中国农业科学 ›› 2017, Vol. 50 ›› Issue (3): 437-450.doi: 10.3864/j.issn.0578-1752.2017.03.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于荧光检测技术的多花黑麦草EST-SSR指纹图谱的构建

刘欢1,张新全1,马啸1,张瑞珍2,何光武2,潘玲1,金梦雅1

 
  

  1. 1四川农业大学草业科学系,成都 611130;2四川省草原工作总站,成都 610041
  • 收稿日期:2016-08-04 出版日期:2017-02-01 发布日期:2017-02-01
  • 通讯作者: 张新全,E-mail:zhangxq@sicau.edu.cn
  • 作者简介:刘欢,Tel:18728157834;E-mail:snlh9231@163.com
  • 基金资助:
    国家现代牧草产业技术体系(CARS-35-05)、农业部草品种DUS测定项目(20130106)、四川省饲草育种攻关(2011NZ0098-11)

Construction of EST-SSR Fingerprinting Based on Fluorescence Detection Technology for Italian Ryegrass

LIU Huan1, ZHANG XinQuan1, MA Xiao1, ZHANG RuiZhen2, HE GuangWu2, PAN Ling1, JIN MengYa1

 
  

  1. 1Department of Grassland Science, Sichuan Agricultural University, Chengdu 611130; 2Grassland Station of Sichuan Province, Chengdu 610041
  • Received:2016-08-04 Online:2017-02-01 Published:2017-02-01

摘要: 【目的】构建基于EST-SSR荧光标记的多花黑麦草(Lolium multiflorum Lam.)DNA指纹鉴定体系,为多花黑麦草品种鉴定提供高通量技术手段,为不同品种的合理应用提供参考依据,有效保护农民利益和育种权益。【方法】利用表型性状差异大的3个品种(特高Tetragold、长江2号ChangjiangNo.2和阿伯德Aubade),通过聚丙烯酰胺凝胶电泳从200对EST-SSR引物中筛选出扩增条带清晰、多态性好、扩增稳定的30对引物。在筛选出的每对引物5′端添加荧光标记FAM后,采用毛细管法通过DNA分析仪检测200个单株不同等位变异的扩增片段,从30对EST-SSR引物中筛选出25对扩增稳定的荧光引物,建立基于高通量荧光SSR标记的多花黑麦草品种鉴定体系。【结果】通过25对EST-SSR引物构建的DNA指纹图谱来进行10个多花黑麦草材料的品种鉴定。25对EST-SSR引物共检测到127个等位基因,等位变异扩增片段长度范围为51—249 bp,每对引物可检测到有效等位基因数为2—11个,特异等位基因最多可检测到11个(N101),平均每对引物4.00个;多态性位点的比率范围为33.33%—100.00%。平均PIC值为0.702,Shannon指数最大为3.322(N101),平均为1.929,基因多样性指数变幅为0.159—0.500,平均0.318,可鉴别的材料数为0—10个;其中14对特征引物在10个品种(系)上可检测出25个特异等位基因。综合来看,引物N101在200对引物中鉴别效率最高,可直接将10个多花黑麦草品种(系)区分开,在长江2号、赣选1号和川农2号上同时检测出特异等位基因。但由于多花黑麦草在品种间与品种内变异均较高,因此为鉴定更多材料,从25对引物中选择了6对扩增和检测效果较好的引物(N54、N101、N146、N151、N154、N156),6对引物可检测到的稳定等位基因数均不小于19个,在达伯瑞和邦德上最多可检测到22个等位基因。通过6对高效引物构建了10个多花黑麦草品种(系)的DNA指纹图谱,包括标准模式图、图谱代码和图谱QR编码。首次利用EST-SSR荧光标记毛细管电泳检测,为10个多花黑麦草材料分别构建了唯一的指纹代码和QR编码。【结论】利用6对高效引物构建了多花黑麦草SSR高通量鉴定体系,其中荧光引物N101多态性最高,可直接鉴别10个多花黑麦草品种(系)。

关键词: 多花黑麦草, EST-SSR分子标记, 指纹图谱, 荧光检测, 毛细管电泳

Abstract: 【Objective】In this study, a Italian ryegrass (Lolium multiflorum Lam.) variety identification system based on fluorescently labeled ETS-SSR markers was developed to provide a high-throughput DNA profiling means for identification of Italian ryegrass varieties, which can provide valuable information for the use of Italian ryegrass production and an effective method of protecting farmers’ benefits and breeders’ rights.【Method】Using three Italian ryegrass varieties (Tetragold, ChangjiangNo.2 and Aubade) with high difference of phenotypic traits, 30 primers were screened from the original 200 EST-SSR primers by polyacrylamide gel electrophoresis, which had clear amplification bands, rich polymorphism and stable amplification. Markers selected were labeled at the 5′ end of forward primer using fluorescent tags FAM, DNA analyzer was employed to detect different allelic variations of 200 individual PCR-amplified fragments by capillary electrophoresis. After further screening, 25 out of 30 fluorescent markers were chosen based on stable amplification to construct a high throughput identification system for Lolium multiflorum L..【Result】DNA fingerprint of ten Italian ryegrass materials were constructed using 25 EST-SSR primers for variety identification. A total of 127 alleles were amplified by 25 primer pairs, and the amplification fragment length ranged from 51 to 249. The effective number of alleles ranged from 2 to 11 on each pair of primers among them, primer N101 with 11 specific alleles was the most of all primers and each pair of primers had 4.00 specific alleles on average. The ratio of polymorphism sites ranged from 33.33% to 100.00%. The average value of polymorphic information content (PIC) was 0.702, the largest Shannon’s value (I) was 3.322 (N101) and the average value was 1.929, the Nei’s genetic diversity (H) ranged from 0.159 to 0.500 and the average value was 0.318. The amount of identified materials was from 0 to 10, and 14 of 25 primer pairs had 25 specific amplification sites among ten varieties (strains). Taken together, the results showed that the N101 fluorescence primer had the highest identification capability, which can identify these varieties (strains) directly. In addition, specific alleles were detected in primer ‘N101’ in three varieties such as Changjiang No.2, Ganxuan No.1 and Chuannong No.2. But due to high variation exists in same variety and different varieties of Lolium multiflorum L., in order to identify other materials, 6 EST-SSR primers (N54, N101, N146, N151, N154, N156) with good amplification and detection effect were chosen from 25 primers, the numbers of stable alleles were no less than 19, and the varieties “Double Barrel” and “Abundant” with 22 alleles were the most of all materials. Six high-efficiency primers were used for construction of DNA fingerprint spectrum including the standard model, fingerprint code and QR encodes of fingerprint spectrum. In this study, the fingerprint code and the unique QR encode for ten varieties of Italian ryegrass were firstly obtained using EST-SSR molecular marker with the aid of capillary electrophoresis and fluorescence labeling technology.【Conclusion】In this study, a SSR high-throughput identification system was constructed according to six pairs high efficiency primers, in which the ‘N101’ fluorescence primer having the most polymorphism can directly apply to identification of 10 Italian ryegrass varieties (strains).

Key words: Italian ryegrass (Lolium multiflorum Lam.), EST-SSR marker, fingerprinting, fluorescence detection, capillary electrophoresis