多花黑麦草DUS测定中SSR标记品种鉴定比较分析

doi:10.3864/j.issn.0578-1752.2015.02.18

摘要/Abstract

摘要： 【目的】多花黑麦草是具世界栽培意义的牧草，但因异花授粉特性，育种材料的频繁交流导致品种间的遗传差异越来越小，传统农艺性状进行品种鉴定变得越来越困难。高效、快速有效地鉴定品种对实现育种目标具有重要作用，可为辅助多花黑麦草品种DUS测试和品种鉴定、知识产权保护等提供科学依据。【方法】基于SSR标记具有稳定性强、多态性高和共显性特点，对6个国审骨干多花黑麦草品种采用30株混合取样策略，每个品种以不同单株构成的30株混合样本量进行3次重复试验，以重复试验中至少出现2次的频率高的强带进行统计，从165对多花黑麦草SSR引物中，筛选出20对引物用于多花黑麦草品种鉴定。并采用30株单株样本扩增与30株混合样本扩增相结合的方法，加之扩增结果的比对，进行品种的鉴定分析。【结果】利用13-07A、01-06D和15-08C引物对6个多花黑麦草品种进行30株单株和30株混合取样对比性分析表明，混合株的某些条带相对于单株条带更为明显，且条带数更多，但也易出现一些弥散带，单株扩增中出现频率在40%及以上的条带则会在混合样本中出现；3组混合取样结果表明，20对SSR引物共扩增出143条清晰可辨条带，其中多态性条带127条，多态性条带的比率为88.81%，每对引物扩增的条带数为5—11，平均为7.15条，平均多态性条带为6.35条，平均多态性信息含量（PIC）为0.571，有的引物虽多态性信息含量较低，但稳定性好，3组混合样本扩增结果几乎一致；6个多花黑麦草品种的遗传相似系数范围为0.4755—0.7552，遗传相似系数最大的为邦德和阿德纳，遗传相似系数最小的为长江2号和特高。在平均遗传相似系数为0.615时可以将6个多花黑麦草品种划分为三大类：第Ⅰ类包括安格斯1号、邦德、阿德纳、达伯瑞；第Ⅱ类是长江2号；第Ⅲ类是特高。20对引物中有9对引物可以直接进行6个品种的鉴定，而其余的引物只能鉴别其中的3个或4个，则可通过不同引物的组合提高鉴别能力。【结论】对于异花授粉植物，多态性并非衡量引物有效性的唯一标准，引物稳定性也是衡量引物有效性的重要指标；相对于单株取样法，采用混合取样法进行品种鉴定更为有效；筛选出的20对SSR引物可以明确区分供试材料，采用SSR分子标记对多花黑麦草进行品种鉴定分析具有可行性。

关键词: 多花黑麦草, 品种鉴定, 取样方法, SSR标记, DUS测定

Abstract: 【Objective】Annual ryegrass is a world-cultivated forage, but frequent exchange of breeding material causes intervarietal genetic differences become smaller due to the cross-pollination characteristics, therefore, cultivar identification using traditional agronomic traits is becoming increasingly difficult. Efficient, rapid identification of cultivar plays an important role in breeding, and also provides a scientific basis for DUS testing and intellectual property protection.【Method】In this study, the cultivar identification among 6 backbone varieties of annual ryegrass was conducted by using 20 pairs of SSR primers from 165 for 3 bulk of DNA samples each attracted from 30 random individual plants equally based on strong stability, high polymorphism and co-dominant characteristics of SSR markers, and the specific bands of bulked samples appeared at least twice could be recorded in amplification profile. And the combination of 30 individuals and bulked samples using SSR markers, and the comparation of amplification profiles were used to identify 6 varieties of annual ryegrass.【Result】Comparative analysis of 6 varieties of annual ryegrass using SSR markers between individuals and bulked samples by using 3 primer pairs (13-07A, 12-10F and 15-08C) showed that the amplification bands of bulked samples were more obvious and more bands than individuals, but also some diffuse bands. And the frequency bands more than 40% in individuals appeared the same bands in bulked samples. The results of identification of 6 varieties of annual ryegrass using 3 bulked samples showed that, 20 primer pairs amplified 143 legible bands, comprising 127 polymorphic bands, and the polymorphism ratio reached 88.81%, and the amplified brands to each pair of primers were 5 to 11, and the average amplified brand, polymorphic bands, and polymorphism information content (PIC) were 7.15, 6.35 and 0.571, respectively. Although some primers polymorphic information content is low, high stability, i.e. three groups had almost same amplification results. The genetic similarity coefficient of 6 varieties varied from 0.4755 to 0.7552, the largest genetic similarity coefficient was between Abundant and Aderenalin, and the smallest genetic similarity coefficient was between Changjiang No.2 and Tetragold. The 6 varieties of annual ryegrass were divided into three groups on the average genetic similarity coefficient of 0.615, one included Angus1, Abundant, Aderenalin and Double Barrel, one was ChangjiangNo.2, and one was Tetragold. Nine of 20 SSR primer pairs could directly identify 6 varieties of ryegrass, however, the other primer pairs could only identify 3 or 4 varieties, the identification ability could be improved by combining with different primer pairs.【Conclusion】For cross-pollinated plants, polymorphism is not the only criterion to measure the effectiveness of primers, the stability of primers is also an important indicator. By comparative analysis of the 2 methods, bulked samples using SSR markers is better than the method of individuals. The screened 20 pairs of SSR primers could clearly distinguish 6 varieties of ryegrass, indicating that the use of SSR markers is feasible and efficient for cultivar identification.

Key words: annual ryegrass (Lolium multiflorum Lam.); cultivar identification, sampling methods, simple sequence repeats markers, DUS testing

黄婷，马啸，张新全，张新跃，张瑞珍，符开欣. 多花黑麦草DUS测定中SSR标记品种鉴定比较分析[J]. 中国农业科学, 2015, 48(2): 381-389.

HUANG Ting, MA Xiao, ZHANG Xin-quan, ZHANG Xin-yue, ZHANG Rui-zhen, FU Kai-xin. Comparation of SSR Molecular Markers Analysis of Annual Ryegrass Varieties in DUS Testing[J]. Scientia Agricultura Sinica, 2015, 48(2): 381-389.

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