杨树新品种的SSR指纹图谱构建和倍性检测

Fingerprints of SSR Markers and Ploidy Detection for New Populus Varieties

【目的】对24份杨树种质进行指纹图谱构建、系谱关系鉴定和遗传多样性分析，并进行倍性检测，为杨树新品种鉴定和知识产权保护提供科学的理论依据，同时为杨树的育种工作奠定坚实的基础。【方法】利用 TP-M13-SSR 技术对24份杨树种质进行指纹图谱构建和系谱关系鉴定。从杨树 SSR 数据库中选取200对 SSR 引物，利用遗传背景差异大的 4份杨树种质进行引物筛选，选出19对扩增条带清晰、具有多态性且重复性好的引物对24份杨树种质进行扩增，利用 GeXP 毛细管电泳对 PCR 荧光产物进行检测，采用非加权组平均法（UPGMA）进行聚类分析，利用流式细胞术（FCM）进行倍性检测。【结果】19对 SSR 引物共扩增出102条条带，多态性条带97条，占95．10%，每个位点的等位基因数为2～11个，平均每对引物为5．37个。19对 SSR 引物均未检测到中林2025杨、中红杨和全红杨的条带差异，这3份种质相对于其他21份种质有特异的共同指纹条带；3对高效引物 ORPM_103，ORPM_180和 GCPM_1255可以将剩余21份杨树种质完全区分开。SSR 标记构建的指纹图谱与其系谱关系基本一致。对24份种质进行聚类分析，相似系数在0.50～1.00之间。当相似系数为0．56时，可分为5大类：第1类包括50号杨、中怀1号、中怀2号、I-69、帝国杨、中林2025杨、中红杨和全红杨；第2类包括青杨、森海 1号和森海 2号；第3类包括36号杨、丹红杨、南杨、239、1-116、中成 1号、中成 2号、中成3号、中成4号和中豫1号；第4类包括北抗杨和创新杨；第5类只有中林46杨。SSR 检测发现中怀 1号、森海 1号、森海 2号、青杨和中林46杨扩增出3个不同等位基因位点，其余19份杨树种质只出现 1个或2个等位基因位点；FCM 检测结果证实这5份种质均为三倍体，与 SSR 检测结果一致。【结论】本研究对24份杨树种质进行指纹图谱构建、遗传多样性分析和倍性测试，证实用 SSR标记可有效地检测亲本与子代之间的系谱关系，并准确地反映植物的倍性。筛选出 3对快速鉴别其中21份杨树种质的 SSR 引物，发现5个杨树种质为三倍体。本研究结果可为杨树新品种鉴定和知识产权保护提供理论依据。

[Objective]Genus Populus,with many species and a long history of cultivation and utilization,is widely used for afforestation,shelterbelt and timber production. In recent decades,with rapid progress in Populus breeding in China,a large number of new varieties with fast growth and superior characteristics have been developed. However,the genetic basis of these new varieties were narrow and genetic differences among parental trees for hybridization was small, and morphological differences among Populus varieties is becoming increasingly smaller,leading to difficulties in variety identification and protection of plant breeders’rights （PBR）. How to distinguish accurately and quickly new Populus varieties has become the top priority. This study was aimed to construct fingerprints,identify pedigree relationships,＆nbsp;analyze genetic diversity and detect ploidy of 24 accessions of Populus germplasms.[Method]Fingerprint construction and pedigree relationships of 24 accessions of Populus germplasms were analyzed using TP-M13-SSR （ simple sequence repeat with tailed primer M13 ） . 200 pairs of SSR primers were selected from Populus SSR database and screened by 4 accessions of genetically distant Populus germplasms. 19 pairs of primers with clear amplification bands,high polymorphism and stable repeatability were selected and used for PRC amplification of the 24 accessions of germplasms. PCR products labeled fluorescent were detected by using GeXP capillary electrophoresis. UPGMA （ unweighted pair group method arithmetic averages） were used for clustering analysis. FCM （ flow cytometry） was used to detect the ploidy.[Result]102 fragments were generated from 19 pairs of SSR primers,including 97 polymorphic fragments,accounting for 95. 10% of the total. The number of alleles at each locus was between 2 and 11,with an average of 5. 37 alleles for each pair of primers. No differences were detected among varieties P. deltoides‘2025’,‘Zhonghong’and‘Quanhong’by the 19 pairs of SSR primers. These 3 accessions of Populus germplasms had a unique fingerprint pattern relative to other 21 accessions of germplasms. The rest 21 accessions of Populus germplasms could be completely distinguished by 3 efficient pairs of SSR primers,ORPM_103,ORPM_180 and GCPM_1255. The fingerprints constructed by SSR markers were basically in consistence with the pedigree relationships. Cluster analysis by NTSYS-pc 2. 10e software showed that the similarity coefficient ranged from 0. 50 to 1. 00. When the similarity coefficient was 0. 56,24 accessions of Populus germplasms were divided into five categories： the first category included P. deltoides ‘55/56’,‘Zhonghuai 1 ’,‘Zhonghuai 2 ’, I-69,‘Imperial’,‘2025’,‘Zhonghong ’ and ‘Quanhong ’; the second category included P. cathayana,‘Senhai 1 ’ and ‘Senhai 2 ’; the third category included ‘2 KEN8’,‘Danhong’,‘Nan’,‘239 ’,‘1-116’,‘Zhongcheng 1’,‘Zhongcheng 2’,‘Zhongcheng 3’,‘Zhongcheng 4 ’and‘Zhongyu 1 ’; the fourth category included‘Beikang’and‘Chuangxin’; the fifth category included‘Zhonglin 46’. Loci with 3 different alleles were amplified in‘Zhonghuai 1’,‘Senhai 1’,‘Senhai 2’,P. cathayana and‘Zhonglin 46’,while loci with only 1 or 2 alleles were amplified in the rest 19 accessions of Populus germplasms. The result of FCM confirmed that these 5 accessions of germplasms were triploid, consistent with the result of SSR detection. [Conclusion]In this study, fingerprint construction,genetic diversity and FCM analysis were carried out for 24 accessions of Populus germplasms. SSR markers could effectively detect pedigree relationships between parents and offspring,and accurately reflect the ploidy of plants. 3 pairs of SSR primers were found to be able to identify 21 accessions of Populus germplasms. 5 accessions of Populus germplasms were found triploid. The results provided a theoretical basis for variety identification and protection of plant breeder’s rights for Populus.