棉花有益突变体的创制及突变性状的分子遗传学鉴定
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摘要
采用EMS化学诱变、~(14)N重离子注入及~(60)Coγ射线辐照3种诱变方式处理冀棉20、农大156和农抗2号棉花品种,利用四分位法和系谱选择法筛选到植物学性状、产量性状和纤维品质性状突变体并采用SSR标记对突变体进行分子水平鉴定。筛选得到的有益突变体不仅可以丰富棉花种质资源同时也为相应基因的分离和克隆提供了研究材料。主要研究内容与结果如下:
1.采用1.5%甲基磺酸乙酯(EMS)处理农大156种子幼胚获得棕纤维、无棉酚腺体突变体。对M_1代浅棕纤维材料的进一步遗传分析,证实纤维颜色是由1对基因控制(1白色:2浅棕色:1深棕);纤维颜色与纤维品质性状明显负相关,随着纤维颜色加深纤维长度逐渐变短、强力逐渐变低、麦克隆值逐渐变大、伸长率逐渐提高。在基因组SSR标记的DNA水平上检测验证了突变体与野生型之间的遗传差异性。
2.采用250Gy的~(60)Coγ射线辐射农抗2号种子,在M_2代获得了超鸡脚叶、无棉酚腺体突变体。该突变体的叶形基因为L°L°、无腺体基因为glgl。利用经典遗传学方法证实超鸡脚叶对阔叶为不完全显性;突变体的无色素腺体性状是由1对隐性基因控制,并且与叶形基因独立遗传。基因组SSR标记的DNA水平上检测验证了遗传差异性。
3.采用0.5%EMS诱变冀棉20种子。通过四分位法结合系谱选择,在M_4代获得了M_4-66-4和M_4-236-5两个突变体。M_4-66-4的绒长为26.58mm较对照降低了1.99mm、比强度为24.8cN/tex较对照下降了1.90 cN/tex;M_4-236-5的绒长为30.44mm较对照提高了1.87mm、比强度为30.00cN/tex较对照提高了3.30 cN/tex。突变体M_(4:5)家系的绒长和比强度表现稳定。在60对与纤维长度相关和52对与纤维强力相关的SSR引物中BNL1414、BNL1434、BNL2821、BNL1421、BNL4030和JESPR300六对引物能够在突变体与冀棉20之间扩增出明显差异的多态性33条。两个突变体的M_(4:5)家系的SSR多态性条带表现稳定。该结果从分子水平验证了突变体M_4-66-4、M_4-236-5的遗传稳定性及其与冀棉20的遗传差异性。
4.采用0.125%EMS对冀棉20进行合子诱变。在M_3代筛选到突变体合诱M_3-4-2,其籽指为13.12g高出对照3.61g,M_(3:4)家系表现稳定。采用0.5%EMS对冀棉20进行种子诱变,在M_4代选择到突变体M_4-97-2-4,其籽指为6.80 g低于对照4.08g,M_(4:5)家系表现稳定。通过对合诱M_3-4-2、M_4-97-2-4和冀棉20的SSR标记多态性分析,在17对与籽指相关的SSR引物中JESPR114和CIR354两对引物在突变体与冀棉20之间共扩增出4条多态性条带。对突变体的后代家系进行DNA标记多态性检测,4条多态性条带表现稳定。该结果从分子水平验证了突变体合诱M_3-4-2、M_4-97-2-4的遗传稳定性及其与冀棉20的遗传差异性。
5.采用1.5%EMS对农大156进行种子诱变。在M_4代筛选到突变体N-156M_4-101-3,其绒长为30.28mm较对照提高了1.38mm、比强度为31.20cN/tex较对照提高了1.1cN/tex、麦克隆值为4.86较对照提高了1.03。M_(4:5)家系表现稳定。采用80 Gy~(14)N重离子注入农大156。在M_3代筛选到突变体~(14)N-156 M_3-100-4,其绒长为27.42mm较对照下降了1.48mm;比强度为23.70cN/tex较对照下降了6.4cN/tex;麦克隆值为5.13较对照提高了1.30。M_(3:4)家系表现稳定。对~(14)N-156 M_3-100-4、N-156 EMSM_4-101-3与农大156进行SSR多态性差异检测。在60对与纤维长度相关、52对与纤维强力相关、42对与麦克隆值相关的SSR引物中,BNL2821、BNL3280、BNL4030、BNL1513和TMG8五对引物能够在突变体与农大156之间扩增出16条多态性条带。两个突变体的后代家系的SSR多态性与两个突变体的品质性状表型一致,在分子水平上验证了两个突变体纤维长度、强力及细度的遗传稳定性及其与农大156的遗传差异性。
Three varieties of upland cotton(Gossypium hirsutum L.),which are G-20,N-156 and NK-20,were induced by three methods including EMS chemical mutation,~(14)N heavy ions implantation and ~(60)Coγ-rays radiation to select botany character,yield trait and fiber quality trait mutants.The acquirement of these mutants will be valuable not only for enriching the germplasm resources but also to the foundation of mutation material foreground for the location and clone of correlative genes.The main results were as followings:
1.Brown colored fiber mutant named N-156 EMS M1-LBF had been selected by 1.5% Ethyl Methane-sulfonate(EMS) treated with germinating seeds.Brown colored fibre was controlled by one pair alleles through genetical analysis(one white colored:two light-brown colored:one deep brown colored).Fibre color correlated with fiber quality negatively.The fiber length(FL) changed shorter,fiber strength(FS) changed lower,fiber micronaire(FM) changed larger and fiber elongation(FE) changed higher along with fiber color deeping.The genetical diversity between mutant and CK was verified by the result of SSR polymorphic diversity comparison.
2.Super-okra leaf,gossypol glandless mutant was obtained on M_2 generation by 250 Gy ~(60)Coγrays radiation to cotton varety NK-2.It was approved that super-okra leaf was incomplete dominance to normal leaf.Gossypol gland was controlled by one pair of recessive genes and heredity independent with leaf shape genes.The genotype of the mutant is L°L°glgl.The genetical diversity of mutant and NK-2 were checked by SSR polymorphic diversity comparison
3.The seeds of variety G-20 were induced by 0.5%EMS.Two mutants named M_4-66-4 and M_4-236-5 were obtained on M4 generation through pedigree selection.The result of phenotype measurement indicated:FL and FS of M_4-66-4 were 26.85 mm and 24.80 cN/tex which were 1.99mm shorter and 1.90 cN/tex lower than that of G-20,meanwhile FL and FS of M_4-236-5 were 30.44 mm and 30.00 cN/tex which were 1.87mm longer and 3.30 cN/tex higher than that of G-20.The phenotype measurement of M_(4:5) family lines were stable.The result of the SSR polymorphic diversity comparison to G-20 indicated:six SSR primers screened from 60 primer pairs correlated with FL and 52 primer pairs correlated with FS could amplify 33 polymorphic bands among mutants and G-20.The genetical stability and the variance among two mutants and G-20 were verified by the result of SSR polymorphic diversity comparison.
4.Zygotes of variety G-20 were implanted by 0.125%EMS.Seed index(SI) of Zygote induced M_3-4-2,which had been selected on M_2 generation through pedigree selection, was13.12g,3.61g heavier than that of G-20.The phenotype measurement of M_(3:4) family line was stable.The seeds of variety G-20 were induced by 0.5%EMS and the SI of M_4-97-2-4 was 6.80g,4.08g lower than that of G-20.SI of M_(4:5) famliy line was stable.The result of SSR polymorphic diversity comparison to G-20 indicated:two SSR primers screened from 17 primer pairs correlated with SI,which were JESPR114 and CIR354, could amplify polymorphic bands among mutants and G-20.The genetical stability and the variance among two mutants and G-20 were verified by the result of SSR polymorphic diversity comparison.
5.The seeds of variety N-156 were induced by 1.5%EMS.N-156 M_4-101-3 were obtained on M_4 generation through pedigree selection.The result of phenotype measurement indicated:FL,FS and FM of the:mutant were 30.28 mm,31.20 cN/tex and 4.86 which were 1.38 mm longer and 1.10 cN/tex higher and 1,03 thicker than that of N-156.The phenotype measurement of FL,FS and FM of M_(4:5) family line were stable. The seeds of variety N-156 were implanted by ~(14)N heavy ions.~(14)N-156 M_3-100-4 was obtained on M_3 generation through pedigree selection.The result of phenotype measurement indicated:FL,LS and FM of ~(14)N-156 M_3-100-4 were 27.42 mm,23.70 cN/tex and 5.13 which are 1.48mm shorter and 6.40 cN/tex lower and 1.30 thicker than that of N-156.The phenotype of FL,FS and FM of M_(4:5) family line were stable.The genetical stability of FL,FS and FM were verified by the above results.The result of SSR polymorphic diversity comparison to N-156 indicated:five SSR primers screened from 60 primer pairs correlated with FL,52 primer pairs correlated with FS,42 primer pairs correlated with FM,which were BNL2821、BNL3280、BNL4030、BNL1513 and TMG8, could amplify 16 polymorphic bands among mutants and N-156.The result of FL,FS and FM of the two mutants on M_4 generation was verified by the variance statistics and DNA marker analysis in M_(4:5) family line.The genetical stability and the variance among two mutants and N-156 were verified by the result of SSR polymorphic diversity comparison.
1.采用1.5%甲基磺酸乙酯(EMS)处理农大156种子幼胚获得棕纤维、无棉酚腺体突变体。对M_1代浅棕纤维材料的进一步遗传分析,证实纤维颜色是由1对基因控制(1白色:2浅棕色:1深棕);纤维颜色与纤维品质性状明显负相关,随着纤维颜色加深纤维长度逐渐变短、强力逐渐变低、麦克隆值逐渐变大、伸长率逐渐提高。在基因组SSR标记的DNA水平上检测验证了突变体与野生型之间的遗传差异性。
2.采用250Gy的~(60)Coγ射线辐射农抗2号种子,在M_2代获得了超鸡脚叶、无棉酚腺体突变体。该突变体的叶形基因为L°L°、无腺体基因为glgl。利用经典遗传学方法证实超鸡脚叶对阔叶为不完全显性;突变体的无色素腺体性状是由1对隐性基因控制,并且与叶形基因独立遗传。基因组SSR标记的DNA水平上检测验证了遗传差异性。
3.采用0.5%EMS诱变冀棉20种子。通过四分位法结合系谱选择,在M_4代获得了M_4-66-4和M_4-236-5两个突变体。M_4-66-4的绒长为26.58mm较对照降低了1.99mm、比强度为24.8cN/tex较对照下降了1.90 cN/tex;M_4-236-5的绒长为30.44mm较对照提高了1.87mm、比强度为30.00cN/tex较对照提高了3.30 cN/tex。突变体M_(4:5)家系的绒长和比强度表现稳定。在60对与纤维长度相关和52对与纤维强力相关的SSR引物中BNL1414、BNL1434、BNL2821、BNL1421、BNL4030和JESPR300六对引物能够在突变体与冀棉20之间扩增出明显差异的多态性33条。两个突变体的M_(4:5)家系的SSR多态性条带表现稳定。该结果从分子水平验证了突变体M_4-66-4、M_4-236-5的遗传稳定性及其与冀棉20的遗传差异性。
4.采用0.125%EMS对冀棉20进行合子诱变。在M_3代筛选到突变体合诱M_3-4-2,其籽指为13.12g高出对照3.61g,M_(3:4)家系表现稳定。采用0.5%EMS对冀棉20进行种子诱变,在M_4代选择到突变体M_4-97-2-4,其籽指为6.80 g低于对照4.08g,M_(4:5)家系表现稳定。通过对合诱M_3-4-2、M_4-97-2-4和冀棉20的SSR标记多态性分析,在17对与籽指相关的SSR引物中JESPR114和CIR354两对引物在突变体与冀棉20之间共扩增出4条多态性条带。对突变体的后代家系进行DNA标记多态性检测,4条多态性条带表现稳定。该结果从分子水平验证了突变体合诱M_3-4-2、M_4-97-2-4的遗传稳定性及其与冀棉20的遗传差异性。
5.采用1.5%EMS对农大156进行种子诱变。在M_4代筛选到突变体N-156M_4-101-3,其绒长为30.28mm较对照提高了1.38mm、比强度为31.20cN/tex较对照提高了1.1cN/tex、麦克隆值为4.86较对照提高了1.03。M_(4:5)家系表现稳定。采用80 Gy~(14)N重离子注入农大156。在M_3代筛选到突变体~(14)N-156 M_3-100-4,其绒长为27.42mm较对照下降了1.48mm;比强度为23.70cN/tex较对照下降了6.4cN/tex;麦克隆值为5.13较对照提高了1.30。M_(3:4)家系表现稳定。对~(14)N-156 M_3-100-4、N-156 EMSM_4-101-3与农大156进行SSR多态性差异检测。在60对与纤维长度相关、52对与纤维强力相关、42对与麦克隆值相关的SSR引物中,BNL2821、BNL3280、BNL4030、BNL1513和TMG8五对引物能够在突变体与农大156之间扩增出16条多态性条带。两个突变体的后代家系的SSR多态性与两个突变体的品质性状表型一致,在分子水平上验证了两个突变体纤维长度、强力及细度的遗传稳定性及其与农大156的遗传差异性。
Three varieties of upland cotton(Gossypium hirsutum L.),which are G-20,N-156 and NK-20,were induced by three methods including EMS chemical mutation,~(14)N heavy ions implantation and ~(60)Coγ-rays radiation to select botany character,yield trait and fiber quality trait mutants.The acquirement of these mutants will be valuable not only for enriching the germplasm resources but also to the foundation of mutation material foreground for the location and clone of correlative genes.The main results were as followings:
1.Brown colored fiber mutant named N-156 EMS M1-LBF had been selected by 1.5% Ethyl Methane-sulfonate(EMS) treated with germinating seeds.Brown colored fibre was controlled by one pair alleles through genetical analysis(one white colored:two light-brown colored:one deep brown colored).Fibre color correlated with fiber quality negatively.The fiber length(FL) changed shorter,fiber strength(FS) changed lower,fiber micronaire(FM) changed larger and fiber elongation(FE) changed higher along with fiber color deeping.The genetical diversity between mutant and CK was verified by the result of SSR polymorphic diversity comparison.
2.Super-okra leaf,gossypol glandless mutant was obtained on M_2 generation by 250 Gy ~(60)Coγrays radiation to cotton varety NK-2.It was approved that super-okra leaf was incomplete dominance to normal leaf.Gossypol gland was controlled by one pair of recessive genes and heredity independent with leaf shape genes.The genotype of the mutant is L°L°glgl.The genetical diversity of mutant and NK-2 were checked by SSR polymorphic diversity comparison
3.The seeds of variety G-20 were induced by 0.5%EMS.Two mutants named M_4-66-4 and M_4-236-5 were obtained on M4 generation through pedigree selection.The result of phenotype measurement indicated:FL and FS of M_4-66-4 were 26.85 mm and 24.80 cN/tex which were 1.99mm shorter and 1.90 cN/tex lower than that of G-20,meanwhile FL and FS of M_4-236-5 were 30.44 mm and 30.00 cN/tex which were 1.87mm longer and 3.30 cN/tex higher than that of G-20.The phenotype measurement of M_(4:5) family lines were stable.The result of the SSR polymorphic diversity comparison to G-20 indicated:six SSR primers screened from 60 primer pairs correlated with FL and 52 primer pairs correlated with FS could amplify 33 polymorphic bands among mutants and G-20.The genetical stability and the variance among two mutants and G-20 were verified by the result of SSR polymorphic diversity comparison.
4.Zygotes of variety G-20 were implanted by 0.125%EMS.Seed index(SI) of Zygote induced M_3-4-2,which had been selected on M_2 generation through pedigree selection, was13.12g,3.61g heavier than that of G-20.The phenotype measurement of M_(3:4) family line was stable.The seeds of variety G-20 were induced by 0.5%EMS and the SI of M_4-97-2-4 was 6.80g,4.08g lower than that of G-20.SI of M_(4:5) famliy line was stable.The result of SSR polymorphic diversity comparison to G-20 indicated:two SSR primers screened from 17 primer pairs correlated with SI,which were JESPR114 and CIR354, could amplify polymorphic bands among mutants and G-20.The genetical stability and the variance among two mutants and G-20 were verified by the result of SSR polymorphic diversity comparison.
5.The seeds of variety N-156 were induced by 1.5%EMS.N-156 M_4-101-3 were obtained on M_4 generation through pedigree selection.The result of phenotype measurement indicated:FL,FS and FM of the:mutant were 30.28 mm,31.20 cN/tex and 4.86 which were 1.38 mm longer and 1.10 cN/tex higher and 1,03 thicker than that of N-156.The phenotype measurement of FL,FS and FM of M_(4:5) family line were stable. The seeds of variety N-156 were implanted by ~(14)N heavy ions.~(14)N-156 M_3-100-4 was obtained on M_3 generation through pedigree selection.The result of phenotype measurement indicated:FL,LS and FM of ~(14)N-156 M_3-100-4 were 27.42 mm,23.70 cN/tex and 5.13 which are 1.48mm shorter and 6.40 cN/tex lower and 1.30 thicker than that of N-156.The phenotype of FL,FS and FM of M_(4:5) family line were stable.The genetical stability of FL,FS and FM were verified by the above results.The result of SSR polymorphic diversity comparison to N-156 indicated:five SSR primers screened from 60 primer pairs correlated with FL,52 primer pairs correlated with FS,42 primer pairs correlated with FM,which were BNL2821、BNL3280、BNL4030、BNL1513 and TMG8, could amplify 16 polymorphic bands among mutants and N-156.The result of FL,FS and FM of the two mutants on M_4 generation was verified by the variance statistics and DNA marker analysis in M_(4:5) family line.The genetical stability and the variance among two mutants and N-156 were verified by the result of SSR polymorphic diversity comparison.
引文
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