白菜类作物开花时间相关基因BraA.FLM.a的CAPS标记开发与利用
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摘要
[目的]本文旨在研究白菜类作物开花时间候选基因BraA.FLM.a的酶切扩增多态性序列(CAPS)标记开发与利用。[方法]PCR扩增2个亲本的BraA.FLM.a基因外显子2到内含子2区域的特异序列,开发CAPS标记BraA.FLM.a-NlaⅢ,用CAPS标记对F2分离群体的基因型进行检测,并进行Pearson相关性分析研究基因型与开花时间表现型的关系。[结果]通过调查亲本早开花油用型品种‘Yellow sarson’材料YS-143和晚开花芜菁品种‘Vegetable turnip’材料VT-044的开花时间和F2群体的开花时间,发现两亲本的开花时间存在显著性差异。通过PCR扩增克隆了两亲本的BraA.FLM.a基因的特异序列,并在突变位点T-A处开发出CAPS标记BraA.FLM.a-NlaⅢ。在136份F_2分离群体上进行此CAPS标记的验证后,发现T基因型开花时间晚于A基因型的开花时间,并且差异显著(P<0.05)。[结论]BraA.FLM.a基因的CAPS标记BraA.FLM.a-NlaⅢ与开花时间表型显著相关,该标记可以用作开花时间的分子选择辅助育种。
[Objectives]The aim of this study was to explore the development and utilization of cleaved amplified polymorphic sequences( CAPS) marker for flowering time gene BraA. FLM. a in Brassica rapa. [Methods]We cloned the sequence of exon 2 to intron 2 region of the BraA.FLM.a gene from the parents. It was confirmed by sequencing that there was a mutation A-T in LE2+13 in exon 2,and subsequently a CAPS marker for BraA.FLM.a was developed. We investigated the flowering time of the double haploid segregating populations and tested their genotypes using the CAPS marker. The correlation between genotypes and flowering time were studied by Pearson correlation analysis. [Results]By investigating the flowering time of early-flowering oil-type‘Yellow sarson'material YS-143,the late-flowering‘Vegetable turnip'material VT-044 and the late flowering vegetable turnip and the flowering time of the F_2 population,it was found that there was a significant difference in the flowering time between two parents. We cloned the specific sequence of the BraA.FLM.a gene of two parents and developed the CAPS marker BraA.FLM.a-Nla Ⅲ at the mutation site T-A. The validation of this CAPS marker on 136 F_2 isolates revealed that the flowering time of T genotype was later than that of A genotype,and there was significant difference( P<0.05). [Conclusions]This CAPS marker BraA.FLM.a-Nla Ⅲ for the BraA.FLM.a gene was found to be significantly associated with flowering time. Thus,the marker BraA.FLM.a-Nla Ⅲ can be subjected to markerassisted selection of flowering time in breeding.
[Objectives]The aim of this study was to explore the development and utilization of cleaved amplified polymorphic sequences( CAPS) marker for flowering time gene BraA. FLM. a in Brassica rapa. [Methods]We cloned the sequence of exon 2 to intron 2 region of the BraA.FLM.a gene from the parents. It was confirmed by sequencing that there was a mutation A-T in LE2+13 in exon 2,and subsequently a CAPS marker for BraA.FLM.a was developed. We investigated the flowering time of the double haploid segregating populations and tested their genotypes using the CAPS marker. The correlation between genotypes and flowering time were studied by Pearson correlation analysis. [Results]By investigating the flowering time of early-flowering oil-type‘Yellow sarson'material YS-143,the late-flowering‘Vegetable turnip'material VT-044 and the late flowering vegetable turnip and the flowering time of the F_2 population,it was found that there was a significant difference in the flowering time between two parents. We cloned the specific sequence of the BraA.FLM.a gene of two parents and developed the CAPS marker BraA.FLM.a-Nla Ⅲ at the mutation site T-A. The validation of this CAPS marker on 136 F_2 isolates revealed that the flowering time of T genotype was later than that of A genotype,and there was significant difference( P<0.05). [Conclusions]This CAPS marker BraA.FLM.a-Nla Ⅲ for the BraA.FLM.a gene was found to be significantly associated with flowering time. Thus,the marker BraA.FLM.a-Nla Ⅲ can be subjected to markerassisted selection of flowering time in breeding.
引文
[1]原玉香,孙日飞,张晓伟,等.芸薹种作物抽薹相关基因Br FLC1的CAPS标记[J].园艺学报,2008,35(11):1635-1640.Yuan Y X,Sun R F,Zhang X W,et al.A CAPS marker linked to bloting related gene Br FLC1 in Brassica rapa[J].Acta Horticulturae Sinica,2008,35(11):1635-1640(in Chinese with English abstract).
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[3]Wang X W,Wang H,Wang J,et al.The genome of the mesopolyploid crop species Brassica rapa[J].Nature Genetics,2011,43(10):1035-1039.
[4]Cheng F,Sun R F,Hou X L,et al.Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea[J].Nature Genetics,2016,48(10):1218-1224.
[5]侯喜林,宋小明.不结球白菜种质资源的研究与利用[J].南京农业大学学报,2012,35(5):35-42.DOI:10.7685/j.issn.1000-2030.2012.05.005.Hou X L,Song X M.Research and utilization of Brassica campestris ssp.chinensis Makino(non-heading Chinese cabbage)germplasm resources[J].Journal of Nanjing Agricultural University,2012,35(5):35-42(in Chinese with English abstract).
[6]户秋稳,吕炜,张蜀宁,等.优质晚抽薹四倍体不结球白菜的创制及特性[J].南京农业大学学报,2015,38(5):757-763.DOI:10.7685/j.issn.1000-2030.2015.05.009.Hu Q W,LüW,Zhang S N,et al.The induction and characteristics of high quality and late-bolting tetraploid non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2015,38(5):757-763(in Chinese with English abstract).
[7]Srikanth A,Schmid M.Regulation of flowering time:all roads lead to Rome[J].Cellular and Molecular Life Sciences,2011,68(12):2013-2037.
[8]Song J,Irwin J,Dean C.Remembering the prolonged cold of winter[J].Current Biology,2013,23(17):807-811.
[9]刘永平,杨静,杨明峰.植物开花调控途径[J].生物工程学报,2015,31(11):1553-1566.Liu Y P,Yang J,Yang M F.Pathways of flowering regulation in plants[J].Chinese Journal of Biotechnology,2015,31(11):1553-1566(in Chinese with English abstract).
[10]Sheldon C C,Rouse D T,Finnegan E J,et al.The molecular basis of vernalization:the central role of FLOWERING LOCUS C(FLC)[J].Proc Natl Acad Sci USA,2000,97(7):3753-3758.
[11]Schranz M E,Quijada P,Sung S B,et al.Characterization and effects of the replicated flowering time gene FLC in Brassica rapa[J].Genetics,2002,162(3):1457-1468.
[12]Okazaki K,Sakamoto K,Kikuchi R,et al.Mapping and characterization of FLC homologs and QTL analysis of flowering time in Brassica oleracea[J].Theoretical and Applied Genetics,2007,114(4):595-608.
[13]荣子龙,侯喜林,史公军,等.不结球白菜晚抽薹Bc FLC1基因克隆及表达分析[J].南京农业大学学报,2010,33(6):23-27.DOI:10.7685/j.issn.1000-2030.2010.06.005.Rong Z L,Hou X L,Shi G J,et al.Cloning and expression analysis of late bolting Bc FLC1 gene from Brassica campestris ssp.chinensis[J].Journal of Nanjing Agricultural University,2010,33(6):23-27(in Chinese with English abstract).
[14]Tadege M,Sheldon C C,Helliwell C A,et al.Control of flowering time by FLC orthologues in Brassica napus[J].Plant Journal,2001,28(5):545-553.
[15]Yuan Y X,Wu J,Sun R F,et al.A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flowering time[J].Journal of Experimental Botany,2009,60(4):1299-1308.
[16]张学铭,刘博,胡云艳,等.白菜类作物Br FLC5与开花时间相关的d CAPs标记开发[J].园艺学报,2014,41(10):2035-2042.Zhang X M,Liu B,Hu Y Y,et al.Development of d CAPs marker for Br FLC5 related with flowering time in Brassica rapa[J].Acta Horticulturae Sinica,2014,41(10):2035-2042(in Chinese with English abstract).
[17]肖栋,王滔,赵建军,等.深度测序白菜类作物Br FLC5基因揭示春化抑制开花时间位点[J].园艺学报,2015,42(S1):2662.Xiao D,Wang T,Zhao J J,et al.Deep sequencing of Br FLC5 gene in Brassica rapa revealed the site of supressing flowering time in vernalization[J].Acta Horticulturae Sinica,2015,42(S1):2662(in Chinese).
[18]Balasubramanian S,Weigel D.Temperature induced flowering in Arabidopsis thaliana[J].Plant Signal Behav,2006,1(5):227-228.
[19]Werner J D,Borevitz J O,Warthmann N,et al.Quantitative trait locus mapping and DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation[J].Proc Natl Acad Sci USA,2005,102(7):2460-2465.
[20]Scortecci K C,Michaels S D,Amasino R M.Identification of a MADS-box gene,FLOWERING LOCUS M,that represses flowering[J].Plant Journal,2001,26(2):229-236.
[21]stergaard L,King G J.Standardized gene nomenclature for the Brassica genus[J].Plant Methods,2008,4:10.
[22]史学晖,李英慧,于佰双,等.大豆胞囊线虫主效抗病基因Rhg4(Gm SHMT)的CAPS/d CAPS标记开发和利用[J].作物学报,2015,41(10):1463-1471.Shi X H,Li Y H,Yu B S,et al.Development and utilization of CAPS/d CAPS markers based on the SNPs lying in soybean cyst nematode resistant genes Rhg4[J].Acta Agronomica Sinica,2015,41(10):1463-1471(in Chinese with English abstract).
[23]王彩芬,刘冬成,马晓玲,等.水稻耐盐基因SKC1特异性CAPS标记的开发与验证[J].分子植物育种,2015,13(11):2437-2440.Wang C F,Liu D C,Ma X L,et al.Development and validation of the specific CAPS markers of rice salt tolerant gene SKC1[J].Molecular Plant Breeding,2015,13(11):2437-2440(in Chinese with English abstract).
[24]于拴仓,柴敏,郑晓鹰,等.番茄叶霉病抗性基因Cf-5的CAPS标记建立[J].分子植物育种,2005,3(1):57-60.Yu S C,Chai M,Zheng X Y,et al.A CAPS marker for leaf mould resistance gene Cf-5 in tomato[J].Molecular Plant Breeding,2005,3(1):57-60(in Chinese with English abstract).
[25]靳哲,张扬勇,方智远,等.结球甘蓝Bo CBF1与Bo CBF2基因的CAPS标记及其对耐寒性的影响[J].中国蔬菜,2012(14):23-30.Jin Z,Zhang Y Y,Fang Z Y,et al.CAPS markers of Bo CBF1 and Bo CBF2 genes in cabbage and their contribution to cold-tolerance[J].China Vegetables,2012(14):23-30(in Chinese with English abstract).
[26]李昱,罗志鹏,赵淑清.拟南芥开花时间调控的整合途径[J].植物生理学通讯,2007,43(5):799-804.Li Y,Luo Z P,Zhao S Q.Integraiton pathway of floweirng time control in Aarbidopsis[J].Plant Physiology Journal,2007,43(5):799-804(in Chinese with English abstract).
[2]Bonnema G,Carpio D C,Zhao J J.Diversity analysis and molecular taxonomy of Brassica vegetable crops[M]//Sadowski J,Kole C,Enfield N H.Genetics,Genomics and Breeding of Vegetable Brassicas.Valencia,USA:Science Publishers,2011:81-124.
[3]Wang X W,Wang H,Wang J,et al.The genome of the mesopolyploid crop species Brassica rapa[J].Nature Genetics,2011,43(10):1035-1039.
[4]Cheng F,Sun R F,Hou X L,et al.Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea[J].Nature Genetics,2016,48(10):1218-1224.
[5]侯喜林,宋小明.不结球白菜种质资源的研究与利用[J].南京农业大学学报,2012,35(5):35-42.DOI:10.7685/j.issn.1000-2030.2012.05.005.Hou X L,Song X M.Research and utilization of Brassica campestris ssp.chinensis Makino(non-heading Chinese cabbage)germplasm resources[J].Journal of Nanjing Agricultural University,2012,35(5):35-42(in Chinese with English abstract).
[6]户秋稳,吕炜,张蜀宁,等.优质晚抽薹四倍体不结球白菜的创制及特性[J].南京农业大学学报,2015,38(5):757-763.DOI:10.7685/j.issn.1000-2030.2015.05.009.Hu Q W,LüW,Zhang S N,et al.The induction and characteristics of high quality and late-bolting tetraploid non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2015,38(5):757-763(in Chinese with English abstract).
[7]Srikanth A,Schmid M.Regulation of flowering time:all roads lead to Rome[J].Cellular and Molecular Life Sciences,2011,68(12):2013-2037.
[8]Song J,Irwin J,Dean C.Remembering the prolonged cold of winter[J].Current Biology,2013,23(17):807-811.
[9]刘永平,杨静,杨明峰.植物开花调控途径[J].生物工程学报,2015,31(11):1553-1566.Liu Y P,Yang J,Yang M F.Pathways of flowering regulation in plants[J].Chinese Journal of Biotechnology,2015,31(11):1553-1566(in Chinese with English abstract).
[10]Sheldon C C,Rouse D T,Finnegan E J,et al.The molecular basis of vernalization:the central role of FLOWERING LOCUS C(FLC)[J].Proc Natl Acad Sci USA,2000,97(7):3753-3758.
[11]Schranz M E,Quijada P,Sung S B,et al.Characterization and effects of the replicated flowering time gene FLC in Brassica rapa[J].Genetics,2002,162(3):1457-1468.
[12]Okazaki K,Sakamoto K,Kikuchi R,et al.Mapping and characterization of FLC homologs and QTL analysis of flowering time in Brassica oleracea[J].Theoretical and Applied Genetics,2007,114(4):595-608.
[13]荣子龙,侯喜林,史公军,等.不结球白菜晚抽薹Bc FLC1基因克隆及表达分析[J].南京农业大学学报,2010,33(6):23-27.DOI:10.7685/j.issn.1000-2030.2010.06.005.Rong Z L,Hou X L,Shi G J,et al.Cloning and expression analysis of late bolting Bc FLC1 gene from Brassica campestris ssp.chinensis[J].Journal of Nanjing Agricultural University,2010,33(6):23-27(in Chinese with English abstract).
[14]Tadege M,Sheldon C C,Helliwell C A,et al.Control of flowering time by FLC orthologues in Brassica napus[J].Plant Journal,2001,28(5):545-553.
[15]Yuan Y X,Wu J,Sun R F,et al.A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flowering time[J].Journal of Experimental Botany,2009,60(4):1299-1308.
[16]张学铭,刘博,胡云艳,等.白菜类作物Br FLC5与开花时间相关的d CAPs标记开发[J].园艺学报,2014,41(10):2035-2042.Zhang X M,Liu B,Hu Y Y,et al.Development of d CAPs marker for Br FLC5 related with flowering time in Brassica rapa[J].Acta Horticulturae Sinica,2014,41(10):2035-2042(in Chinese with English abstract).
[17]肖栋,王滔,赵建军,等.深度测序白菜类作物Br FLC5基因揭示春化抑制开花时间位点[J].园艺学报,2015,42(S1):2662.Xiao D,Wang T,Zhao J J,et al.Deep sequencing of Br FLC5 gene in Brassica rapa revealed the site of supressing flowering time in vernalization[J].Acta Horticulturae Sinica,2015,42(S1):2662(in Chinese).
[18]Balasubramanian S,Weigel D.Temperature induced flowering in Arabidopsis thaliana[J].Plant Signal Behav,2006,1(5):227-228.
[19]Werner J D,Borevitz J O,Warthmann N,et al.Quantitative trait locus mapping and DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation[J].Proc Natl Acad Sci USA,2005,102(7):2460-2465.
[20]Scortecci K C,Michaels S D,Amasino R M.Identification of a MADS-box gene,FLOWERING LOCUS M,that represses flowering[J].Plant Journal,2001,26(2):229-236.
[21]stergaard L,King G J.Standardized gene nomenclature for the Brassica genus[J].Plant Methods,2008,4:10.
[22]史学晖,李英慧,于佰双,等.大豆胞囊线虫主效抗病基因Rhg4(Gm SHMT)的CAPS/d CAPS标记开发和利用[J].作物学报,2015,41(10):1463-1471.Shi X H,Li Y H,Yu B S,et al.Development and utilization of CAPS/d CAPS markers based on the SNPs lying in soybean cyst nematode resistant genes Rhg4[J].Acta Agronomica Sinica,2015,41(10):1463-1471(in Chinese with English abstract).
[23]王彩芬,刘冬成,马晓玲,等.水稻耐盐基因SKC1特异性CAPS标记的开发与验证[J].分子植物育种,2015,13(11):2437-2440.Wang C F,Liu D C,Ma X L,et al.Development and validation of the specific CAPS markers of rice salt tolerant gene SKC1[J].Molecular Plant Breeding,2015,13(11):2437-2440(in Chinese with English abstract).
[24]于拴仓,柴敏,郑晓鹰,等.番茄叶霉病抗性基因Cf-5的CAPS标记建立[J].分子植物育种,2005,3(1):57-60.Yu S C,Chai M,Zheng X Y,et al.A CAPS marker for leaf mould resistance gene Cf-5 in tomato[J].Molecular Plant Breeding,2005,3(1):57-60(in Chinese with English abstract).
[25]靳哲,张扬勇,方智远,等.结球甘蓝Bo CBF1与Bo CBF2基因的CAPS标记及其对耐寒性的影响[J].中国蔬菜,2012(14):23-30.Jin Z,Zhang Y Y,Fang Z Y,et al.CAPS markers of Bo CBF1 and Bo CBF2 genes in cabbage and their contribution to cold-tolerance[J].China Vegetables,2012(14):23-30(in Chinese with English abstract).
[26]李昱,罗志鹏,赵淑清.拟南芥开花时间调控的整合途径[J].植物生理学通讯,2007,43(5):799-804.Li Y,Luo Z P,Zhao S Q.Integraiton pathway of floweirng time control in Aarbidopsis[J].Plant Physiology Journal,2007,43(5):799-804(in Chinese with English abstract).