detected 6-18(average 13.06)polymorphic loci,with an average expected heterozygosity of 0.799,average observed heterozygosity of 0.689,and average polymorphism information content of 0.769.The 18 primers amplified 70 unique specific loci,with 1-9(average 3.89)specific loci per primer pair,and a specificity rate of 31.63%.The UPGMA cluster analysis showed that the genetic similarity coefficients among the 35 Populus germplasm accessions ranged from 0.753 to 0.979(average 0.814),indicating high genetic similarity.When the genetic similarity coefficient was 0.815,the tested samples could be divided into 4 categories,with theⅠcategory could be divided into 3 subcategories.The clustering results were generally consistent with the genealogical relationship and phenotypic characteristics classification of the germplasm accessions,effectively reflecting the genetic relationships among the accessions.The SSR fingerprint profiles of the 35 Populus germplasm accessions were constructed using the primers GCPM-1255,PMGC-2525,ORPM-103,and WPMS14,which could distinguish all the germplasm accessions.