红三叶遗传图谱构建及抗白粉病基因QTL定位

doi:10.11686/cyxb2017213

摘要/Abstract

摘要： 本研究以感(岷山红三叶)、抗(澳大利亚红三叶品种♀Sensation×Renegade♂杂交新品系“甘农PR1”)白粉病红三叶材料为父母本杂交并种植成苗经人工接菌后筛选出抗、感白粉病的F₁群体为作图群体,利用AFLP标记构建红三叶高密度遗传图谱,并利用区间作图法对抗白粉病QTL进行了定位分析,可以为红三叶抗白粉病基因克隆和转基因等分子辅助育种奠定基础。结果表明,149个AFLP标记构建得到的遗传图谱包含7个连锁群(LG1,LG2,LG3,LG4,LG5,LG6和LG7),遗传图谱的总距离为640.5 cM。其中,LG1连锁群的遗传距离(140.6 cM)和标记间平均距离(9.4 cM)均最大;LG4连锁群的遗传距离(55.2 cM)和标记间平均距离(1.8 cM)最小。应用区间作图法对红三叶抗白粉病基因进行QTL分析定位,共检测到5个抗白粉病相关QTL位点(qrp-1,qrp-2,qrp-3,qrp-4和qrp-5),其中qrp-1、qrp-2、qrp-3和qrp-4位于LG4连锁群上,qrp-5位于LG5连锁群上。5个QTL位点对抗白粉病的贡献率为29%~90%,qrp-1对红三叶白粉病抗性的贡献率最大(90%),为主效QTL。

关键词: 红三叶, AFLP分子标记, 遗传连锁图谱, 抗白粉病, QTL分析

Abstract: In this study, F₁ generation red clover plants, obtained from crossing red clover varieties resistant (Gannong PR1, and Sensation and Renegade from Australia) or sensitive (Minshan) to powdery mildew, were artificially inoculated with powdery mildew and used as a quantitative trait locus (QTL) mapping population. AFLP markers were used to construct the genetic linkage map and the interval mapping method was used to locate the QTL for resistance to powdery mildew. This study will establish foundation to gene location and transgenosis of red clover. It was found that the linkage map based on 149 AFLP markers included 7 linkage groups (LG1, LG2, LG3, LG4, LG5, LG6 and LG7). The map covers a total distance of 640.5 cM. LG1 was found to be the largest (140.6 cM with the average distance between two markers being 9.4 cM), while LG4 was found to be the smallest yet with the highest marker density of any LG (55.2 cM in length with 1.8 cM on average between markers. Five QTLs (qrp-1, qrp-2, qrp-3, qrp-4 and qrp-5) related to powdery mildew resistance, and located on LG4 and LG5, were detected. The phenotypic variance explained by the 5 QTLs for powdery mildew resistance ranged from 29% to 90%, with the largest contribution being from qrp-1.Therefore, qrp-1 was identified as the main gene controlling powdery mildew resistance in red clover.

Key words: red clover (Trifolium pratense), AFLP marker, genetic linkage map, powdery mildew resistance, QTL analysis

蒲小剑,田久胜,田新会,杜文华. 红三叶遗传图谱构建及抗白粉病基因QTL定位[J]. 草业学报, 2018, 27(4): 79-88.

PU Xiao-jian, TIAN Jiu-sheng, TIAN Xin-hui, DU Wen-hua. Construction of the AFLP linkage map and QTL analysis of powdery mildew resistance in red clover[J]. Acta Prataculturae Sinica, 2018, 27(4): 79-88.

参考文献

[1] Jiang Y B, Wang C Z, Cui G W.Effect of red clover isoflavone on immune organs, igs and anti-oxidation activity of broiler chicks. Acta Agrestia Sinica, 2011, 19(3): 520-524.
姜义宝, 王成章, 崔国文. 红车轴草异黄酮对肉鸡免疫器官、免疫球蛋白及抗氧化性能的影响. 草地学报, 2011, 19(3): 520-524.
[2] Du W H.Red clover. Lanzhou: Gansu Science and Technology Press, 2006: 7-8.
杜文华. 红三叶. 兰州:甘肃科学技术出版社, 2006: 7-8.
[3] Liu X L, Du W H, Song C.Effects of nitrogen and phosphorus fertilization on isoflavone content in red clover. Acta Agriculture Boreali-occidentalis Sinica, 2010, 19(7): 159-163.
刘晓玲, 杜文华, 宋超. 氮磷肥施用量对红三叶中异黄酮含量的影响. 西北农业学报, 2010, 19(7): 159-163.
[4] Wang Z M, Yue M Q, Du W H, et al. Excellent legume for feeding and medicine-Minshan Trifolium pretense. Pratacultural Science, 2005, 22(4): 33-35.
王志明, 岳民勤, 杜文华, 等. 集饲用和药用价值于一体的牧草新秀——岷山红三叶. 草业科学, 2005, 22(4): 33-35.
[5] Liu X L, Song C, Du W H.Study on mechanism of resistance to powdery mildew in Minshan red clover. Grassland and Turf, 2011, 31(3): 73-76.
刘晓玲, 宋超, 杜文华. 红三叶对白粉病抗性机理研究. 草原与草坪, 2011, 31(3): 73-76.
[6] Wang Z H, Zhu D L, Yu C.Application in germplasm improvement of medical plants via biotechnology. Bulletin of Science and Technology, 2010, 26(1): 88-92.
王忠华, 朱东亮, 俞超. 现代育种技术在药用植物品种改良中的应用. 科技通报, 2010, 26(1): 88-92.
[7] Jia X X, Qi E F, Ma S, et al. Analysis of drought tolerance and herbicide resistance in transgenic potato plants over-expressing DREB1A/Bar. Acta Prataculturae Sinica, 2015, 24(11): 58-64.
贾小霞, 齐恩芳, 马胜, 等. 转DREB1A/Bar双价基因马铃薯的耐旱性及除草剂抗性分析. 草业学报, 2015, 24(11): 58-64.
[8] Li D M.Constructions on the linkage genetic map and QTL mapping for forage yield and rust resistance related traits in triticale. Lanzhou: Gansu Agricultural University, 2016.
李冬梅. 饲草型小黑麦的遗传图谱构建及草产量和抗锈病相关基因的QTL定位. 兰州: 甘肃农业大学, 2016.
[9] Xu Z, Mei L H, Li Z Y, et al. A genetic linkage map of Blume riagram f.sp. tritici based on AFLP molecular markers and a virulence genes. Acta Phytopathologica Sinica, 2009, 39(1): 16-22.
徐志, 梅丽宏, 李志英, 等. 利用AFLP分子标记和无毒基因构建小麦白粉菌遗传连锁图谱. 植物病理学报, 2009, 39(1): 16-22.
[10] Xu J Q, He F, Cui F, et al. Powdery mildew resistant locus in Tritileymus translocation line shannong 6343 mapped by molecular markers. Molecular Plant Breeding, 2012, 10(1): 86-91.
徐金秋, 何方, 崔法, 等. 小滨麦易位系山农6343抗白粉病基因的分子标记定位. 分子植物育种, 2012, 10(1): 86-91.
[11] Liu L Z, Li H, Yang R, et al. Mapping of gene resistance to wheat powdery mildew using SSR markers. Acta Botanica Boreali-Occidentalia Sinica, 2012, 32(7): 1322-1327.
刘联正, 李华, 杨睿, 等. 小麦抗白粉病基因SSR标记定位. 西北植物学报, 2012, 32(7): 1322-1327.
[12] Liu S L, Wang C Y, Wang Q Y, et al. SSR analysis of powdery mildew resistance gene in a new GERMPLASM N9628-2 of Triticum aestivum L. Acta Agronomica Sinica, 2008, 34(1): 84-88.
刘素兰, 王长有, 王秋英, 等. 小麦新种质N9628-2抗白粉病基因的SSR分析. 作物学报, 2008, 34(1): 84-88.
[13] Li Q Y, Zhang X L, Zhang Z G, et al. Advance of studies on constructing linkage map in Chinese cabbage using molecular marker. Biotechnology Bulletin, 2012, (6): 18-24.
李巧云, 张晓亮, 张志刚, 等. 大白菜分子遗传图谱构建研究进展. 生物技术通报, 2012, (6): 18-24.
[14] Zhu J, Wang T, Zhao Y J, et al. Identification of apple varieties with AFLP molecular markers. Acta Horticulturae Sinica, 2000, 27(2): 102-106.
祝军, 王涛, 赵玉军, 等. 应用AFLP分子标记鉴定苹果品种. 园艺学报, 2000, 27(2): 102-106.
[15] Gao J Q, Zhou J C, Wang X C, et al. Construction of AFLP fingerprint of partial high resistance to low phosphorus stress genotypic in Sugarbeet (Beta vulgaris L.). Jiangsu Agricultural Sciences, 2017, 45(6): 36-38.
高金秋, 周建朝, 王孝纯, 等. 部分高抗低磷胁迫基因型甜菜AFLP指纹图谱的构建. 江苏农业科学, 2017, 45(6): 36-38.
[16] Wang D, Yu X X, Yu Z, et al. AFLP analysis of genetic differences among elite hybrid potato lines. Acta Prataculturae Sinica, 2016, 25(9): 117-124.
王丹, 于肖夏, 于卓, 等. 马铃薯杂种优良株系遗传差异的AFLP分析. 草业学报, 2016, 25(9): 117-124.
[17] Xie L B, Wang X, Chen L X, et al. QTL mapping of Vitamin C in pepper fruit based on AFLP and SSR. Chinese Agricultural Science Bulletin, 2017, 33(16): 41-45.
谢立波, 王雪, 陈立新, 等. 基于AFLP和SSR技术对辣椒果实维生素C含量的QTL定位. 中国农学通报, 2017, 33(16): 41-45.
[18] Wang Z W, Wei L M, Xie X M, et al. Establishment of cotton genetic map and QTL analysis of fiber quality traits by using AFLP. Jiangsu Agricultural Sciences, 2015, 43(7): 71-74.
王志伟, 魏利民, 谢晓美, 等. 利用AFLP构建棉花遗传图谱及纤维品质性状QTL分析. 江苏农业科学, 2015, 43(7): 71-74.
[19] Wang X Z, Zhou R, Zhang X J, et al. Map construction and QTL analysis of oil content in soybean. Chinese Journal of Oil Crop Sciences, 2008, 30(3): 272-278.
王贤智, 周蓉, 张晓娟, 等. 大豆遗传图谱的构建和含油量的QTL分析. 中国油料作物学报, 2008, 30(3): 272-278.
[20] Li C X, Xu W G, Wang G S, et al. Molecular marker mapping and genetic analysis of a novel powdery mildew resistance gene PmHNK. Scientia Agricultura Sinica, 2009, 42(8): 2771-2777.
李春鑫, 许为钢, 王根松, 等. 小麦白粉病抗病新基因PmHNK的遗传分析和分子标记定位. 中国农业科学, 2009, 42(8): 2771-2777.
[21] Alfandi M, Ji Y, Shen L P, et al. Construction of genetic linkage map and localization of QTLs for powdery mildew resistance in cucumber (Cucumis sativus). IV International Symposium on Cucurbits, 2010, 871: 33-41.
[22] Fernando Y L, Carmen C, Emilio S, et al. Genetic linkage map of melon (Cucumis melon L.) and localization of a major QTL for powdery mildew resistance. Molecular Breeding, 2011, 27(2): 181-192.
[23] Ren J Z.Scientific research methods of pratacultural science. Beijing: China Agricultural Press, 1998: 214-236.
任继周. 草业科学研究方法. 北京: 中国农业出版社, 1998: 214-236.
[24] Vos P, Hogers R, Bleeker M, et al. AFLP a new technique for DNA fingerprinting. Nucleic Acids Research, 1995, 23: 4407-4414.
[25] Liu S X.Practical biological histological techniques. Beijing: Science Press, 2003: 222-224.
刘世新. 实用生物组织学技术. 北京: 科学出版社, 2003: 222-224.
[26] Jiang Z Y, Yu X X, Yu Z, et al. Construction of a genetic linkage map for tetraploid hybrid wheat grass using a SSR molecular marker. Acta Prataculturae Sinica, 2016, 25(6): 94-101.
姜志艳, 于肖夏, 于卓, 等. 利用SSR分子标记构建四倍体杂交冰草的遗传连锁图谱. 草业学报, 2016, 25(6): 94-101.
[27] Ding C L, Liu Y, Xu N X, et al. QTL analysis of traits relating to cold resistance of Zoysia japonica. Acta Agrestia Sinica, 2010, 18(5): 703-707.
丁成龙, 刘颖, 许能祥, 等. 日本结缕草抗寒相关性状的QTL分析. 草地学报, 2010, 18(5): 703-707.
[28] Cai C F, Liu G X, Cheng F Y, et al. Selecting optimal F₁ segregation population for genetic linkage mapping in tree peony. Journal of Beijing Forestry University, 2015, 37(3): 140-147.
蔡长福, 刘改秀, 成仿云, 等. 牡丹遗传作图最适F₁分离群体的选择. 北京林业大学学报, 2015, 37(3): 140-147.
[29] Guo J B, Huang L, Cheng L Q, et al. An integrated genetic linkage map from three F₂ population of cultivated peanut (Arachis hypogaea L.). Acta Agronomica Sinica, 2016, 42(2): 159-169.
郭建斌, 黄莉, 成良强, 等. 利用3个F₂群体整合高密度栽培种花生遗传连锁图. 作物学报, 2016, 42(2): 159-169.
[30] Du X L, Zhang Z W, Wu B, et al. Construction and analysis of genetic linkage map in tartary buckwheat (Fagopyrum tataricum) using SSR. Chinese Agricultural Science Bulletin, 2013, 29(21): 61-65.
杜晓磊, 张宗文, 吴斌, 等. 苦荞SSR分子遗传图谱的构建及分析. 中国农学通报, 2013, 29(21): 61-65.
[31] Jiang Z Y, Yu X X, Yu Z, et al. Construction of an AFLP-based genetic linkage map of tetraploid hybrid wheatgrass. Journal of Triticeae Crops, 2015, 35(4): 457-463.
姜志艳, 于肖夏, 于卓, 等. 四倍体杂交冰草AFLP遗传连锁图谱的构建. 麦类作物学报, 2015, 35(4): 457-463.
[32] Fang Y Y, Yu X X, Yu Z, et al. QTL mapping analysis of 10 important traits in Sorghum-Sudan grass hybrid. Journal of Northwest A&F University (Nature Science Edition), 2015, 43(4): 26-34, 43.
房永雨, 于肖夏, 于卓, 等. 高丹草10个重要性状的QTL定位分析. 西北农林科技大学学报(自然科学版), 2015, 43(4): 26-34, 43.
[33] Li L, Chen Z W, Du Z Z, et al. Genetic map preliminary structure of low-nitrogen tolerance used DH population in barley. Journal of Triticeae Crops, 2012, 32(6): 1021-1025.
李梁, 陈志伟, 杜志钊, 等. 大麦DH群体在耐低氮相关遗传连锁图谱初步构建中的运用. 麦类作物学报, 2012, 32(6): 1021-1025.
[34] Studer B, Jensen L B, Hentrup S, et al. Genetic characterisation of seed yield and fertility traits in perennial ryegrass (Lolium perenne L.). Theoretical and Applied Genetics, 2008, 117(5): 781-791.
[35] Wang M Y, Zhang T J, Long R C, et al. Preliminary construction of genetic map in tetraploid alfalfa. Acta Agrestia Sinica, 2015, 23(6): 1247-1251.
王梦颖, 张铁军, 龙瑞才, 等. 四倍体紫花苜蓿遗传图谱的初步构建. 草地学报, 2015, 23(6): 1247-1251.
[36] Isobe S, Kölliker R, Hisano H, et al. Construction of a consensus linkage map for red clover (Trifolium pretense L.). BMC Plant Biology, 2009, (9): 57.
[37] Xie W G.Genetic linkage map and flowering time gene mapping in orchard grass (Dactylis glomerata L.). Ya’an: Sichuan Agricultural University, 2012.
谢文刚. 鸭茅分子遗传连锁图谱构建及开花基因定位. 雅安: 四川农业大学, 2012.
[38] 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.
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.
[39] Zhong L, Yang C Y, Shu J H, et al. Research progress on genetic breeding of tall fescue (Festuca arundinacea Schreb.). Agricultural Science & Technology, 2014, 15(2): 265-268.
[40] Li S, Zhao G F.AFLP molecular marker and its application. Acta Botanica Boreali-Occidentalia Sinica, 2003, 23(5): 830-836.
李珊, 赵桂仿. AFLP分子标记及其应用. 西北植物学报, 2003, 23(5): 830-836.
[41] Hu Z H, Guo H Y, Hu X L, et al. Genetic diversity of Cannabis sativa L. based on AFLP analysis. Journal of Plant Genetic Resources, 2012, 13(4): 555-561.
胡尊红, 郭鸿彦, 胡学礼, 等. 大麻品种遗传多样性的AFLP分析. 植物遗传资源学报, 2012, 13(4): 555-561.
[42] Li H Y, Li Z Y, Xin X, et al. Genetic diversity in Medicago ruthenica (L.) Trautv. populations using AFLP marker. Journal of Plant Genetic Resources, 2016, 17(1): 78-83.
李鸿雁, 李志勇, 辛霞, 等. 野生扁蓿豆种质资源AFLP 遗传多样性的分析. 植物遗传资源学报, 2016, 17(1): 78-83.
[43] Wang B, Weng M L.The principle and application of AFLP. Hybrid Rice, 1996, (5): 27-30.
王斌, 翁曼丽. AFLP的原理及其应用. 杂交水稻, 1996, (5): 27-30.
[44] Bert P F, Charmet G, Sourdille P, et al. A high-density molecular map for ryegrass (Lolium perenne) using AFLP markers. Theoretical and Applied Genetics, 1999, 99(3): 445-452.
[45] Jaime S S, Daniza M U, Leonardo A R.Molecular description and similarity relationships among native germplasm potatoes (Solanum tuberosum ssp. tuberosum L.) using morphological data and AFLP markers. Electronic Journal of Biotechnology, 2007, 10(3): 436-443.
[46] Xie W G, Liu W X, Zhang J Q, et al. Molecular genetic linkage map and its application in forage crops. Pratacultural Science, 2014, 31(6): 1147-1159.
谢文刚, 刘文献, 张建全, 等. 牧草分子遗传连锁图谱及其应用. 草业科学, 2014, 31(6): 1147-1159.
[47] Jing Z B, Yu L, Wei L, et al. Research advances in genomics and transgenic of forage in abroad. Biotechnology Bulletin, 2011, (11): 12-25.
井赵斌, 俞靓, 魏琳, 等. 国外牧草基因组学和转基因技术研究进展. 生物技术通报, 2011, (11): 12-25.
[48] Zhu Y W, Lin Y R, Chen L.Research progress of rice molecular breeding in China. Journal of Xiamen University (Natural Science), 2016, 55(5): 661-671.
朱义旺, 林雅容, 陈亮. 我国水稻分子育种研究进展. 厦门大学学报(自然科学版), 2016, 55(5): 661-671.
[49] Hirata M, Cai H W, Inoue M, et al. 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): 270-279.
[50] Tian L B.Constructing of genetic maps and QTL mapping of powdery mildew resistance in bitter melon. Shenyang: Shenyang Agricultural University, 2015.
田丽波. 苦瓜遗传图谱构建及白粉病抗性的QTL定位. 沈阳: 沈阳农业大学, 2015.
[51] Shang L G.Genetic analysis of heterosis using backcross population in upland cotton (Gossypium hirsutum L.). Beijing: China Agricultural University, 2016.
商连光. 陆地棉回交群体杂种优势遗传基础研究. 北京: 中国农业大学, 2016.
[52] Zhang J.Construction of high-density genetic map and QTL analysis of ornamental traits in Mei. Beijing: Beijing Forestry University, 2016.
张杰. 梅花高密度遗传图谱构建及部分观赏性状QTL分析. 北京: 北京林业大学, 2016.
[53] Wang L.Construction of wheat molecular genetic map and QTL analysis for agronomic and quanlity traits. Taian: Shandong Agricultural University, 2012.
王霖. 小麦遗传连锁图谱构建及主要农艺和品质性状QTL定位. 泰安: 山东农业大学, 2012.