短枝木麻黄无性系鉴定及其指纹图谱构建

目的]构建木麻黄无性系的DNA指纹图谱鉴定体系,为今后短枝木麻黄的品种鉴定、品种权保护、新品种的选育提供依据。方法]从71对EST-SSR引物中筛选出扩增条带清晰、多态性好、扩增稳定的12对引物,采用降落PCR和毛细管电泳的基因分型方法,以采集的9个短枝木麻黄标准无性系的基因分型结果为参照,对华南沿海地区采集的109个短枝木麻黄无性系单株进行鉴定,并结合引物组合法构建基于EST-SSR分子标记的短枝木麻黄无性系鉴定体系。结果]12对EST-SSR引物对109个短枝木麻黄无性系单株进行PCR扩增,共检测到50个等位基因,每个位点的等位基因数为3～6个,平均为4.2个,每对引物可区分2～5个短枝木麻黄无性系。根据鉴定结果,最终所有样品被划分为22个无性系,除已知的9个标准无性系外,另有13个无性系为不知名无性系,说明不同地区的无性系存在重复命名现象;利用引物组合法进一步优化后,发现只需7对引物就可将22个短枝木麻黄无性系完全区分,并依此建立了基于7对EST-SSR引物的22个短枝木麻黄无性系的指纹图谱,每个无性系都获得了一个7位数的指纹图谱编码。结论]利用7对EST-SSR引物构建的22个短枝木麻黄无性系的DNA指纹图谱可用于短枝木麻黄无性系的鉴别,研究表明,华南沿海地区部分短枝木麻黄无性系存在命名混乱、同物异名、无性系数量少的现象,新品种选育工作亟待开展和加强。

Identification and Fingerprinting Construction of Casuarina equisetifolia Clones

Objective] To construct DNA fingerprints of Casuarina equisetifolia clones and to provide the references for cultivar identification, cultivar variety right protection, and genetic breeding.Method] Twelve EST-SSR primers with clear and stable amplified products were selected from 71 primers. The 12 primer pairs were used to identify the cultivars of 109 clonal samples collected from coastal shelterbelts of South China using touchdown PCR and capillary electrophoresis genotyping method through matching genotyping results of 9 standard reference cultivars. A DNA fingerprint identification system of C. equisetifolia clones based on EST-SSR molecular markers and multi-primer combination method was established.Result] In this study, totally 50 alleles were detected from 12 primers, where the allele number of each locus ranged from 3-6 and the average value was 4.2, and 2-5 clones can be identified by single locus. According to identification results, 109 clone samples were identified as 22 clones, including 9 reference cultivars and 13 unknown clones, suggesting that repetitive naming of clones was common in different regions. After further optimization by multi-primer combination method the valid molecular marker decreased to 7 primers, that a 7-digit numbers fingerprinting was established to identify the clones of C. equisetifolia, in which each clone can obtain a 7-digit numbers fingerprinting code.Conclusion] The DNA fingerprints of 22 clones can be used for the identification of C. equisetifolia clones. This study demonstrates that some clones are repetitive named and the reality of insufficient clone for afforestation. South China will enforce to strengthen breeding and selection work of new clone cultivars of C. equisetifolia.