甜瓜单性花相关基因CmACS-7的分子标记开发与应用
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摘要
甜瓜(Cucumismelo L.)性型中主要是雄花、两性花同株型(雄全同株,简称两性花),雌雄异花同株型(单性花)比较少见。甜瓜育种工作中为提高制种效率,往往将母本系转育成单性花。传统方法筛选单性花植株耗时长,占地面积大。根据CmACS-7基因序列设计引物进行PCR扩增,甜瓜9份材料均可获得188 bp的片段。多序列比对发现,材料单性花性状与扩增片段的碱基变异相关联,关联位点为限制性内切酶AluⅠ的识别位点。扩增产物进行酶切,甜瓜单性花材料CmACS-7基因的PCR扩增产物均可以被酶切成两条带(160 bp和28 bp),两性花材料CmACS-7基因的PCR扩增产物不能被酶切(188 bp),因此建立CmACS-7基因的CAPS分子标记。利用此标记对58份甜瓜材料进行验证,甜瓜单性花性状与分子标记结果显著相关。基于甜瓜单性花决定基因CmACS-7的CAPS分子标记可用于单性花的选育。
Type of Cucumis melo L.is mainly male,or bisexual flowers with plant type(monoecism,referred to as bisexual flowers),monoecious plant(with unisexual flowers) is relatively rare.In order to improve the seed production efficiency of melon breeding,maternal plants are generally bred to monoecious.Traditional method of monoecious plant screening usually cost long time and cover large area.According to the PCR amplification of primers designed from the sequence of CmACS-7 gene,9 materials of Cucumis melo L.could all get a fragment of 188 bp.Multiple sequence alignment revealed that monoecious character of the material was associated with the base variation of amplified fragment,and the association site was the recognition site of restriction enzyme AluⅠ.The result of enzyme digestion showed that the PCR amplification product of CmACS-7 gene from melon unisexual flower could be digested into two bands(160 bp and 28 bp),while that from bisexual flower could not be digested(188 bp).Therefore,the study developed a CAPS molecular maker of CmACS-7 gene.The maker was applied to verify in 58 melon materials,displaying a significant association between melon monoecious character and molecular marker result.The CAPS makers based on the identity gene CmACS-7 of unisexual flower in melon could be used for the selection and breeding of unisexual flower.
Type of Cucumis melo L.is mainly male,or bisexual flowers with plant type(monoecism,referred to as bisexual flowers),monoecious plant(with unisexual flowers) is relatively rare.In order to improve the seed production efficiency of melon breeding,maternal plants are generally bred to monoecious.Traditional method of monoecious plant screening usually cost long time and cover large area.According to the PCR amplification of primers designed from the sequence of CmACS-7 gene,9 materials of Cucumis melo L.could all get a fragment of 188 bp.Multiple sequence alignment revealed that monoecious character of the material was associated with the base variation of amplified fragment,and the association site was the recognition site of restriction enzyme AluⅠ.The result of enzyme digestion showed that the PCR amplification product of CmACS-7 gene from melon unisexual flower could be digested into two bands(160 bp and 28 bp),while that from bisexual flower could not be digested(188 bp).Therefore,the study developed a CAPS molecular maker of CmACS-7 gene.The maker was applied to verify in 58 melon materials,displaying a significant association between melon monoecious character and molecular marker result.The CAPS makers based on the identity gene CmACS-7 of unisexual flower in melon could be used for the selection and breeding of unisexual flower.
引文
Boualem A.,Fergany M.,Fernandez R.,Troadec C.,Martin A.,Morin H.,Sari M.A.,Collin F.,Flowers J.M.,Pitrat M.,Purugganan M.D.,Dogimont C.,and Bendahmane A.,2008,Aconserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons,Science,321(5890):836-838
Chen X.J.,Liu G.S.,Chen Z.K.,Zhi Y.B.,Yi M.L.,and Liu H.X.,2002,A review of the pathway of ethylene biosynthesis and the relevant genetic engineering,Redai Yaredai Zhiwu Xuebao(Journal of Tropical and Subtropical Botany),10(1):83-98(陈新建,刘国顺,陈占宽,郅玉宝,易明林,刘鸿先,2002,乙烯生物合成途径及其相关基因工程的研究进展,热带亚热带植物学报,10(1):83-98)
Chernys J.T.,Zeevaart J.A.D.,2000,Characterization of the9-cis-epoxycarotenoid dioxygenase gene family and the regu lation of abscisic acid biosynthesis in avocado,Plant Physiol.,124(1):343-353
Gao M.L.,Zhu Z.C.,Gao P.,and Luan F.S.,2011,A microsatellite-based genetic map of melon and localization of gene for gynoecious sex expression using recombinant inbred lines,Yuanyi Xuebao(Acta Horticulturae Sinica),38(7):1308-1316(高美玲,朱子成,高鹏,栾非时,2011,甜瓜重组自交系群体SSR遗传图谱构建及纯雌性基因定位,园艺学报,38(7):1308-1316)
Gong H.,Luo J.N.,Luo S.B.,Zheng X.M.,He X.L.,and Wu H.B.,2014,Progress of study on the molecular mechanism of sex-determination in melon and cucumber,Yuanyi Xuebao(Acta Horticulturae Sinica),41(2):382-388(龚浩,罗剑宁,罗少波,郑晓明,何晓莉,吴海滨,2014,黄瓜与甜瓜的性别决定分子机制研究进展,园艺学报,41(2):382-388)
Han Y.Y.,Ma Y.Q.,Li D.Z.,and Xu Z.Q.,2015,The influences of Arabidopsis Pri-mi R156a on flowering time of Nicotianatabacum,Jiyingzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(2):350-356(韩瑶瑶,马燕勤,李典珍,徐子勤,2015,拟南芥Pri-mi R156a基因对烟草开花时间的影响,基因组学与应用生物学,34(2):350-356)
Ke Y.N.,2007,Studies on inheritance and molecular marker linked to monoecious gene of melon,Thesis for M.S.,Tianjin University,Supervisor:Guo F.C.,pp.19-22(柯妍柠,2007,甜瓜(Cucumis melo L.)单性花性状遗传规律及分子标记的研究,硕士学位论文,天津大学,导师:郭富常,pp.19-22)
Martin A.,Troadec C.,Boualem A.,Rajab M.,Fernandez R.,Morin H.,Pitrat M.,Dogimont C.,and Bendahmane A.,2009,A transposon-induced epigenetic change leads to sex determination in melon,Nature,461(7267):1135-1138
Sheng Y.Y.,2009,Research on inheritance of monoecious and gene map on melon,Thesis for M.S.,Northeast Agricultural University,Supervisor:Luan F.S.,pp.46-53(盛云燕,2009,甜瓜雌雄异花同株遗传分析与基因定位的研究,硕士学位论文,东北农业大学,导师:栾非时,pp.46-53)
Tang D.,2007,Molecular mapping of gene aconferring monoe cious phenotype in melon(Cucumis melo L.),Thesis for M.S.,Shanghai Jiaotong University,Supervisor:Wang Z.M.,pp.23-30(唐棣,2007,控制甜瓜花性型基因“A”的分子标记,硕士学位论文,上海交通大学,导师:王志民,pp.23-30)
Wang H.M.,Chen J.,Shi Y.F.,Pan G.,Shen H.C.,and Wu J.L.,2012,Development and validation of CAPS markers for marker-assisted selection of rice blast resistance gene%Pi25,Zuowu Xuebao(Acta Agronomica Sinica),38(11):1960-1968(王惠梅,陈洁,施勇烽,潘刚,沈海超,吴建利,2012,稻瘟病抗性基因Pi25特异性CAPS标记的开发与验证,作物学报,38(11):1960-1968)
Ye L.H.,2013,CAPS molecular maker development and assisted selection breeding for ipa1 in rice(Oryza sativa L.),Thesis for M.S.,Jiangxi Agricultural University,Supervisor:He X.P.,pp.22-24(叶丽华,2013,基于ipa1基因的CAPS分子标记开发与辅助选择育种,硕士学位论文,江西农业大学,导师:贺晓鹏,pp.22-24)
Yu R.,Zhang X.,Yu Y.,Yang Y.M.,and Yang J.Q.,2006,Studies on selective breeding of monoecious muskmelon and analysis of heredity regular,Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest Sci-Technology University of A-gricultural and Forestry),34(11):160-162(于蓉,张显,于远,杨玉梅,杨建强,2006,厚皮甜瓜单性花系的选育及其遗传分析,西北农林科技大学学报(自然科学版),34(11):160-162)
Chen X.J.,Liu G.S.,Chen Z.K.,Zhi Y.B.,Yi M.L.,and Liu H.X.,2002,A review of the pathway of ethylene biosynthesis and the relevant genetic engineering,Redai Yaredai Zhiwu Xuebao(Journal of Tropical and Subtropical Botany),10(1):83-98(陈新建,刘国顺,陈占宽,郅玉宝,易明林,刘鸿先,2002,乙烯生物合成途径及其相关基因工程的研究进展,热带亚热带植物学报,10(1):83-98)
Chernys J.T.,Zeevaart J.A.D.,2000,Characterization of the9-cis-epoxycarotenoid dioxygenase gene family and the regu lation of abscisic acid biosynthesis in avocado,Plant Physiol.,124(1):343-353
Gao M.L.,Zhu Z.C.,Gao P.,and Luan F.S.,2011,A microsatellite-based genetic map of melon and localization of gene for gynoecious sex expression using recombinant inbred lines,Yuanyi Xuebao(Acta Horticulturae Sinica),38(7):1308-1316(高美玲,朱子成,高鹏,栾非时,2011,甜瓜重组自交系群体SSR遗传图谱构建及纯雌性基因定位,园艺学报,38(7):1308-1316)
Gong H.,Luo J.N.,Luo S.B.,Zheng X.M.,He X.L.,and Wu H.B.,2014,Progress of study on the molecular mechanism of sex-determination in melon and cucumber,Yuanyi Xuebao(Acta Horticulturae Sinica),41(2):382-388(龚浩,罗剑宁,罗少波,郑晓明,何晓莉,吴海滨,2014,黄瓜与甜瓜的性别决定分子机制研究进展,园艺学报,41(2):382-388)
Han Y.Y.,Ma Y.Q.,Li D.Z.,and Xu Z.Q.,2015,The influences of Arabidopsis Pri-mi R156a on flowering time of Nicotianatabacum,Jiyingzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(2):350-356(韩瑶瑶,马燕勤,李典珍,徐子勤,2015,拟南芥Pri-mi R156a基因对烟草开花时间的影响,基因组学与应用生物学,34(2):350-356)
Ke Y.N.,2007,Studies on inheritance and molecular marker linked to monoecious gene of melon,Thesis for M.S.,Tianjin University,Supervisor:Guo F.C.,pp.19-22(柯妍柠,2007,甜瓜(Cucumis melo L.)单性花性状遗传规律及分子标记的研究,硕士学位论文,天津大学,导师:郭富常,pp.19-22)
Martin A.,Troadec C.,Boualem A.,Rajab M.,Fernandez R.,Morin H.,Pitrat M.,Dogimont C.,and Bendahmane A.,2009,A transposon-induced epigenetic change leads to sex determination in melon,Nature,461(7267):1135-1138
Sheng Y.Y.,2009,Research on inheritance of monoecious and gene map on melon,Thesis for M.S.,Northeast Agricultural University,Supervisor:Luan F.S.,pp.46-53(盛云燕,2009,甜瓜雌雄异花同株遗传分析与基因定位的研究,硕士学位论文,东北农业大学,导师:栾非时,pp.46-53)
Tang D.,2007,Molecular mapping of gene aconferring monoe cious phenotype in melon(Cucumis melo L.),Thesis for M.S.,Shanghai Jiaotong University,Supervisor:Wang Z.M.,pp.23-30(唐棣,2007,控制甜瓜花性型基因“A”的分子标记,硕士学位论文,上海交通大学,导师:王志民,pp.23-30)
Wang H.M.,Chen J.,Shi Y.F.,Pan G.,Shen H.C.,and Wu J.L.,2012,Development and validation of CAPS markers for marker-assisted selection of rice blast resistance gene%Pi25,Zuowu Xuebao(Acta Agronomica Sinica),38(11):1960-1968(王惠梅,陈洁,施勇烽,潘刚,沈海超,吴建利,2012,稻瘟病抗性基因Pi25特异性CAPS标记的开发与验证,作物学报,38(11):1960-1968)
Ye L.H.,2013,CAPS molecular maker development and assisted selection breeding for ipa1 in rice(Oryza sativa L.),Thesis for M.S.,Jiangxi Agricultural University,Supervisor:He X.P.,pp.22-24(叶丽华,2013,基于ipa1基因的CAPS分子标记开发与辅助选择育种,硕士学位论文,江西农业大学,导师:贺晓鹏,pp.22-24)
Yu R.,Zhang X.,Yu Y.,Yang Y.M.,and Yang J.Q.,2006,Studies on selective breeding of monoecious muskmelon and analysis of heredity regular,Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest Sci-Technology University of A-gricultural and Forestry),34(11):160-162(于蓉,张显,于远,杨玉梅,杨建强,2006,厚皮甜瓜单性花系的选育及其遗传分析,西北农林科技大学学报(自然科学版),34(11):160-162)