燕麦全基因组SSR位点鉴定及多态性标记开发

doi:10.13560/j.cnki.biotech.bull.1985.2023-0697

摘要/Abstract

摘要：

【目的】了解燕麦基因组SSR位点分布情况并开发燕麦全基因组SSR标记，为燕麦皮裸基因克隆、遗传图谱构建、群体多样性分析等提供科学的参考依据。【方法】利用已公布的“三分三”裸燕麦参考基因组，通过TBtools软件对燕麦全基因组的SSR位点进行检索并利用EXCEL和SPSS软件对检索数据分析其分布特征。根据检索结果设计引物，在构建的F₂遗传群体中小规模进行非变性聚丙烯酰胺凝胶电泳验证，并克隆测序其有效性与真实性。【结果】在燕麦基因组中，SSR数量众多，类型丰富。全基因组共检索到828 138个SSR位点，SSR平均密度为78.16个/Mb，平均距离为12.90 kb。单、双、三核苷酸重复类型占主导优势。基元重复次数主要集中在5次和6次，A/T、AG/CT和AAC/GTT重复基元为优势基元。燕麦基因组SSR位点呈现中度多态性，长度变化范围为11-1 587 bp。并由此开发设计52对多态性引物在试验材料中进行扩增验证，在构建的遗传群体中均具有多态性。【结论】在“三分三”裸燕麦参考基因组中开发SSR引物有效可行，可以用于后续进一步试验研究。

关键词: 燕麦, 皮裸, SSR标记, 引物开发, 多态性

Abstract:

【Objective】 Due to the limited number of SSR markers developed in oat, it is difficult to meet the needs of functional gene mapping and polymorphism marker development in oat.【Method】 Using the published reference genome, the SSR loci of the whole oat genome were retrieved by TBtools software, and their distribution characteristics were analyzed by Excel and SPSS software.【Result】 The SSR was abundant and varied in oat genome. A total of 828 138 SSR loci were identified in the whole genome, with an average density of 78.16 SSR/Mb and an average distance of 12.90 kb. Single, double and trinucleotide repeat types were dominant. The repetitions of the primitives were mainly 5 and 6, and the A/T, AG/CT and AAC/GTT repetitions were the dominant primitives. The SSR loci of oat genome showed moderate polymorphism, with length ranging from 11 to 1 587 bp. Through the systematic identification and analysis of the whole oat gene SSR locus, 52 pairs of polymorphic primers were developed and validated in the test material.【Conclusion】 The development of SSR primers in the reference genome of “Sanfensan” naked oat is effective and feasible, and can be used for further experimental research.

Key words: oats, skin, SSR markers, primer development, polymorphism

陈凯凌, 武涛, 徐逸群, 高佳, 张美俊, 李欣, 贾举庆. 燕麦全基因组SSR位点鉴定及多态性标记开发[J]. 生物技术通报, 2024, 40(2): 120-129.

CHEN Kai-ling, WU Tao, XU Yi-qun, GAO Jia, ZHANG Mei-jun, LI Xin, JIA Ju-qing. Identification of SSR Loci and Development of Polymorphic Markers in Whole Genome of Oat[J]. Biotechnology Bulletin, 2024, 40(2): 120-129.

图/表 9

参考文献 32

[1]	吴斌, 郑殿升, 严威凯, 等. 燕麦分子育种研究进展[J]. 植物遗传资源学报, 2019, 20(3): 485-495.
	Wu B, Zheng DS, Yan KW, et al. Advances in molecular breeding of oats[J]. Journal of Plant Genetic Resources, 2019, 20(3): 485-495.
[2]	Rasane P, Jha A, Sabikhi L, et al. Nutritional advantages of oats and opportunities for its processing as value added foods-areview[J]. J Food Sci Technol, 2015, 52(2): 662-675.
[3]	潘莹, 程时锋. 燕麦基因组学研究进展[J]. 植物遗传资源学报, 2021, 22(2): 304-308. doi: 10.13430/j.cnki.jpgr.20200910001
	Pan Y, Cheng SF. Research progress on oat genomics study[J]. J Plant Genet Res, 2021, 22(2): 304-308.
[4]	Kamal N, Tsardakas Renhuldt N, Bentzer J, et al. The mosaic oat genome gives insights into a uniquely healthy cereal crop[J]. Nature, 2022, 606(7912): 113-119. doi: 10.1038/s41586-022-04732-y
[5]	Peng YY, Yan HH, Guo LC, et al. Reference genome assemblies reveal the origin and evolution of allohexaploid oat[J]. Nat Genet, 2022, 54(8): 1248-1258. doi: 10.1038/s41588-022-01127-7
[6]	Wu JZ, Zhao Q, Wu GW, et al. Development of novel SSR markers for flax(Linum usitatissimum L.)using reduced-representation genome sequencing[J]. Front Plant Sci, 2017, 7: 2018.
[7]	Guo R, Landis JB, Moore MJ, et al. Development and application of transcriptome-derived microsatellites in Actinidia eriantha(Actinidiaceae)[J]. Front Plant Sci, 2017, 8: 1383. doi: 10.3389/fpls.2017.01383 URL
[8]	Malausa T, Gilles A, Meglecz E, et al. High-throughput microsatellite isolation through 454 GS-FLX titanium pyrosequencing of enriched DNA libraries[J]. Mol Ecol Resour, 2011, 11(4): 638-644. doi: 10.1111/j.1755-0998.2011.02992.x pmid: 21676194
[9]	Sowa S, Paczos-Grzęda E. Identification of molecular markers for the PC39 gene conferring resistance to crown rust in oat[J]. Theor Appl Genet, 2020, 133(4): 1081-1094. doi: 10.1007/s00122-020-03533-z pmid: 31927607
[10]	Zhao J, Kebede AZ, Menzies JG, et al. Chromosomal location of the crown rust resistance gene Pc98 in cultivated oat(Avena sativa L.)[J]. Theor Appl Genet, 2020, 133(4): 1109-1122. doi: 10.1007/s00122-020-03535-x pmid: 31938813
[11]	Yan H, Yu K, Xu Y, et al. Position validation of the dwarfing gene dw6 in oat(Avena sativa L.)and its correlated effects on agronomic traits[J]. Front Plant Sci, 2021, 12: 668847. doi: 10.3389/fpls.2021.668847 URL
[12]	吴斌, 张茜, 宋高原, 等. 裸燕麦SSR标记连锁群图谱的构建及β-葡聚糖含量QTL的定位[J]. 中国农业科学, 2014, 47(6): 1208-1215. doi: 10.3864/j.issn.0578-1752.2014.06.017
	Wu B, Zhang Q, Song GY, et al. Construction of SSR genetic linkage map and analysis of QTLs related to β-glucan Content of naked oat(Avena nuda L.)[J]. Sci Agric Sin, 2014, 47(6): 1208-1215.
[13]	Isabel LP, Park JR, Lee GS, et al. Development of EST-SSR markers and analysis of genetic relationship it's resources in hexaploid oats[J]. J Crop Sci Biotechnol, 2019, 22(3): 243-251. doi: 10.1007/s12892-019-0158-0
[14]	Chen CJ, Chen H, Zhang Y, et al. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Mol Plant, 2020, 13(8): 1194-1202. doi: S1674-2052(20)30187-8 pmid: 32585190
[15]	Ubert IP, Zimmer CM, Pellizzaro K, et al. Genetics and molecular mapping of the naked grains in hexaploid oat[J]. Euphytica, 2017, 213(2): 41. doi: 10.1007/s10681-017-1836-1
[16]	Zhou Y, Zhao XB, Li YW, et al. Triticum population sequencing provides insights into wheat adaptation[J]. Nat Genet, 2020, 52(12): 1412-1422. doi: 10.1038/s41588-020-00722-w pmid: 33106631
[17]	原志敏. 玉米全基因组SSRs分子标记开发与特征分析[D]. 雅安: 四川农业大学, 2013.
	Yuan ZM. Development and characterization of SSRs molecular markers in maize genome[D]. Ya'an: Sichuan Agricultural University, 2013.
[18]	马名川, 刘龙龙, 刘璋, 等. 苦荞全基因组SSR位点特征分析与分子标记开发[J]. 作物杂志, 2021(1): 38-46.
	Ma MC, Liu LL, Liu Z, et al. Analysis of SSR loci in whole genome and development of molecular markers in tartary buckwheat[J]. Crops, 2021(1): 38-46.
[19]	张晗, 王雪梅, 王东建, 等. 谷子基因组SSR信息分析和标记开发[J]. 分子植物育种, 2013, 11(1): 30-36.
	Zhang H, Wang XM, Wang DJ, et al. Survey of SSRs in foxtail millet genome and development of SSR markers[J]. Mol Plant Breed, 2013, 11(1): 30-36.
[20]	仇静静. 野生花生全基因组SSR标记的开发与应用[D]. 济南: 山东师范大学, 2018.
	QIU JJ. Development and application of SSR markers in wild peanut genome[D]. Jinan: Shandong Normal University, 2018.
[21]	Cavagnaro PF, Senalik DA, Yang LM, et al. Genome-wide characterization of simple sequence repeats in cucumber(Cucumis sativus L.)[J]. BMC Genomics, 2010, 11: 569. doi: 10.1186/1471-2164-11-569 pmid: 20950470
[22]	关玲, 章镇, 王新卫, 等. 苹果基因组SSR位点分析与应用[J]. 中国农业科学, 2011, 44(21): 4415-4428. doi: 10.3864/j.issn.0578-1752.2011.21.010
	Guan L, Zhang Z, Wang XW, et al. Evaluation and application of the SSR loci in apple genome[J]. Sci Agric Sin, 2011, 44(21): 4415-4428. doi: 10.3864/j.issn.0578-1752.2011.21.010
[23]	李乔乔, 王宇晴, 刘蕊, 等. 甜菜全基因组SSR引物的筛选与评价[J]. 中国农学通报, 2022, 38(12): 95-99. doi: 10.11924/j.issn.1000-6850.casb2021-0726
	Li QQ, Wang YQ, Liu R, et al. The whole genome SSR primers of sugar beet: screening and evaluation[J]. Chin Agric Sci Bull, 2022, 38(12): 95-99. doi: 10.11924/j.issn.1000-6850.casb2021-0726
[24]	Han B, Wang CB, Tang ZH, et al. Genome-wide analysis of microsatellite markers based on sequenced database in chinese spring wheat(Triticum aestivum L.)[J]. PLoS One, 2015, 10(11): e0141540. doi: 10.1371/journal.pone.0141540 URL
[25]	Morgante M, Hanafey M, Powell W. Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J]. Nat Genet, 2002, 30(2): 194-200. doi: 10.1038/ng822 pmid: 11799393
[26]	张霞, 于卓, 金兴红, 等. 马铃薯SSR引物的开发、特征分析及在彩色马铃薯材料中的扩增研究[J]. 作物学报, 2022, 48(4): 920-929. doi: 10.3724/SP.J.1006.2022.14065
	Zhang X, Yu Z, Jin XH, et al. Development and characterization analysis of potato SSR primers and the amplification research in colored potato materials[J]. Acta Agron Sin, 2022, 48(4): 920-929. doi: 10.3724/SP.J.1006.2022.14065
[27]	杜磊, 蒙秋伊, 尚昆, 等. 薏苡基因组SSR标记开发与应用[J]. 分子植物育种, 2022, 20(3): 887-894.
	Du L, Meng QY, Shang K, et al. Development and application of genomic SSR markers in coix lachryma-jobi genome[J]. Mol Plant Breed, 2022, 20(3): 887-894.
[28]	姚嘉瑜, 张立武, 赵捷, 等. 黄麻全基因组SSR鉴定与特征分析[J]. 作物学报, 2019, 45(1): 10-17. doi: 10.3724/SP.J.1006.2019.84072
	Yao JY, Zhang LW, Zhao J, et al. Evaluation and characteristic analysis of SSRs from the whole genome of jute(Corchorus capsularis)[J]. Acta Agron Sin, 2019, 45(1): 10-17. doi: 10.3724/SP.J.1006.2019.84072 URL
[29]	童治军, 焦芳婵, 肖炳光. 普通烟草及其祖先种基因组SSR位点分析[J]. 中国农业科学, 2015, 48(11): 2108-2117. doi: 10.3864/j.issn.0578-1752.2015.11.003
	Tong ZJ, Jiao FC, Xiao BG. Analysis of SSR loci in nicotina tabacum genome and its two ancestral species genome[J]. Sci Agric Sin, 2015, 48(11): 2108-2117.
[30]	叶卫军, 陈圣男, 杨勇, 等. 绿豆SSR标记的开发及遗传多样性分析[J]. 作物学报, 2019, 45(8): 1176-1188. doi: 10.3724/SP.J.1006.2019.84155
	Ye WJ, Chen SN, Yang Y, et al. Development of SSR markers and genetic diversity analysis in mung bean[J]. Acta Agron Sin, 2019, 45(8): 1176-1188.
[31]	Oliveira EJ, Pádua JG, Zucchi MI, et al. Origin, evolution and genome distribution of microsatellites[J]. Genet Mol Biol, 2006, 29(2): 294-307. doi: 10.1590/S1415-47572006000200018 URL
[32]	Luo MC, Gu YQ, Puiu D, et al. Genome sequence of the progenitor of the wheat D genome Aegilops tauschii[J]. Nature, 2017, 551(7681): 498-502. doi: 10.1038/nature24486 URL

亚组 Subgroup	染色体 Chromosome	染色体大小 Size of chromosome/Mb	SSR数量 Number of SSRs	SSR密度 SSR density/(SSR·Mb^-1)	SSR距离 SSR distance/(kb·SSR^-1)
A	1A	551.55	46 585	84.46	11.84
	2A	462.81	41 810	90.34	11.07
	3A	418.65	34 491	82.39	12.14
	4A	468.67	41 615	88.79	11.26
	5A	489.91	40 918	83.52	11.97
	6A	456.82	40 619	88.92	11.25
	7A	497.83	41 202	82.76	12.08
C	1C	459.16	30 835	67.15	14.89
	2C	574.16	38 944	67.83	14.74
	3C	616.20	49 132	79.73	12.54
	4C	701.82	46 713	66.56	15.02
	5C	601.56	41 519	69.02	14.49
	6C	610.20	45 292	74.22	13.47
	7C	530.95	37 060	69.80	14.33
D	1D	473.71	36 983	78.07	12.80
	2D	532.98	41 375	77.63	12.88
	3D	464.06	33 680	72.58	13.78
	4D	451.84	37 661	83.35	12.00
	5D	500.51	39 941	79.80	12.53
	6D	293.84	22 043	75.02	13.33
	7D	498.92	39 720	79.61	12.56
合计Total		10 656.15	828 138	-	-

亚组 Subgroup	染色体 Chromosome	染色体大小 Size of chromosome/Mb	SSR数量 Number of SSRs	SSR密度 SSR density/(SSR·Mb^-1)	SSR距离 SSR distance/(kb·SSR^-1)
A	1A	551.55	46 585	84.46	11.84
	2A	462.81	41 810	90.34	11.07
	3A	418.65	34 491	82.39	12.14
	4A	468.67	41 615	88.79	11.26
	5A	489.91	40 918	83.52	11.97
	6A	456.82	40 619	88.92	11.25
	7A	497.83	41 202	82.76	12.08
C	1C	459.16	30 835	67.15	14.89
	2C	574.16	38 944	67.83	14.74
	3C	616.20	49 132	79.73	12.54
	4C	701.82	46 713	66.56	15.02
	5C	601.56	41 519	69.02	14.49
	6C	610.20	45 292	74.22	13.47
	7C	530.95	37 060	69.80	14.33
D	1D	473.71	36 983	78.07	12.80
	2D	532.98	41 375	77.63	12.88
	3D	464.06	33 680	72.58	13.78
	4D	451.84	37 661	83.35	12.00
	5D	500.51	39 941	79.80	12.53
	6D	293.84	22 043	75.02	13.33
	7D	498.92	39 720	79.61	12.56
合计Total		10 656.15	828 138	-	-

项目 Item	平均值 Average	标准差 Standard deviation	染色体大小 Size of chromosome	SSR密度 SSR density	SSR距离 SSR distance	SSR数量 Number of SSRs
染色体大小Size of chromosome	507.44	85.19	1
SSR密度SSR density	78.16	7.38	-0.38	1
SSR距离SSR distance	12.90	1.24	0.40	-0.99**	1
SSR数量Number of SSRs	39 435.14	5 945.73	0.81**	0.21	-0.20	1

项目 Item	平均值 Average	标准差 Standard deviation	染色体大小 Size of chromosome	SSR密度 SSR density	SSR距离 SSR distance	SSR数量 Number of SSRs
染色体大小Size of chromosome	507.44	85.19	1
SSR密度SSR density	78.16	7.38	-0.38	1
SSR距离SSR distance	12.90	1.24	0.40	-0.99**	1
SSR数量Number of SSRs	39 435.14	5 945.73	0.81**	0.21	-0.20	1

基因组Genome	亚组Subgroup	染色体长度Chromosome length/Mb	SSR数目Number of SSR	SSR密度SSR density/(SSR·Mb^-1)
Sang	A	3 277.34	242 707	73.99
	C	3 867.70	260 585	67.30
	D	3 125.40	223 135	71.32
三分三	A	3 346.24	287 240	85.88
	C	4 094.05	289 495	70.62
	D	3 215.86	251 403	78.01