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Scientia Silvae Sinicae ›› 2015, Vol. 51 ›› Issue (2): 69-79.doi: 10.11707/j.1001-7488.20150209

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Fingerprints of SSR Markers and Ploidy Detection for New Populus Varieties

Jia Huixia1,2, Ji Huijuan1, Hu Jianjun1, Lu Mengzhu1,2   

  1. 1. State Key Laboratory of Tree Genetics and Breeding Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration Research Institute of Forestry, CAF Beijing 100091;
    2. Key Laboratory of Forest Genetics & Biotechnology of Ministry of Education Nanjing Forestry University Nanjing 210037
  • Received:2014-02-26 Revised:2014-04-22 Online:2015-02-25 Published:2015-03-11

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Abstract:

【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, 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 ‘2KEN8’, ‘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.

Key words: Populus, TP-M13-SSR, fingerprint, pedigree analysis, ploidy detection

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