基于荧光检测技术的多花黑麦草EST-SSR指纹图谱的构建

doi:10.3864/j.issn.0578-1752.2017.03.003

摘要/Abstract

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

刘欢，张新全，马啸，张瑞珍，何光武，潘玲，金梦雅. 基于荧光检测技术的多花黑麦草EST-SSR指纹图谱的构建[J]. 中国农业科学, 2017, 50(3): 437-450.

LIU Huan, ZHANG XinQuan, MA Xiao, ZHANG RuiZhen, HE GuangWu, PAN Ling, JIN MengYa. Construction of EST-SSR Fingerprinting Based on Fluorescence Detection Technology for Italian Ryegrass[J]. Scientia Agricultura Sinica, 2017, 50(3): 437-450.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.03.003

https://www.chinaagrisci.com/CN/Y2017/V50/I3/437

参考文献

[1] 董宽虎, 沈益新. 饲草生产学. 北京: 中国农业出版社, 2003: 113-117.

DONG K H, SHEN Y X. Forage Production. Beijing: China Agriculture Press, 2003: 113-117. (in Chinese)

[2] BOLARIC S, BARTH S, MELCHINGER A E, POSSELT U K. Genetic diversity in European perennial ryegrass cultivars investigated with RAPD makers. Plant Breeding, 2005, 124(2): 161-166.

[3] ROLDAN-RUIZ I, VAN EUWIJK F A, GILLILAND T J, DUBREUIL P, SILLMANN C, LALLEMAND J, LOOSE M D, BARIL C P. A comparative study of molecular and morphological methods of describing relationships between perennial ryegrass (Lolium perenne L.)varieties. Theoretical and Applied Genetics, 2001, 103(8): 1138-1150.

[4] GUSTAVO. DNA Markers. New York: Wiley-vch Press, 1998.

[5] International Union for the Protection of New Varieties of Plants. Guidelines for DNA-profiling: Molecular markers selection and database construction. Geneva: UPOV, 2006.

[6] International Union for the Protection of New Varieties of Plants. Possible use of molecular markers in the examination of distinctness, uniformity and stability. Geneva: UPOV, 2011.

[7] HIRATA M, CAI H W, INOUE M, YUYAMA N, MIURA Y, KOMATSU T, TAKAMIZO T, FUJIMORI M. Development of simple sequence repeat (SSR) markers and construction of an SSR-based linkage map in Italian ryegrass (Lolium multiflorum Lam.). Theoretical and Applied Genetics, 2006, 113(2): 42-47.

[8] PASAKINSKENE I, GRIFFITHS C M, BETTANY A J E. Simple-sequence repeats as primers to generate species-specific DNA markers in Lolium and Festuca grasses. Theoretical and Applied Genetics, 2000, 100(4): 384-390.

[9] 赵欣欣, 张新全, 苗佳敏, 黄琳凯, 季杨. 多花黑麦草杂交种SSR分子标记鉴定及表型比较分析. 农业生物技术学报, 2013(7): 811-819.

ZHAO X X, ZHANG X Q, MIAO J M, HUANG L K, JI Y. Identification by SSR molecular markers and comparative analysis in morphological characterization in Lolium multiflorum hybrid progenies. Journal of Agricultural Biotechnology, 2013(7): 811-819. (in Chinese)

[10] 罗永聪, 马啸, 张新全. 利用SSR技术构建多花黑麦草品种指纹图谱. 农业生物技术学报, 2013, 21(7): 799-810.

LUO Y C, MA X, ZHANG X Q. DNA Fingerprinting of annual ryegrass (Lolium multiflorum Lam.) varieties using SSR makers. Journal of Agricultural Biotechnology, 2013, 21(7): 799-810. (in Chinese)

[11] 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. 中国农业科学, 2015, 48(2): 381-389.

HUANG T, MA X, ZHANG X Q, ZHANG X Y, ZHANG R Z, FU K X. Comparation of SSR molecular markers analysis of annual ryegrass varieties in DUS testing. Scientia Agricultura Sinica, 2015, 48(2): 381-389. (in Chinese)

[12] PRAKASH P G, GOWDA M B, KUNDUR P J, VIMARSHA H S, SHASHIDHAR H E. Upgraded horizontal polyacrylamide gel units for DNA marker genotyping. Indian Journal of Science and Technology, 2015, 8(9): 822.

[13] 程本义, 夏俊辉, 龚俊义, 杨仕华. SSR荧光标记毛细管电泳检测法在水稻DNA指纹鉴定中的应用. 中国水稻科学, 2011, 25(6): 672-676.

CHENG B Y, XIA J H, GONG J Y, YANG S H. Application of capillary electrophoresis detection with fluorescent SSR markers in rice DNA fingerprint identification. Chinese Journal of Rice Science, 2011, 25(6): 672-676. (in Chinese)

[14] 陈雅琼, 李凤霞, 李锡坤, 徐军, 张磊, 王绍美, 孙玉合. 烟草SSR荧光标记与毛细管电泳检测技术研究. 中国烟草科学, 2011, 32(2): 66-70.

CHEN Y Q, LI F X, LI X K, XU J, ZHANG L, WANG S M, SUN Y H. Fluorescent detection and capillary electrophoresis in tobacco SSR. Chinese Tobacco Science, 2011, 32(2): 66-70. (in Chinese)

[15] 易红梅. 基于毛细管电泳荧光标记的玉米品种SSR高通量检测体系的建立研究[D]. 北京: 首都师范大学, 2006.

YI H M. Establishment a high throughput detection system of SSR markers fitting for maize variety based on capillary electrophoresis detection with fluorescent[D]. Beijing: Capital Normal University, 2006. (in Chinese)

[16] 许鲲, 李锋, 吴金锋, 谷铁城, 陈碧云, 高桂珍, 闫贵欣, 李俊, 乔江伟, 汪念, 伍晓明. SSR荧光标记毛细管电泳法与国家冬油菜区试指纹鉴定平台的构建. 中国油料作物学报, 2014, 36(2): 150-159.

XU K, LI F, WU J F, GU T C, CHEN B Y, GAO G Z, YAN G X, LI J, QIAO J W, WANG N, WU X M. Fingerprint identification platform of capillary electrophoresis detection with fluorescent SSR markers on national winter rapeseed varieties (lines) field trials. Chinese Journal of Oil Crop Sciences, 2014, 36(2): 150-159. (in Chinese)

[17] SANCHEZ-PEREZ R, BALLESTER J, DICENTA F, ARUS P, MARTINEZ-GOMEZ P. Comparison of SSR polymorphisms using automated capillary sequencers, and polyacrylamide and agarose gel electrophoresis: Implications for the assessment of genetic diversity and relatedness in almond. Scientia Horticulturae, 2006, 108(3): 310-316.

[18] 张利达, 唐克轩. 植物EST-SSR标记开发及其应用. 基因组学与应用生物学, 2010, 29(3): 534-541.

ZHANG L D, TANG K X. Development of plant EST-SSR markers and its application. Genomics and Applied Biology, 2010, 29(3): 534-541. (in Chinese)

[19] 赵岩, 孔凡美, 丁承强, 李斯深. EST-SSR标记在结缕草中的通用性. 中国草地学报, 2008, 30(3): 69-73.

ZHAO Y, KONG F M, DING C Q, LI S S. Transferability of EST-SSR markers in zoysiagrasses. Chinese Journal of Grassland, 2008, 30(3): 69-73. (in Chinese)

[20] 黄秀, 曾捷, 聂刚, 刘欢, 黄琳凯, 张新全, 蒋晓梅, 张瑜, 张博涛. 牛鞭草品种EST-SSR指纹图谱构建及遗传多样性分析. 热带亚热带植物学报, 2014, 22(2): 165-171.

HUANG X, ZENG J, NIE G, LIU H, HUANG L K, ZHANG X Q, JIANG X M, ZHANG Y, ZHANG B T. Construction of fingerprinting and genetic diversity analysis of Hemarthia cultivars by EST-SSR. Journal of Tropical and Subtropical Botany, 2014, 22(2): 165-171. (in Chinese)

[21] 温莹, 逯晓萍, 任锐, 米福贵, 韩平安, 薛春雷. 高丹草EST-SSR标记的开发及其遗传多样性. 遗传, 2013, 35(2): 225-232.

WEN Y, LU X P, REN R, MI F G, HAN P A, XUE C L. Development of EST-SSR marker and genetic diversity analysis in Sorghum bicolor×Sorghum sudanenes. Hereditas, 2013, 35(2): 225-232. (in Chinese)

[22] 王凤格, 易红梅, 赵久然, 吕波, 堵苑苑, 田红丽. 植物品种鉴定DNA指纹方法总则(NY/T2594-2014). 北京: 中国人民共和国农业部, 2014.

WANG F G, YI H M, ZHAO J R, LÜ B, DU Y Y, TIAN H L. General guideline for identification of plant varieties by DNA fingerprinting(NY/T2594-2014). Beijing: Ministry of Agriculture of the People's Republic of China, 2014. (in Chinese)

[23] 梁宏伟, 王长忠, 李忠, 罗相忠, 邹桂. 聚丙烯酰胺凝胶快速、高效银染方法的建立. 遗传, 2008, 30(10): 1379-1382.

LIANG H W, WANG C Z, LI Z, LUO X Z, ZOU G. Improvement of the silver-stained technique of polyacrylamide gel electrophoresis. Hereditas, 2008, 30(10): 1379-1382. (in Chinese)

[24] 张富民, 葛颂. 群体遗传学研究中的数据处理方法I.RAPD数据的AMOVA分析. 生物多样性, 2002, 10(4): 438-444.

ZHANG F M, GE S. Data analysis in population genetics I. analysis of RAPD data with AMOVA. Biodiversity Science, 2002, 10(4): 438-444. (in Chinese)

[25] YEH F C, YANG R C, BOYLE T. Pop gene, Version 1.31 Microsoft Windows-based Freeware for Population Genetic Analysis, Quick User-guide. University of Alberta: Center for the International Forestry Research, 1999.

[26] NEI M. Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences of the USA, 1973, 70(12): 2223-3321.

[27] 陈昌文, 曹珂, 王力荣, 朱更瑞, 方伟超. 中国桃主要品种资源及其野生近缘种的分子身份证构建. 中国农业科学, 2011, 44(10): 2081-2093.

CHEN C W, CAO K, WANG L R, ZHU G R, FANG W C. Molecular ID establishment of main China peach varieties and peach related species. Scientia Agricultura Sinica, 2011, 44(10): 2081-2093. (in Chinese)

[28] 宋海斌, 崔喜波, 马鸿艳, 朱子成, 栾非时. 基于SSR标记的甜瓜品种(系)DNA指纹图谱库的构建. 中国农业科学, 2012, 45(13): 2676-2689.

SONG H B, CUI X B, MA H Y, ZHU Z C, LUAN F S. Construction of DNA fingerprint database based on SSR marker for varieties (lines) of Cucumis melo L.. Scientia Agricultura Sinica, 2012, 45(13): 2676-2689. (in Chinese)

[29] 王凤格, 赵久然, 郭景伦, 佘花娣, 陈刚. 比较三种DNA指纹分析方法在玉米品种纯度及真伪鉴定中的应用. 分子植物育种, 2003, 5(1): 655-661.

WANG F G, ZHAO J R, GUO J L, SHE H D, CHEN G. Comparison of three DNA fingerprint analyzing methods for maize cultivars' identification. Molecular Plant Breeding, 2003, 5(1): 655-661. (in Chinese)

[30] 岳静, 朱志成, 申雅娟, 张卉, 王汝宝. 利用SSR标记技术鉴定玉米品种真实性的研究. 中国农学通报, 2011, 27(12): 201-204.

YUE J, ZHU Z C, SHEN Y J, ZHANG H, WANG R B. Study on identifying the authenticity of maize varieties by SSR markers. Chinese Agricultural Science Bulletin, 2011, 27(12): 201-204. (in Chinese)

[31] MORGANTE M, OLIVIERI A M. PCR-amplified microsatellites as markers in plant genetics. The Plant Journal, 1993, 3(1): 175-182.

[32] ROJAS G, MENDEZ M A, MUNOZ C, LEMUS G, HINRICHSEN P. Identification of a minimal microsatellite marker panel for the fingerprinting of peach and nectarine cultivars. Electronic Journal of Biotechnology, 2008, 11(5): 4-5.

[33] DANIN-POLEGY, REIS N, BAUDRACCO-ARNAS S, PITRAT M, STAUB J E, OLIVER M, ARUS P, DE VICENTE C M, KATZIR N. Simple sequence repeats in Cucumis mapping and map merging. Genome, 2000, 43: 963-974.

[34] AKKAYA M S, SHOEMAKER R C, SPECHT J E, BHAGWAT A A, CREGAN P B. Integration of simple sequence repeat DNA markers into a soybean linkage map. Crop Science, 1995, 35(5): 1439-1445.

[35] 葛亚英, 张飞, 沈晓岚, 刘建新, 王炜勇, 田丹青, 张智. 丽穗凤梨ISSR遗传多样性分析与指纹图谱构建. 中国农业科学, 2012, 45(4): 726-733.

GE Y Y, ZHANG F, SHEN X L, LIU J X, WANG W Y, TIAN D Q, ZHANG Z. Analysis of genetic diversity and construction of fingerprint of Vriesea by ISSR. Scientia Agricultura Sinica, 2012, 45(4): 726-733. (in Chinese)

[36] HECKENBERGER M, BOHN M, ZIEGLE J S, JOE L K, HAUSER J D, HUTTON M, MELCHINGER A E. Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties: I. Genetic and technical sources of variation in SSR data. Molecular Breeding, 2002, 10: 181-191.

[37] 徐宗大, 赵兰勇, 张玲, 杨志莹. 玫瑰SRAP遗传多样性分析与品种指纹图谱构建. 中国农业科学, 2011, 44(8): 1662-1669.

XU Z D, ZHAO L Y, ZHANG L, YANG Z Y. Analysis of genetic diversity and construction of fingerprint of Rosa rugosa by SRAP. Scientia Agricultura Sinica, 2011, 44(8): 1662-1669. (in Chinese)

[38] 蒋林峰, 张新全, 黄琳凯, 马啸, 罗登, 张宗瑜, 蒙芬. 中国鸭茅主栽品种DNA指纹图谱构建. 植物遗传资源学报, 2014, 15(3): 604-614.

JIANG L F, ZHANG X Q, HUANG L K, MA X, LUO D, ZHANG Z Y, MENG F. Construction of DNA fingerprinting of dominant orchardgrass (Dactylis glomerata) varieties of China. Journal of Plant Genetic Resources, 2014, 15(3): 604-614. (in Chinese)

[39] ARCHAK S, GAIKWARDA B, GAUTAM D, RAO E V V B, SWAMY K R M, KARIHALOO J L. DNA fingerprinting of Indian cashew (Anacardium occodentale L.) varieties using RAPD and ISSR techniques. Euphytica, 2003, 130(3): 397-404.

[40] FANG D Q, ROOSE M L. Identification of closely related citrus cultivars with inter-simple sequence repeat markers. Theoretical and Applied Genetics, 1997, 95(3): 408-417.