谷子矮秆基因的等位性和GA_3敏感性测定
摘要
矮秆基因的利用是小麦、水稻等作物抗倒伏高产育种成功的关键,国内外许多实验室已经对多种作物的矮秆基因开展了系统深入的研究。但对谷子矮秆种质的系统和深入研究至少未有报道,障碍了矮秆基因在谷子育种中的应用。本论文对现有谷子矮秆材料相关基因的等位性、GA敏感性等方面进行了深入研究。
(1)谷子矮秆基因等位性测定。为了解不同的谷子矮秆材料矮秆基因的等位性,按照半双列杂交设计,我们获得了不同矮秆谷子品种间的58个杂交组合。测定结果表明,45个组合含有非等位基因,7个组合含等位基因,6个组合有待进一步确定含有等位基因或亲本中含有显性矮秆基因。另外,在47个被测定的矮秆谷子品种中,34个品种含有隐性矮秆基因,其它品种待定。赤矮9号、1066A和矮丰1号含有等位的隐性矮秆基因;安矮13号与矮88,创19-11与延矮2号,宽九与矮协1号,红谷1与呼早谷1等4个组合都有等位的隐性矮秆基因。
(2)谷子矮秆基因对GA_3敏感性的测定。矮秆突变体可分为对外源GA敏感和不敏感两类,本实验对48份谷子矮秆突变体进行了GA_3敏感性的鉴定。在48份矮秆谷子材料中,苗长对GA_3敏感的有46份,不敏感的有2份。不同品种的不同器官对GA_3的敏感性表现出多样性。如矮丰1号、大青秸和延矮2号的叶长对GA_3不敏感,而中胚轴、胚芽鞘和根对GA_3敏感;矮88的胚芽鞘对GA_3不敏感,而叶长、中胚轴和根对GA_3敏感。这说明谷子矮秆材料受多个矮秆基因控制,或者它们所处的遗传背景差异很大。两个对GA_3不敏感的材料84133纯合和显矮,它们对GA_3处理的不敏感表现在幼苗期的各个器官,同含Rht-D1b和Rht-D1b基因的小麦品种在苗期对GA_3不敏感性相似。用GA_3鉴定矮秆谷子敏感性时,中胚轴为鉴定的首选器官,所用GA_3在30-50ppm之间最适宜。
(3)谷子矮秆材料中高秆个体来源的ISSR标记鉴定。我们对14个矮秆谷子品系中出现的35个高秆单株进行了研究,并用12个在谷子中稳定扩增的ISSR引物对这些高秆单株进行了高秆出现原因的鉴定。NTSYS2.1软件聚类分析表明,这些高秆个体绝大多数为杂交后代,同谷子是严格自交授粉作物是一致的。安矮8号-高-2的F_2群体性状没有分离,且分子鉴定与安矮8号只有一条带的差异,可以初步确定其为安矮8号的回复突变体,但结果有待进一步验证。
(4)谷子GAI基因的克隆。为了解84133对GA不敏感的分子机理,我们用PCR技术分别在昭谷1号及其矮秆突变体“84133纯合”中克隆得到了2072bp的谷子的GAI基因序列,结果昭谷1号与84133纯合的序列完全一致,说明该材料矮秆的机理是该基因其他部分(调控区)或其他基因变异的结果。NCBI-B1AST比对结果表明,谷子GAI基因序列与玉米的dwarf8基因、甘蔗的GAI基因高度同源性。
Utilization of dwarf genes was the key factor to increase lodging resistance and grain yield in wheat and rice,and systematic research on dwarf materials of many crops were carried out worldwide.However,the detailed study of dwarf materials of foxtail millet was rarely reported,which severely restricts the application of dwarf foxtail millet genes in breeding.The main goal of this paper was to identify allelism and gibberellin-sensitivity of foxtail millet materials collected.
(1) Allelism detection of dwarf foxtail millet genes.Using half diallel cross design, crosses were made and 58 ridized combinations were obtained,of which 45 nonallelic combinations,7 allelic combinations,6 allelic combinations or parents had dominant dwarf gene combinations.Among the 47 dwarf foxtail millet varieties determined,33 varieties had recessive dwarf gene,the others were undetermined.Chiai 9,1066A and Aifeng 1 had recessive dwarf gene combinations.Moreover,Chuang 19-11 and Yanai 2,Ai88 and Anai 13,Kuanjiu and Aixie 1,Honggu 1 and Huzaogu 1 had allelic recessive dwarf gene.
(2) Identification of gibberellin-sensitivity of foxtail millet dwarf genes.Dwarf mutants could be divided into gibberellin-insensitive and gibberellin-sensitive,so we identified the gibberellin-sensitivity of 48 foxtail millet dwarf mutants.The result shows: Seedling length of 46 samples were gibberellin-sensitive,the other two 84133chunhe and Xianai were gibberellin-insensitive.Also,gibberellin-sensitivity exhibited diversity in different organs of different varieties.Such as leaf length of Aifengl,Daqingjie and Yanai2 was gibberellin-insensitive,but their mesocotyl coleoptiles and root length were gibberellin-sensitive;coleoptiles of Ai88 was gibberellin-insensitive,but its leaf length, mesocotyl and root length were gibberellin-sensitive.It suggests that different dwarf materials may carry different dwarf genes and they have a different mechanism.All organs of the two gibberellin-insensitive varieties 84133chunhe and Xianai were gibberellin-insensitive in the seedling stage,which was similar to wheat varieties containing Rht-D1 and Rht-D1b genes.Mesocotyl was the critical organ to identify the gibberellin-sensitivity of dwarf foxtail millet,the suitable concentration of GA_3 was 30-50ppm.
(3) Detection of the origin mechanism of high clum individuals in dwarf foxtail millet lines with ISSR technique.Using 12 ISSR primers,genetic variation of 35 high clum individuals from 14 dwarf foxtail millet lines were studied and compared with their original lines.The NTSYS2.1 cluster analysis indicated that most of those individual plants were outcross hybrids,it shows that foxtail millet was not a strictly self pollination species. F_2 generation of Anai8-Gao-2 had not segregation of populations characters,and the molecular identification shows that it had onlyl different band with its original Anai8,so it could be a reverse mutation of Anai8 and which need further experiment to proof.
(4) Clone of Foxtail millet GAI gene.The GAI gene sequence of Zhaogul and 84133chunhe which obtained by the PCR method was identical,and so its dwarf mechanism need more work to mine.According to NCBI-BLAST,the sequence of Foxtail millet GAI gene was highly homology with maize's dwarf8 gene and Saccharum officinarum GAI gene.
(1)谷子矮秆基因等位性测定。为了解不同的谷子矮秆材料矮秆基因的等位性,按照半双列杂交设计,我们获得了不同矮秆谷子品种间的58个杂交组合。测定结果表明,45个组合含有非等位基因,7个组合含等位基因,6个组合有待进一步确定含有等位基因或亲本中含有显性矮秆基因。另外,在47个被测定的矮秆谷子品种中,34个品种含有隐性矮秆基因,其它品种待定。赤矮9号、1066A和矮丰1号含有等位的隐性矮秆基因;安矮13号与矮88,创19-11与延矮2号,宽九与矮协1号,红谷1与呼早谷1等4个组合都有等位的隐性矮秆基因。
(2)谷子矮秆基因对GA_3敏感性的测定。矮秆突变体可分为对外源GA敏感和不敏感两类,本实验对48份谷子矮秆突变体进行了GA_3敏感性的鉴定。在48份矮秆谷子材料中,苗长对GA_3敏感的有46份,不敏感的有2份。不同品种的不同器官对GA_3的敏感性表现出多样性。如矮丰1号、大青秸和延矮2号的叶长对GA_3不敏感,而中胚轴、胚芽鞘和根对GA_3敏感;矮88的胚芽鞘对GA_3不敏感,而叶长、中胚轴和根对GA_3敏感。这说明谷子矮秆材料受多个矮秆基因控制,或者它们所处的遗传背景差异很大。两个对GA_3不敏感的材料84133纯合和显矮,它们对GA_3处理的不敏感表现在幼苗期的各个器官,同含Rht-D1b和Rht-D1b基因的小麦品种在苗期对GA_3不敏感性相似。用GA_3鉴定矮秆谷子敏感性时,中胚轴为鉴定的首选器官,所用GA_3在30-50ppm之间最适宜。
(3)谷子矮秆材料中高秆个体来源的ISSR标记鉴定。我们对14个矮秆谷子品系中出现的35个高秆单株进行了研究,并用12个在谷子中稳定扩增的ISSR引物对这些高秆单株进行了高秆出现原因的鉴定。NTSYS2.1软件聚类分析表明,这些高秆个体绝大多数为杂交后代,同谷子是严格自交授粉作物是一致的。安矮8号-高-2的F_2群体性状没有分离,且分子鉴定与安矮8号只有一条带的差异,可以初步确定其为安矮8号的回复突变体,但结果有待进一步验证。
(4)谷子GAI基因的克隆。为了解84133对GA不敏感的分子机理,我们用PCR技术分别在昭谷1号及其矮秆突变体“84133纯合”中克隆得到了2072bp的谷子的GAI基因序列,结果昭谷1号与84133纯合的序列完全一致,说明该材料矮秆的机理是该基因其他部分(调控区)或其他基因变异的结果。NCBI-B1AST比对结果表明,谷子GAI基因序列与玉米的dwarf8基因、甘蔗的GAI基因高度同源性。
Utilization of dwarf genes was the key factor to increase lodging resistance and grain yield in wheat and rice,and systematic research on dwarf materials of many crops were carried out worldwide.However,the detailed study of dwarf materials of foxtail millet was rarely reported,which severely restricts the application of dwarf foxtail millet genes in breeding.The main goal of this paper was to identify allelism and gibberellin-sensitivity of foxtail millet materials collected.
(1) Allelism detection of dwarf foxtail millet genes.Using half diallel cross design, crosses were made and 58 ridized combinations were obtained,of which 45 nonallelic combinations,7 allelic combinations,6 allelic combinations or parents had dominant dwarf gene combinations.Among the 47 dwarf foxtail millet varieties determined,33 varieties had recessive dwarf gene,the others were undetermined.Chiai 9,1066A and Aifeng 1 had recessive dwarf gene combinations.Moreover,Chuang 19-11 and Yanai 2,Ai88 and Anai 13,Kuanjiu and Aixie 1,Honggu 1 and Huzaogu 1 had allelic recessive dwarf gene.
(2) Identification of gibberellin-sensitivity of foxtail millet dwarf genes.Dwarf mutants could be divided into gibberellin-insensitive and gibberellin-sensitive,so we identified the gibberellin-sensitivity of 48 foxtail millet dwarf mutants.The result shows: Seedling length of 46 samples were gibberellin-sensitive,the other two 84133chunhe and Xianai were gibberellin-insensitive.Also,gibberellin-sensitivity exhibited diversity in different organs of different varieties.Such as leaf length of Aifengl,Daqingjie and Yanai2 was gibberellin-insensitive,but their mesocotyl coleoptiles and root length were gibberellin-sensitive;coleoptiles of Ai88 was gibberellin-insensitive,but its leaf length, mesocotyl and root length were gibberellin-sensitive.It suggests that different dwarf materials may carry different dwarf genes and they have a different mechanism.All organs of the two gibberellin-insensitive varieties 84133chunhe and Xianai were gibberellin-insensitive in the seedling stage,which was similar to wheat varieties containing Rht-D1 and Rht-D1b genes.Mesocotyl was the critical organ to identify the gibberellin-sensitivity of dwarf foxtail millet,the suitable concentration of GA_3 was 30-50ppm.
(3) Detection of the origin mechanism of high clum individuals in dwarf foxtail millet lines with ISSR technique.Using 12 ISSR primers,genetic variation of 35 high clum individuals from 14 dwarf foxtail millet lines were studied and compared with their original lines.The NTSYS2.1 cluster analysis indicated that most of those individual plants were outcross hybrids,it shows that foxtail millet was not a strictly self pollination species. F_2 generation of Anai8-Gao-2 had not segregation of populations characters,and the molecular identification shows that it had onlyl different band with its original Anai8,so it could be a reverse mutation of Anai8 and which need further experiment to proof.
(4) Clone of Foxtail millet GAI gene.The GAI gene sequence of Zhaogul and 84133chunhe which obtained by the PCR method was identical,and so its dwarf mechanism need more work to mine.According to NCBI-BLAST,the sequence of Foxtail millet GAI gene was highly homology with maize's dwarf8 gene and Saccharum officinarum GAI gene.
引文
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