马铃薯红色薯肉调控基因的精细定位与候选基因分析
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摘要
【目的】薯肉颜色是马铃薯重要的农艺性状,它直接影响马铃薯的营养和商品价值,一直是马铃薯遗传研究和育种改良的重要目标。本研究通过对二倍体红色薯肉分离群体的混池分析、基因精细定位和候选基因表达分析,确定调控红色薯肉的候选基因,为下一步基因功能、遗传调控研究及彩色马铃薯的分子育种奠定基础。【方法】本研究通过向二倍体红色薯肉亲本导入自交不亲和抑制基因Sli获得BC1S1群体,从300个单株中挑选18株红色薯肉和21株黄色薯肉个体提取基因组DNA,分别测序进行混池分析。通过集团分离分析法(bulked segregation analysis,BSA)对基因进行初步定位;在定位区间内开发分子标记,对796份BC1S1植株进行基因型分析,筛选交换单株,并结合表型对基因进行精细定位;借助参考基因组注释信息和qRT-PCR表达量分析确定候选基因。【结果】本研究通过构建薯肉颜色分离的二倍体BC1S1群体,利用BSA-seq分析把调控薯肉花青素合成的主效位点定位在第10号染色体48.70—52.20 Mb。最终,利用分子标记将该基因定位于51.47—51.85Mb的377 kb区间内。基于参考基因组注释信息,此区间包括5个基因,其中2个基因注释为MYB类转录因子,结合表达量数据推测这2个基因为候选基因,编号分别为PGSC0003DMG400013966、PGSC0003DMG400013965。【结论】本研究将调控马铃薯薯肉花青素积累的一个主效位点定位于第10号染色体51.47—51.85 Mb之间,推测PGSC0003DMG400013966和PGSC0003DMG400013965为候选基因。
【Objective】 The color of tuber flesh is an important agronomic trait, which directly affects the nutritional and commercial value of potatoes. Accordingly, it is an important goal for potato genetic research and breeding improvement. In this study, we mapped the locus controlling red flesh color by bulked segregant analysis(BSA) and fine mapping, and predicted the candidate genes aided by expression analysis, which provides a foundation for gene function characterization, regulation analysis as well as the molecular breeding of colored potatoes. 【Method】 We crossed a red flesh diploid clone with the Sli gene donor to produce F1, and the F1 was backcrossed with the red flesh clone, and finally generated a BC1 S1 diploid population. From the BC1 S1 segregation population with 300 individuals, the genomic DNA of 18 red-colored flesh and 21 yellow-colored flesh individuals were extracted respectively and used for sequencing, and the locus controlling red flesh was preliminarily mapped by BSA-seq. The locus was fined mapped through genotyping and phenotyping of the recombination plants from 796 BC1 S1 individuals. Based on reference genomic annotation and qRT-PCR expression analysis, candidate genes were predicted. 【Result】 In this study, BC1 S1 segregating population with red and yellow-colored tuber flesh was constructed. The major locus which regulates related anthocyanin synthesis in potato flesh was mapped on chromosome 10 within a physical distance between 48.70 Mb and 52.20 Mb. Finally, molecular markers were used to locate the gene into a 377 kb genomic interval from 51.47 Mb to 51.85 Mb. There are five annotated genes in this genomic region based on reference genome annotation information, two of them were MYB transcription factors. We predicted that the two genes, PGSC0003 DMG400013966 and PGSC0003 DMG400013965, were candidate genes controlling red tuber flesh.【Conclusion】 In this research, the major locus which regulates accumulation of anthocyanin in potato tuber flesh finally was mapped to chromosome 10 with an interval from 51.47 Mb to 51.85 Mb and predicted the candidate genes, PGSC0003 DMG400013966 and PGSC0003 DMG400013965.
【Objective】 The color of tuber flesh is an important agronomic trait, which directly affects the nutritional and commercial value of potatoes. Accordingly, it is an important goal for potato genetic research and breeding improvement. In this study, we mapped the locus controlling red flesh color by bulked segregant analysis(BSA) and fine mapping, and predicted the candidate genes aided by expression analysis, which provides a foundation for gene function characterization, regulation analysis as well as the molecular breeding of colored potatoes. 【Method】 We crossed a red flesh diploid clone with the Sli gene donor to produce F1, and the F1 was backcrossed with the red flesh clone, and finally generated a BC1 S1 diploid population. From the BC1 S1 segregation population with 300 individuals, the genomic DNA of 18 red-colored flesh and 21 yellow-colored flesh individuals were extracted respectively and used for sequencing, and the locus controlling red flesh was preliminarily mapped by BSA-seq. The locus was fined mapped through genotyping and phenotyping of the recombination plants from 796 BC1 S1 individuals. Based on reference genomic annotation and qRT-PCR expression analysis, candidate genes were predicted. 【Result】 In this study, BC1 S1 segregating population with red and yellow-colored tuber flesh was constructed. The major locus which regulates related anthocyanin synthesis in potato flesh was mapped on chromosome 10 within a physical distance between 48.70 Mb and 52.20 Mb. Finally, molecular markers were used to locate the gene into a 377 kb genomic interval from 51.47 Mb to 51.85 Mb. There are five annotated genes in this genomic region based on reference genome annotation information, two of them were MYB transcription factors. We predicted that the two genes, PGSC0003 DMG400013966 and PGSC0003 DMG400013965, were candidate genes controlling red tuber flesh.【Conclusion】 In this research, the major locus which regulates accumulation of anthocyanin in potato tuber flesh finally was mapped to chromosome 10 with an interval from 51.47 Mb to 51.85 Mb and predicted the candidate genes, PGSC0003 DMG400013966 and PGSC0003 DMG400013965.
引文
[1]李颖,李广存,李灿辉,屈冬玉,黄三文.二倍体杂种优势马铃薯育种的展望.中国马铃薯,2013,27(2):96-99.LI Y,LI G C,LI C H,QU D Y,HUANG S W.Prospects of diploid hybrid breeding in potato.Chinese Potato Journal,2013,27(2):96-99.(in Chinese)
[2]LIU Y,TIKUNOV Y,SCHOUTEN R E,MARCELIS L F M,VISSERR G F,BOVY A.Anthocyanin biosynthesis and degradation mechanisms in Solanaceous vegetables:A review.Frontiers in Chemistry,2018,6:52.
[3]LACHMAN J,HAMOUZ K.Red and purple coloured potatoes as a significant antioxidant source in human nutrition-A review.Plant Soil and Environment,2005,51(11):477-482.
[4]ZHANG Y F,CHENG S P,DE JONG D,GRIFFITHS H,HALITSCHKER,DE JONG W.The potato R locus codes for dihydroflavonol4-reductase.Theoretical and Applied Genetics,2009,119(5):931-937.
[5]JUNG C S,GRIFFITHS H M,DE JONG D M,CHENG S P,BODISM,DE JONG W S.The potato P locus codes for flavonoid 3',5'-hydroxylase.Theoretical and Applied Genetics,2005,110(2):269-275.
[6]DE JONG W S,DE JONG D M,DE JONG H,KALAZICH J,BODISM.An allele of dihydroflavonol 4-reductase associated with the ability to produce red anthocyanin pigments in potato(Solanum tuberosum L.).Theoretical and Applied Genetics,2003,107(8):1375-1383.
[7]JIANG P,RAUSHER M.Two genetic changes in cis-regulatory elements caused evolution of petal spot position in Clarkia.Nature Plants,2018,4(1):14-22.
[8]HUANG D,WANG X,TANG Z Z,YUAN Y,XU Y T,HE J X,JIANG X L,PENG S A,LI L,BUTELLI E,DENG X X,XU Q.Subfunctionalization of the Ruby2-Ruby1 gene cluster during the domestication of citrus.Nature Plants,2018,4(11):930-941.
[9]DODDS K S,LONG D H.The inheritance of colour in diploid potatoes.I.Types of anthocyanidins and their genetic loci.Journal of Genetics,1955,53(1):136-149.
[10]DODDS K S,LONG D H.The inheritance of colour in diploid potatoes.II.A three-factor linkage group.Journal of Genetics,1956,54(1):27-41.
[11]JUNG C S,GRIFFITHS H M,DE JONG D M,CHENG S P,BODISM,KIM T S,DE JONG W S.The potato developer(D)locus encodes an R2R3 MYB transcription factor that regulates expression of multiple anthocyanin structural genes in tuber skin.Theoretical and Applied Genetics,2009,120(1):45-57.
[12]DE JONG H.Inheritance of pigmented tuber flesh in cultivated diploid potatoes.American Potato Journal,1987,64(7):337-343.
[13]ZHANG Y F,JUNG C S,DE JONG W S.Genetic analysis of pigmented tuber flesh in potato.Theoretical and Applied Genetics,2009,119(1):143-150.
[14]ZHANG H L,YANG B,LIU J,GUO D L,HOU J,CHEN S C,SONGB T,XIE C H.Analysis of structural genes and key transcription factors related to anthocyanin biosynthesis in potato tubers.Scientia Horticulturae,2017,225:310-316.
[15]刘秀丽,吕红,范小峰.两种茄科植物基因组提取方法的比较.陇东学院学报,2017,28(1):53-56.LIU X L,LüH,FAN X F.A Comparison on the methods for genome extraction on two plant species of solanaceae.Journal of Longdong University,2017,28(1):53-56.(in Chinese)
[16]The Potato Genome Sequencing Consortium.Genome sequence and analysis of the tuber crop potato.Nature,2011,475(7355):189-195.
[17]HUANG W J,KHALDUN A B M,LüH Y,DU L W,ZHANG C J,WANG Y.Isolation and functional characterization of a R2R3-MYBregulator of the anthocyanin biosynthetic pathway from Epimedium sagittatum.Plant Cell Reports,2016,35(4):883-894.
[18]PEREZ-DIAZ J R,PEREZ-DIAZ J,MADRID-ESPINOZA J,GONZALEZ-VILLANUEVA E,MORENO Y,RUIZ-LARA S.New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco.Plant Molecular Biology,2016,90(1/2):63-76.
[19]LIU F,YANG Y J,GAO J W,MA C L,BI Y P.A comparative transcriptome analysis of a wild purple potato and its red mutant provides insight into the mechanism of anthocyanin transformation.PLoS ONE,2018,13(1):e0191406.
[20]LIU Y H,LIN-WANG K,DENG C,WARRAN B,WANG L,YU B,YANG H Y,WANG J,ESPLEY R V,ZHANG J L,WANG D,ALLAN A C.Comparative transcriptome analysis of white and purple potato to identify genes involved in anthocyanin biosynthesis.PLoSONE,2015,10(6):e0129148.
[21]HUANG W J,KHALDUN A B M,CHEN J J,ZHANG C J,LüH Y,YUAN L,WANG Y.A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional Chinese medicinal plant,Epimedium sagittatum.Frontiers in Plant Science,2016,7:1089.
[22]KOES R E,QUATTROCCHIO F,MOL J N M.The flavonoid biosynthetic pathway in plants:function and evolution.Bioessays,1994,16(2):123-132.
[23]HOLTON T A,BRUGLIERA F,LESTER D R,TANAKA Y,HYLAND C D,MENTING J G T,LU C Y,FARCY E,STEVENSONT W.Cloning and expression of cytochrome P450 genes controlling flower colour.Nature,1993,366(6452):276-279.
[24]HOLTON T A,CORNISH E C.Genetics and biochemistry of anthocyanin biosynthesis.Plant Cell,1995,7(7):1071-1083.
[25]BOSS P K,DAVIES C,ROBINSON S P.Expression of anthocyanin biosynthesis pathway genes in red and white grapes.Plant Molecular Biology,1996,32(3):565-569.
[26]LISTER C E,LANCASTER J E,WALKER J R L.Developmental changes in enzymes of flavonoid biosynthesis in the skins of red and green apple cultivars.Journal of the Science of Food&Agriculture,1996,71(3):313-320.
[27]DE JONG H.Inheritance of anthocyanin pigmentation in the cultivated potato:A critical review.American Potato Journal,1991,68(9):585-593.
[28]ENDELMAN J B,JANSKY S H.Genetic mapping with an inbred line-derived F2 population in potato.Theoretical and Applied Genetics,2016,129(5):935-943.
[29]SALAMAN R N.The inheritance of colour and other characters in the potato.Journal of Genetics,1911,5(1):192-193.
[2]LIU Y,TIKUNOV Y,SCHOUTEN R E,MARCELIS L F M,VISSERR G F,BOVY A.Anthocyanin biosynthesis and degradation mechanisms in Solanaceous vegetables:A review.Frontiers in Chemistry,2018,6:52.
[3]LACHMAN J,HAMOUZ K.Red and purple coloured potatoes as a significant antioxidant source in human nutrition-A review.Plant Soil and Environment,2005,51(11):477-482.
[4]ZHANG Y F,CHENG S P,DE JONG D,GRIFFITHS H,HALITSCHKER,DE JONG W.The potato R locus codes for dihydroflavonol4-reductase.Theoretical and Applied Genetics,2009,119(5):931-937.
[5]JUNG C S,GRIFFITHS H M,DE JONG D M,CHENG S P,BODISM,DE JONG W S.The potato P locus codes for flavonoid 3',5'-hydroxylase.Theoretical and Applied Genetics,2005,110(2):269-275.
[6]DE JONG W S,DE JONG D M,DE JONG H,KALAZICH J,BODISM.An allele of dihydroflavonol 4-reductase associated with the ability to produce red anthocyanin pigments in potato(Solanum tuberosum L.).Theoretical and Applied Genetics,2003,107(8):1375-1383.
[7]JIANG P,RAUSHER M.Two genetic changes in cis-regulatory elements caused evolution of petal spot position in Clarkia.Nature Plants,2018,4(1):14-22.
[8]HUANG D,WANG X,TANG Z Z,YUAN Y,XU Y T,HE J X,JIANG X L,PENG S A,LI L,BUTELLI E,DENG X X,XU Q.Subfunctionalization of the Ruby2-Ruby1 gene cluster during the domestication of citrus.Nature Plants,2018,4(11):930-941.
[9]DODDS K S,LONG D H.The inheritance of colour in diploid potatoes.I.Types of anthocyanidins and their genetic loci.Journal of Genetics,1955,53(1):136-149.
[10]DODDS K S,LONG D H.The inheritance of colour in diploid potatoes.II.A three-factor linkage group.Journal of Genetics,1956,54(1):27-41.
[11]JUNG C S,GRIFFITHS H M,DE JONG D M,CHENG S P,BODISM,KIM T S,DE JONG W S.The potato developer(D)locus encodes an R2R3 MYB transcription factor that regulates expression of multiple anthocyanin structural genes in tuber skin.Theoretical and Applied Genetics,2009,120(1):45-57.
[12]DE JONG H.Inheritance of pigmented tuber flesh in cultivated diploid potatoes.American Potato Journal,1987,64(7):337-343.
[13]ZHANG Y F,JUNG C S,DE JONG W S.Genetic analysis of pigmented tuber flesh in potato.Theoretical and Applied Genetics,2009,119(1):143-150.
[14]ZHANG H L,YANG B,LIU J,GUO D L,HOU J,CHEN S C,SONGB T,XIE C H.Analysis of structural genes and key transcription factors related to anthocyanin biosynthesis in potato tubers.Scientia Horticulturae,2017,225:310-316.
[15]刘秀丽,吕红,范小峰.两种茄科植物基因组提取方法的比较.陇东学院学报,2017,28(1):53-56.LIU X L,LüH,FAN X F.A Comparison on the methods for genome extraction on two plant species of solanaceae.Journal of Longdong University,2017,28(1):53-56.(in Chinese)
[16]The Potato Genome Sequencing Consortium.Genome sequence and analysis of the tuber crop potato.Nature,2011,475(7355):189-195.
[17]HUANG W J,KHALDUN A B M,LüH Y,DU L W,ZHANG C J,WANG Y.Isolation and functional characterization of a R2R3-MYBregulator of the anthocyanin biosynthetic pathway from Epimedium sagittatum.Plant Cell Reports,2016,35(4):883-894.
[18]PEREZ-DIAZ J R,PEREZ-DIAZ J,MADRID-ESPINOZA J,GONZALEZ-VILLANUEVA E,MORENO Y,RUIZ-LARA S.New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco.Plant Molecular Biology,2016,90(1/2):63-76.
[19]LIU F,YANG Y J,GAO J W,MA C L,BI Y P.A comparative transcriptome analysis of a wild purple potato and its red mutant provides insight into the mechanism of anthocyanin transformation.PLoS ONE,2018,13(1):e0191406.
[20]LIU Y H,LIN-WANG K,DENG C,WARRAN B,WANG L,YU B,YANG H Y,WANG J,ESPLEY R V,ZHANG J L,WANG D,ALLAN A C.Comparative transcriptome analysis of white and purple potato to identify genes involved in anthocyanin biosynthesis.PLoSONE,2015,10(6):e0129148.
[21]HUANG W J,KHALDUN A B M,CHEN J J,ZHANG C J,LüH Y,YUAN L,WANG Y.A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional Chinese medicinal plant,Epimedium sagittatum.Frontiers in Plant Science,2016,7:1089.
[22]KOES R E,QUATTROCCHIO F,MOL J N M.The flavonoid biosynthetic pathway in plants:function and evolution.Bioessays,1994,16(2):123-132.
[23]HOLTON T A,BRUGLIERA F,LESTER D R,TANAKA Y,HYLAND C D,MENTING J G T,LU C Y,FARCY E,STEVENSONT W.Cloning and expression of cytochrome P450 genes controlling flower colour.Nature,1993,366(6452):276-279.
[24]HOLTON T A,CORNISH E C.Genetics and biochemistry of anthocyanin biosynthesis.Plant Cell,1995,7(7):1071-1083.
[25]BOSS P K,DAVIES C,ROBINSON S P.Expression of anthocyanin biosynthesis pathway genes in red and white grapes.Plant Molecular Biology,1996,32(3):565-569.
[26]LISTER C E,LANCASTER J E,WALKER J R L.Developmental changes in enzymes of flavonoid biosynthesis in the skins of red and green apple cultivars.Journal of the Science of Food&Agriculture,1996,71(3):313-320.
[27]DE JONG H.Inheritance of anthocyanin pigmentation in the cultivated potato:A critical review.American Potato Journal,1991,68(9):585-593.
[28]ENDELMAN J B,JANSKY S H.Genetic mapping with an inbred line-derived F2 population in potato.Theoretical and Applied Genetics,2016,129(5):935-943.
[29]SALAMAN R N.The inheritance of colour and other characters in the potato.Journal of Genetics,1911,5(1):192-193.