控制高粱分蘖与主茎株高一致性的基因定位
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摘要
用分蘖与主茎株高一致的高粱品系K35-Y5与分蘖明显高于主茎的高粱恢复系1383杂交, F1自交获得F2分离群体,构建两混池,采用BSA (bulked segregation analysis)和SLAF (specific length amplified fragment sequencing)技术将高粱分蘖与主茎株高一致基因定位。遗传分析表明,分蘖与主茎株高一致性状由1对隐性核基因控制。参考已公布高粱基因组设计酶切方案,构建SLAF文库并测序。对高粱参考基因组序列进行电子酶切预测,确定限制性内切酶为Rsa I+Hae III,酶切片段长度为364～414 bp;测序Q30为91.70%, GC含量为45.79%,达到测序要求;与水稻的测序数据相比,高粱的双端比对效率为93.35%,酶切效率为90.60%, SLAF建库正常。共获得30.80 M reads,开发出133,246个SLAF标签,再通过分析SLAF标签的多态性,检测到319,428个SNP位点。利用SNP-index法和Euclideandistance法及取两者交集进行关联分析,最后得到一个关联区域,位于第9染色体上的54,788,026～56,740,873区间内,关联区域长度1.95 Mb。分析关联区域内的基因在2个亲本之间SNP,对这些SNP进行变异的注释,发现4个非同义突变的SNP。经验证,这4个SNP位点和分蘖与主茎株高一致性状相关。对应到Sobic.009G197901.1、Sobic.009G213300.1和Sobic.009G221200.1三个基因上,这些基因可能是与性状直接相关的功能基因。
In this study, an F2 population derived from a cross between two sorghum lines with same height and different heights of tiller and main stem respectively was used to construct pools. In order to map genes related to same height of tiller and main stem, BSA and SLAF-seq technique were developed. Genetic analysis showed that the trait of same height of tiller and main stem was controlled by a single recessive nuclear gene. Reference genome of sorghum was used to design markers by simulating the number of markers produced by different enzymes. The SLAF library was conducted and sequenced by paired-end sequencing. The restriction enzyme was Rsa I + Hae III. The fragment length was 364-414 bp. The quality of Q30 was up to 91.70% and the GC content(45.79%) was low enough to perform sequencing. Compared with the sequencing data of rice, the construction of SLAF library fitted well to the standard, with its paired-end mapped reads reaching to 93.35% and normal digestion ratio reaching to 90.60% in sorghum. In total of 30.80 M reads and 133,246 SLAF labels were obtained and 319,428 SNPs were found. The associated region was located by SNP-index, Euclidean distance, and their combination. The candidate regions had a size of 1.95 Mb at nucleotides 54,788,026-56,740,873 on Chr.9. The SNPs locating at the associated region were analyzed between the two parents. Four non-synonymous-coding SNPs were found in this region. By verification, these SNPs were considered to be related to same height of tiller and main stem. Corresponding to three candidate genes(Sobic.009 G197901.1, Sobic.009 G213300.1, and Sobic.009 G221200.1), these genes may be functional genes directly related to the traits.
In this study, an F2 population derived from a cross between two sorghum lines with same height and different heights of tiller and main stem respectively was used to construct pools. In order to map genes related to same height of tiller and main stem, BSA and SLAF-seq technique were developed. Genetic analysis showed that the trait of same height of tiller and main stem was controlled by a single recessive nuclear gene. Reference genome of sorghum was used to design markers by simulating the number of markers produced by different enzymes. The SLAF library was conducted and sequenced by paired-end sequencing. The restriction enzyme was Rsa I + Hae III. The fragment length was 364-414 bp. The quality of Q30 was up to 91.70% and the GC content(45.79%) was low enough to perform sequencing. Compared with the sequencing data of rice, the construction of SLAF library fitted well to the standard, with its paired-end mapped reads reaching to 93.35% and normal digestion ratio reaching to 90.60% in sorghum. In total of 30.80 M reads and 133,246 SLAF labels were obtained and 319,428 SNPs were found. The associated region was located by SNP-index, Euclidean distance, and their combination. The candidate regions had a size of 1.95 Mb at nucleotides 54,788,026-56,740,873 on Chr.9. The SNPs locating at the associated region were analyzed between the two parents. Four non-synonymous-coding SNPs were found in this region. By verification, these SNPs were considered to be related to same height of tiller and main stem. Corresponding to three candidate genes(Sobic.009 G197901.1, Sobic.009 G213300.1, and Sobic.009 G221200.1), these genes may be functional genes directly related to the traits.
引文
[1]邹剑秋,朱凯,张志鹏,黄先伟.国内外高粱深加工研究现状与发展前景.杂粮作物,2002,22(5):296-298.Zou J Q,Zhu K,Zhang Z P,Huang X W.Status and prospects of research on sorghum deep processing at home and abroad.Rain Fed Crops,2002,22(5):296-298(in Chinese with English abstract).
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[3]白文斌,张福跃,焦晓燕,董良利,柳青山,平俊爱.中国高粱产业工程技术研究的定位思考.中国农学通报,2013,29(11):107-110.Bai W B,Zhang F Y,Jiao X Y,Dong L L,Liu Q S,Ping J A.The fixed position thought of sorghum engineering technology research in china.Chin Agric Sci Bull,2013,29(11):107-110(in Chinese with English abstract).
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[5]焦少杰,王黎明,姜艳喜,严洪冬,苏德峰,孙广全.粒用高粱机械化栽培品种选择.园艺与种苗,2012,(12):1-2.Jiao S J,Wang L M,Jiang Y X,Yan H D,Su D F,Sun G Q.Varieties selection of grain sorghum for mechanized cultivation.Hortic Seed,2012,(12):1-2(in Chinese with English abstract).
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[9]Rami J F,Dufour P,Trouch G,Fliedel G,Mestres C,Davrieux F,Blanchard P,Hamon P.Quantitative trait loci for grain quality,productivity,morphological and agronomical traits in sorghum(Sorghum bicolor L.Moench).Theor Appl Genet,1998,97:605-616.
[10]Klein R R,Rodrigyez-Herrera R,Schlueter J A.Identification of genomic regions that affect grain-mould incidence and other traits of agronomic importance in sorghum.Theor Appl Genet,2001,102:307-319.
[11]Upadhyaya H D,Wang Y H,Sharma S,Singh S.Association mapping of height and maturity across five environments using the sorghum mini core collection.Genome,2012,55:471-479.
[12]Upadhyaya H D,Wang Y H,Gowda C L,Sharma S.Association mapping of maturity and plant height using SNP markers with the sorghum mini core collection.Theor Appl Genet,2013,126:2003-2015.
[13]Harris-Shultz K R,Davis R F,Knoll J E,Anderson W,Wang H.Inheritance and identification of a major quantitative trait locus(QTL)that confers resistance to Meloidogyne incognita and a novel QTL for plant height in sweet sorghum.Phytopathology,2015,105:1522-1528.
[14]苏舒.高粱形态学农艺性状的QTL定位研究.南京大学硕士学位论文,江苏南京,2012.Su S.QTL Mapping of Agronomic Traits of Morphology in Sorghum.MS Thesis of Nanjing University,Nanjing,Jiangsu,China,2012(in Chinese with English abstract).
[15]刘娟.高粱株高和抗蚜连锁标记的发掘与验证.河北农业大学硕士学位论文,河北保定,2014.Liu J.Discover and Validation of Markers Linkage with Plant Height and Resistance to Aphid of Sorghum.MS Thesis of Agricultural University of Hebei,Baoding,Hebei,China,2014(in Chinese with English abstract).
[16]Lafarge T A,Broad J,Hammer G L.Tillering in grain sorghum over a wide range of population densities:identification of a common hierarchy for tiller emergence,leaf area development and fertility.Ann Bot,2002,90:87-98.
[17]Feltus F A,Hart G E,Schertz K F,Casa A M,Kresovich S,Abraham S,Klein P E,Brown P J,Paterson A H.Alignment of genetic maps and QTLs between inter-and intra-specific sorghum populations.Theor Appl Genet,2006,112:1295-1305.
[18]Shehzad T,Iwata H,Okuno K.Genome-wide association mapping of quantitative traits in sorghum(Sorghum bicolor(L.)Moench)by using multiple models.Breed Sci,2009,59:217-227.
[19]Shiringani A L,Frisch M,Friedt W.Genetic mapping of QTLs for sugar-related traits in a RIL population of Sorghum bicolor L.Moench.Theor Appl Genet,2010,121:323-336.
[20]董维,苏舒,游录鹏,黄守程,戚金亮,陆桂华,黄应华,杨永华.高粱F6代群体分蘖数的QTL定位.南京林业大学学报(自然科学版),2013,37(2):55-58.Dong W,Su S,You L P,Huang S C,Qi J L,Lu G H,Huang Y H,Yang Y H.QTLs analysis of tillers number in F6 sorghum population.J Nanjing For Univ(Nat Sci Edn),2013,37(2):55-58(in Chinese with English abstract).
[21]Kozich J J,Westcott S L,Baxter N T,Highlander S K,Schloss PD.Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the miseq illumina sequencing platform.Appl Environ Microbiol,2013,79:5112-5120.
[22]International Rice Genome Sequencing Project.The map-based sequence of the rice genome.Nature,2005,436:793-800.
[23]Abe A,Kosugi S,Yoshida K,Natsume S,Takagi H,Kanzaki H,Matsumura H,Yoshida K,Mitsuoka C,Tamiru M,Innan H,Cano L,Kamoun S,Terauchi R.Genome sequencing reveals agronomically important loci in rice using MutMap.Nat Biotechnol,2012,30:174-178.
[24]Hill J T,Demarest B L,Bisgrove B W,Gorsi B,Su Y C,Yost H J.MMAPPR:mutation mapping analysis pipeline for pooled RNA-seq.Genome Res,2013,23:687-697.
[25]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics,2009,25:1754-1760.
[26]Takagi H,Abe A,Yoshida K,Kosugi S,Natsume S,Mitsuoka C,Uemura A,Utsushi H,Tamiru M,Takuno S,Innan H,Cano LM,Kamoun S,Terauchi R.QTL-seq:rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations.Plant J,2013,74:174-183.
[27]贺捷.甜高粱分蘖特性国内研究进展.中国糖料,2017,39(2):65-67.He J.Research progresses on tillering characteristics of sweet sorghum in China.Sugar Crops China,2017,39(2):65-67(in Chinese with English abstract).
[28]詹鹏杰,张福耀,王瑞,于纪珍,李燕.适宜机械化生产酿造高粱汾酒粱1号的选育.安徽农业科学,2016,44(31):13-14.Zhan P J,Zhang F Y,Wang R,Yu J Z,Li Y.Breeding of Fenjiuliang No.1:a brewing sorghum suitable for mechanized production.J Anhui Agric Sci,2016,44(31):13-14(in Chinese with English abstract).
[29]Brown P J,Klein P E,Bortiri E,Acharya C B,Rooney W L,Kresovich S.Inheritance of inflorescence architecture in sorghum.Theor Appl Genet,2006,113:931-942.
[30]Shiringani A L,Frisch M,Friedt W.Genetic mapping of QTLs for sugar-related traits in a RIL population of Sorghum bicolor L.Moench.Theor Appl Genet,2010,121:323-336.
[31]王柏柯,李宁,唐亚萍,王强,杨涛,杨生保,帕提古丽,余庆辉,高杰.基于简化基因组测序技术的番茄雄性不育基因定位.西北农林科技大学学报(自然科学版),2017,45(6):177-184.Wang B K,Li N,Tang Y P,Wang Q,Yang T,Yang S B,Pati G L,Yu Q H,Gao J.Mapping male-sterile gene in tomato by specific length amplified fragment sequencing.J Northwest A&F Univ(Nat Sci Edn).2017,45(6):177-184(in Chinese with English abstract).
[32]王伟,刘凡,任莉,徐理,陈旺,曾令益,黄炳文,方小平.采用SLAF-seq技术开发甘蓝型油菜霜霉病抗性SNP位点.中国油料作物学报,2016,38:555-562.Wang W,Liu F,Ren L,Xu L,Chen W,Zeng L Y,Huang B W,Fang X P.Resistance SNP development to downy mildew in Brassica napus using SLAF-seq technique.Chin J Oil Crop Sci,2016,38:555-562(in Chinese with English abstract).
[33]Geng X,Jiang C,Yang J,Wang L,Wu X,Wei W.Rapid identification of candidate genes for seed weight using the SLAF-Seq method in Brassica napus.PLoS One,2016,11:e0147580.
[2]吕富堂,韩爱清,杜秀兰,张福耀,李团银.建国以来中国高粱发展历程及发展趋势.山西农业科学,2002,30(3):20-24.Lyu F T,Han A Q,Du X L,Zhang F Y,Li T Y.Development and tendency of Chinese sorghum since the founding of P R China.JShanxi Agric Sci,2002,30(3):20-24(in Chinese with English abstract).
[3]白文斌,张福跃,焦晓燕,董良利,柳青山,平俊爱.中国高粱产业工程技术研究的定位思考.中国农学通报,2013,29(11):107-110.Bai W B,Zhang F Y,Jiao X Y,Dong L L,Liu Q S,Ping J A.The fixed position thought of sorghum engineering technology research in china.Chin Agric Sci Bull,2013,29(11):107-110(in Chinese with English abstract).
[4]张福耀,平俊爱.高粱的根本出路在于机械化.农业技术与装备,2012,(20):19-21.Zhang F Y,Ping J A.The fundamental way of sorghum is mechanization.Agric Technol Equip,2012,(20):19-21(in Chinese with English abstract).
[5]焦少杰,王黎明,姜艳喜,严洪冬,苏德峰,孙广全.粒用高粱机械化栽培品种选择.园艺与种苗,2012,(12):1-2.Jiao S J,Wang L M,Jiang Y X,Yan H D,Su D F,Sun G Q.Varieties selection of grain sorghum for mechanized cultivation.Hortic Seed,2012,(12):1-2(in Chinese with English abstract).
[6]Quinby J R,Karper R E.Inheritance of height in sorghum.Agronomy,1954,46:212-216.
[7]Pereira M G,Lee M,Bramel-Cox P,Woodman W,Doebley J,Whitkus R.Construction of an RFLP map in sorghum and comparative mapping in maize.Genome,1994,37:236-243.
[8]Lin Y R,Schertz K F,Paterson A H.Comparative analysis of QTLs affecting plant height and maturity across the Poaceae,in reference to an interspecific sorghum population.Genetics,1995,141:391-411.
[9]Rami J F,Dufour P,Trouch G,Fliedel G,Mestres C,Davrieux F,Blanchard P,Hamon P.Quantitative trait loci for grain quality,productivity,morphological and agronomical traits in sorghum(Sorghum bicolor L.Moench).Theor Appl Genet,1998,97:605-616.
[10]Klein R R,Rodrigyez-Herrera R,Schlueter J A.Identification of genomic regions that affect grain-mould incidence and other traits of agronomic importance in sorghum.Theor Appl Genet,2001,102:307-319.
[11]Upadhyaya H D,Wang Y H,Sharma S,Singh S.Association mapping of height and maturity across five environments using the sorghum mini core collection.Genome,2012,55:471-479.
[12]Upadhyaya H D,Wang Y H,Gowda C L,Sharma S.Association mapping of maturity and plant height using SNP markers with the sorghum mini core collection.Theor Appl Genet,2013,126:2003-2015.
[13]Harris-Shultz K R,Davis R F,Knoll J E,Anderson W,Wang H.Inheritance and identification of a major quantitative trait locus(QTL)that confers resistance to Meloidogyne incognita and a novel QTL for plant height in sweet sorghum.Phytopathology,2015,105:1522-1528.
[14]苏舒.高粱形态学农艺性状的QTL定位研究.南京大学硕士学位论文,江苏南京,2012.Su S.QTL Mapping of Agronomic Traits of Morphology in Sorghum.MS Thesis of Nanjing University,Nanjing,Jiangsu,China,2012(in Chinese with English abstract).
[15]刘娟.高粱株高和抗蚜连锁标记的发掘与验证.河北农业大学硕士学位论文,河北保定,2014.Liu J.Discover and Validation of Markers Linkage with Plant Height and Resistance to Aphid of Sorghum.MS Thesis of Agricultural University of Hebei,Baoding,Hebei,China,2014(in Chinese with English abstract).
[16]Lafarge T A,Broad J,Hammer G L.Tillering in grain sorghum over a wide range of population densities:identification of a common hierarchy for tiller emergence,leaf area development and fertility.Ann Bot,2002,90:87-98.
[17]Feltus F A,Hart G E,Schertz K F,Casa A M,Kresovich S,Abraham S,Klein P E,Brown P J,Paterson A H.Alignment of genetic maps and QTLs between inter-and intra-specific sorghum populations.Theor Appl Genet,2006,112:1295-1305.
[18]Shehzad T,Iwata H,Okuno K.Genome-wide association mapping of quantitative traits in sorghum(Sorghum bicolor(L.)Moench)by using multiple models.Breed Sci,2009,59:217-227.
[19]Shiringani A L,Frisch M,Friedt W.Genetic mapping of QTLs for sugar-related traits in a RIL population of Sorghum bicolor L.Moench.Theor Appl Genet,2010,121:323-336.
[20]董维,苏舒,游录鹏,黄守程,戚金亮,陆桂华,黄应华,杨永华.高粱F6代群体分蘖数的QTL定位.南京林业大学学报(自然科学版),2013,37(2):55-58.Dong W,Su S,You L P,Huang S C,Qi J L,Lu G H,Huang Y H,Yang Y H.QTLs analysis of tillers number in F6 sorghum population.J Nanjing For Univ(Nat Sci Edn),2013,37(2):55-58(in Chinese with English abstract).
[21]Kozich J J,Westcott S L,Baxter N T,Highlander S K,Schloss PD.Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the miseq illumina sequencing platform.Appl Environ Microbiol,2013,79:5112-5120.
[22]International Rice Genome Sequencing Project.The map-based sequence of the rice genome.Nature,2005,436:793-800.
[23]Abe A,Kosugi S,Yoshida K,Natsume S,Takagi H,Kanzaki H,Matsumura H,Yoshida K,Mitsuoka C,Tamiru M,Innan H,Cano L,Kamoun S,Terauchi R.Genome sequencing reveals agronomically important loci in rice using MutMap.Nat Biotechnol,2012,30:174-178.
[24]Hill J T,Demarest B L,Bisgrove B W,Gorsi B,Su Y C,Yost H J.MMAPPR:mutation mapping analysis pipeline for pooled RNA-seq.Genome Res,2013,23:687-697.
[25]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics,2009,25:1754-1760.
[26]Takagi H,Abe A,Yoshida K,Kosugi S,Natsume S,Mitsuoka C,Uemura A,Utsushi H,Tamiru M,Takuno S,Innan H,Cano LM,Kamoun S,Terauchi R.QTL-seq:rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations.Plant J,2013,74:174-183.
[27]贺捷.甜高粱分蘖特性国内研究进展.中国糖料,2017,39(2):65-67.He J.Research progresses on tillering characteristics of sweet sorghum in China.Sugar Crops China,2017,39(2):65-67(in Chinese with English abstract).
[28]詹鹏杰,张福耀,王瑞,于纪珍,李燕.适宜机械化生产酿造高粱汾酒粱1号的选育.安徽农业科学,2016,44(31):13-14.Zhan P J,Zhang F Y,Wang R,Yu J Z,Li Y.Breeding of Fenjiuliang No.1:a brewing sorghum suitable for mechanized production.J Anhui Agric Sci,2016,44(31):13-14(in Chinese with English abstract).
[29]Brown P J,Klein P E,Bortiri E,Acharya C B,Rooney W L,Kresovich S.Inheritance of inflorescence architecture in sorghum.Theor Appl Genet,2006,113:931-942.
[30]Shiringani A L,Frisch M,Friedt W.Genetic mapping of QTLs for sugar-related traits in a RIL population of Sorghum bicolor L.Moench.Theor Appl Genet,2010,121:323-336.
[31]王柏柯,李宁,唐亚萍,王强,杨涛,杨生保,帕提古丽,余庆辉,高杰.基于简化基因组测序技术的番茄雄性不育基因定位.西北农林科技大学学报(自然科学版),2017,45(6):177-184.Wang B K,Li N,Tang Y P,Wang Q,Yang T,Yang S B,Pati G L,Yu Q H,Gao J.Mapping male-sterile gene in tomato by specific length amplified fragment sequencing.J Northwest A&F Univ(Nat Sci Edn).2017,45(6):177-184(in Chinese with English abstract).
[32]王伟,刘凡,任莉,徐理,陈旺,曾令益,黄炳文,方小平.采用SLAF-seq技术开发甘蓝型油菜霜霉病抗性SNP位点.中国油料作物学报,2016,38:555-562.Wang W,Liu F,Ren L,Xu L,Chen W,Zeng L Y,Huang B W,Fang X P.Resistance SNP development to downy mildew in Brassica napus using SLAF-seq technique.Chin J Oil Crop Sci,2016,38:555-562(in Chinese with English abstract).
[33]Geng X,Jiang C,Yang J,Wang L,Wu X,Wei W.Rapid identification of candidate genes for seed weight using the SLAF-Seq method in Brassica napus.PLoS One,2016,11:e0147580.