小麦多子房性状细胞质和核基因的SSR分析
摘要
小麦是世界上种植最广泛的农作物之一,也是我国北方最重要的粮食作物。杂种优势普遍存在于各种农作物和园艺作物中,小麦与其它农作物一样,也具有明显的杂种优势。因此杂交小麦的研究应用具有重大的经济效益和社会效益。杂交小麦经过几十年的研究,已经取得了巨大的进步,但目前仍未走向大规模生产,其中一个重要制约因素就是小麦的种子繁殖系数低。多子房性状是我国新发现的一种特殊性状:小花内有2-3个雌蕊、3个雄蕊,结实比较稳定,具有明显的多粒性状。多子房性状可有效提高小麦的繁殖系数,在提高小麦杂交种繁育制种效益方面具有巨大前景。本文对具有多子房性状的部分材料的细胞核基因和细胞质基因进行了SSR分子标记和遗传多样性分析,得出以下主要结论:
1.通过正交试验设计和退火温度梯度实验,获得适合多子房性状SSR-PCR的体系和退火温度。体系:10×Buffer2.0μl,Mg~(2+)(25mM)1.5μl,dNTP(2.5mM) 1.6μl,SSR引物(2μmol/L)2.0μl,模板DNA(50ng/μl) 2.0μl,ddH2O10.9μl。合适的引物退火温度应比Tm值低4-6℃。
2.以矮败小麦为杂交工具材料、多子房小麦多II为供体亲本、单子房小麦77(2)为轮回亲本,经过连续回交5代和自交,初步培育成多子房近等基因系。应用SSR分子标记技术对近等基因系中23个材料及其轮回亲本的遗传相似性和遗传距离进行了比较分析,结果表明:(1)42对SSR引物共扩增出92条谱带,其中53条具有多态性,多态性条带率57.61%;30对引物具有多态性,多态性引物率71.43%;(2)23个材料与轮回亲本的遗传相似系数在0.77-0.95,平均遗传相似系数为0.88;(3)聚类分析将23个材料和轮回亲本的相似系数在0.84处分为7大类,其中70.83%的材料归入第一类;第一类在0.87处又分为3个亚类,其中多子房材料2008H69与轮回亲本差异最小,在0.93处就聚为一类。
3.将多子房小麦多Ⅱ与西藏半野生小麦正反杂交,发现F1全部表现多子房,F2出现性状分离,且多子房植株与单子房植株比例经卡平方测验后符合3:1,说明多子房性状是由一对显性等位基因控制。采用混合群体分组分离法(bulked segregation analysis,BSA)将F2单株建立多子房基因池和单子房基因池,用80对引物进行SSR分子标记,结果发现有2对引物没有任何扩增产物,其它78对引物共扩增出202条谱带。其中10对引物具有多态性,共扩增出31条多态性谱带,占总谱带的15.35%。这10对引物均可作为鉴别两亲本材料的分子标记。
4.将多子房小麦与11个不同类型细胞质材料杂交并调查田间性状。结果表明7个山羊草属细胞质小麦的细胞质和一个普通小麦细胞质材料对多子房性状具有明显的细胞质效应,使F1杂合显性多子房基因表达受阻。
5.提取多子房小麦与异源细胞质材料mtDNA并进行SSR分子标记和多态性分析,结果表明:(1)本研究提取mtDNA纯度高、质量好,但浓度较低;说明田间小麦幼苗替代黄化苗可以作为mtDNA提取材料;(2)21对SSR引物共扩增出57条谱带,其中13对多态性引物扩增出43条多态性条带,多态性条带率78.95%,多态性引物率61.90%;找到两个多子房性状标记引物WMt1和WMt8;(3)聚类分析将12个亲本材料在0.71水平分为两类,其中无细胞质效应的山羊草属细胞质小麦02-11-29、02-11-30和多子房小麦多II聚为一类;其他9个存在细胞质效应的亲本聚为第二类;(4)12个亲本材料与其作母本的F1的遗传相似度都很高,均在0.89水平以上。
Wheat is one of the most widely grown crop in the world and it is the most important food crop of northern China. Heterosis exists in crops and horticultural crops generally. Wheat is conferred with observable heterosis as well as other crops. Therefore, research and application of hybrid wheat has great economic and social benefits. The research on wheat heterosis has made great advance after several decades, but it is still difficult to extend hybrid wheat in a larger area because of low propagation coefficient of wheat seed breeding. Multi-ovary character as a new properties of wheat was discovered in our country, has 2-3 pistils and three stamens in its flower. It has the advantage of stable seed-setting and multiple grain leading to the improvement of propagation coefficient. Multi-ovary wheat has a great future in improving the benefit of hybrid wheat seed production. In this study, part of multi-ovary wheat’s nuclear gene and cytoplasmic gene was used to SSR molecular marker and genetic diversity analysis. The following achievements were obtained.
1. A suitable SSR-PCR amplification system for multi-ovary wheat’s gene was established by orthogonal experimental design and gradient PCR. The optimized SSR-PCR reaction system was: 10×Buffer2.0μl, Mg~(2+)(25mM)1.5μl, dNTP(2.5mM) 1.6μl, SSR primers (2μmol/L) 2.0μl, DNA templates(50ng/μl) 2.0μl, ddH2O10.9μl.The appropriate primer annealing temperature should be lower than its Tm value of 4-6℃.
2. In this study, dwarfing sterile wheat was used as instrumental material, duo II was used as donor parent, and 77(2)was used as recurrent male parent. The multi-ovary near-isogenic lines (NIL) was preliminary cultivated after successive backcrossing(5 times) and crossing. Genetic similarity and genetic distance of 23 NIL materials and their recurrent parents were compared and analysised by using SSR. The results showed:(1)All the selected 42 pairs of SSR primers had amplified 92 bands, 53 bands(57.61%) were polymorphic and 30 pairs(71.43%) of primers were polymorphic. (2)The genetic similarity coefficient of 23 materials and their recurrent parents was between 0.77 and 0.95, with an average of 0.88. (3)Cluster analysis revealed 23 NIL materials and their recurrent parents into 7 classes with the similarity coefficient of 0.84. 70.83% of them were clustered into the first class; the first class included 3 subclasses. The multi-ovary material 2008H69 was the least different from the recurrent parent, it was clustered into a small class with the similarity coefficient of 0.93.
3. Multi-ovary wheat duo II was reciprocal crossed with Zangye. All plants F1 were multi-ovary, the ratio of multi-ovary: mono-ovary in F2 was suitable 3:1. The result indicates that on the premise of cytoplasm of common wheat, the multi-ovary character of wheat was controlled by one dominant nuclei-gene. A multi-ovary and a mono-ovary gene pools were established from F2 segregation population by BSA method. 80 primers were used to SSR and the result showed that there wasn’t any amplification products of two pairs of primers. Other 78 pairs of primers had amplified 202 bands, 31(15.35%) were polymorphic and 10 pairs of primers were polymorphic. All of 10 pairs of primers can be used as the molecular marker between the two parents.
4. Multi-ovary wheat duo II was reciprocal crossed with 11 kinds of wheat with different types of cytoplasm, the result showes: 7 kinds of alloplasmic wheat and a material with common wheat cytoplasm have cytoplasm effects, make the multi-ovary gene expression obstruct in F1 heterozygous dominant gene type.
5. mtDNA of multi-ovary wheat and alloplasmic wheat was extracted and used to SSR and polymorphism analysis. The result shows: (1) mtDNA extracted by our method has the character of high purity, good quality, but low concentration. So we can use wheat leaves of the field in stead of etiolated tissues. (2) All the selected 21 pairs of SSR primers had amplified 57 bands, 43 bands (78.95%) were polymorphic and 13 pairs of primers (61.90%) were polymorphic. Two primers WMt1 and WMt8 which can marker the multi-ovary character were fround. (3) Cluster analysis revealed 12 kinds of wheat into 2 classes with the similarity coefficient of 0.71. Two alloplasmic wheat with Ae.gilops cytoplasm which had no cytoplasm effects 02-11-29、20-11-30 clustered together with multi-ovary wheat duo II, the other nine kinds of wheat existing cytoplasm effects in the second category. (4) F1 population have high genetic similarity with their donor parents, both at 0.89 level or above.
1.通过正交试验设计和退火温度梯度实验,获得适合多子房性状SSR-PCR的体系和退火温度。体系:10×Buffer2.0μl,Mg~(2+)(25mM)1.5μl,dNTP(2.5mM) 1.6μl,SSR引物(2μmol/L)2.0μl,模板DNA(50ng/μl) 2.0μl,ddH2O10.9μl。合适的引物退火温度应比Tm值低4-6℃。
2.以矮败小麦为杂交工具材料、多子房小麦多II为供体亲本、单子房小麦77(2)为轮回亲本,经过连续回交5代和自交,初步培育成多子房近等基因系。应用SSR分子标记技术对近等基因系中23个材料及其轮回亲本的遗传相似性和遗传距离进行了比较分析,结果表明:(1)42对SSR引物共扩增出92条谱带,其中53条具有多态性,多态性条带率57.61%;30对引物具有多态性,多态性引物率71.43%;(2)23个材料与轮回亲本的遗传相似系数在0.77-0.95,平均遗传相似系数为0.88;(3)聚类分析将23个材料和轮回亲本的相似系数在0.84处分为7大类,其中70.83%的材料归入第一类;第一类在0.87处又分为3个亚类,其中多子房材料2008H69与轮回亲本差异最小,在0.93处就聚为一类。
3.将多子房小麦多Ⅱ与西藏半野生小麦正反杂交,发现F1全部表现多子房,F2出现性状分离,且多子房植株与单子房植株比例经卡平方测验后符合3:1,说明多子房性状是由一对显性等位基因控制。采用混合群体分组分离法(bulked segregation analysis,BSA)将F2单株建立多子房基因池和单子房基因池,用80对引物进行SSR分子标记,结果发现有2对引物没有任何扩增产物,其它78对引物共扩增出202条谱带。其中10对引物具有多态性,共扩增出31条多态性谱带,占总谱带的15.35%。这10对引物均可作为鉴别两亲本材料的分子标记。
4.将多子房小麦与11个不同类型细胞质材料杂交并调查田间性状。结果表明7个山羊草属细胞质小麦的细胞质和一个普通小麦细胞质材料对多子房性状具有明显的细胞质效应,使F1杂合显性多子房基因表达受阻。
5.提取多子房小麦与异源细胞质材料mtDNA并进行SSR分子标记和多态性分析,结果表明:(1)本研究提取mtDNA纯度高、质量好,但浓度较低;说明田间小麦幼苗替代黄化苗可以作为mtDNA提取材料;(2)21对SSR引物共扩增出57条谱带,其中13对多态性引物扩增出43条多态性条带,多态性条带率78.95%,多态性引物率61.90%;找到两个多子房性状标记引物WMt1和WMt8;(3)聚类分析将12个亲本材料在0.71水平分为两类,其中无细胞质效应的山羊草属细胞质小麦02-11-29、02-11-30和多子房小麦多II聚为一类;其他9个存在细胞质效应的亲本聚为第二类;(4)12个亲本材料与其作母本的F1的遗传相似度都很高,均在0.89水平以上。
Wheat is one of the most widely grown crop in the world and it is the most important food crop of northern China. Heterosis exists in crops and horticultural crops generally. Wheat is conferred with observable heterosis as well as other crops. Therefore, research and application of hybrid wheat has great economic and social benefits. The research on wheat heterosis has made great advance after several decades, but it is still difficult to extend hybrid wheat in a larger area because of low propagation coefficient of wheat seed breeding. Multi-ovary character as a new properties of wheat was discovered in our country, has 2-3 pistils and three stamens in its flower. It has the advantage of stable seed-setting and multiple grain leading to the improvement of propagation coefficient. Multi-ovary wheat has a great future in improving the benefit of hybrid wheat seed production. In this study, part of multi-ovary wheat’s nuclear gene and cytoplasmic gene was used to SSR molecular marker and genetic diversity analysis. The following achievements were obtained.
1. A suitable SSR-PCR amplification system for multi-ovary wheat’s gene was established by orthogonal experimental design and gradient PCR. The optimized SSR-PCR reaction system was: 10×Buffer2.0μl, Mg~(2+)(25mM)1.5μl, dNTP(2.5mM) 1.6μl, SSR primers (2μmol/L) 2.0μl, DNA templates(50ng/μl) 2.0μl, ddH2O10.9μl.The appropriate primer annealing temperature should be lower than its Tm value of 4-6℃.
2. In this study, dwarfing sterile wheat was used as instrumental material, duo II was used as donor parent, and 77(2)was used as recurrent male parent. The multi-ovary near-isogenic lines (NIL) was preliminary cultivated after successive backcrossing(5 times) and crossing. Genetic similarity and genetic distance of 23 NIL materials and their recurrent parents were compared and analysised by using SSR. The results showed:(1)All the selected 42 pairs of SSR primers had amplified 92 bands, 53 bands(57.61%) were polymorphic and 30 pairs(71.43%) of primers were polymorphic. (2)The genetic similarity coefficient of 23 materials and their recurrent parents was between 0.77 and 0.95, with an average of 0.88. (3)Cluster analysis revealed 23 NIL materials and their recurrent parents into 7 classes with the similarity coefficient of 0.84. 70.83% of them were clustered into the first class; the first class included 3 subclasses. The multi-ovary material 2008H69 was the least different from the recurrent parent, it was clustered into a small class with the similarity coefficient of 0.93.
3. Multi-ovary wheat duo II was reciprocal crossed with Zangye. All plants F1 were multi-ovary, the ratio of multi-ovary: mono-ovary in F2 was suitable 3:1. The result indicates that on the premise of cytoplasm of common wheat, the multi-ovary character of wheat was controlled by one dominant nuclei-gene. A multi-ovary and a mono-ovary gene pools were established from F2 segregation population by BSA method. 80 primers were used to SSR and the result showed that there wasn’t any amplification products of two pairs of primers. Other 78 pairs of primers had amplified 202 bands, 31(15.35%) were polymorphic and 10 pairs of primers were polymorphic. All of 10 pairs of primers can be used as the molecular marker between the two parents.
4. Multi-ovary wheat duo II was reciprocal crossed with 11 kinds of wheat with different types of cytoplasm, the result showes: 7 kinds of alloplasmic wheat and a material with common wheat cytoplasm have cytoplasm effects, make the multi-ovary gene expression obstruct in F1 heterozygous dominant gene type.
5. mtDNA of multi-ovary wheat and alloplasmic wheat was extracted and used to SSR and polymorphism analysis. The result shows: (1) mtDNA extracted by our method has the character of high purity, good quality, but low concentration. So we can use wheat leaves of the field in stead of etiolated tissues. (2) All the selected 21 pairs of SSR primers had amplified 57 bands, 43 bands (78.95%) were polymorphic and 13 pairs of primers (61.90%) were polymorphic. Two primers WMt1 and WMt8 which can marker the multi-ovary character were fround. (3) Cluster analysis revealed 12 kinds of wheat into 2 classes with the similarity coefficient of 0.71. Two alloplasmic wheat with Ae.gilops cytoplasm which had no cytoplasm effects 02-11-29、20-11-30 clustered together with multi-ovary wheat duo II, the other nine kinds of wheat existing cytoplasm effects in the second category. (4) F1 population have high genetic similarity with their donor parents, both at 0.89 level or above.
引文
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Tsunewaki K.1980.Genetic diversity of the cytoplasm in Triticum and Aegilous.Japan:Japan Sociery fothe Promotion of Science.49-209
Weining,S.and Langridge,P.1991. Identification and mapping of polymorphisms in cereales based on the polymerase chain reaction.Teor.Appl.Gent. (82)209-216.
Welsh J,Mcclelland M.1990.Finger printing genomes using PCR with arbitary priers.Nucleic Acids Research,18:7213-7218
Williams JGK,Kubelik AR,Livak KJ. 1990.DNA Polymorphisms amplified by arbitrary Primers are useful as genetic markers. Nucleic Acids Research,18(22):6531-6535
Wilson J.A and W.M. Ross.1962.Cross breeding in Wheat(Triticum aestivum L.)Ⅱ.Hybrid seed set on a cytoplasmic male-sterile winterwheat composite subjected to cross- pollination.Crop SCience,2:415-417.
Zabeau M and Vos R .1992.Selective restriction fragment amplification:a general method for DNA finger printing. European Application 92402629.7(Publication No.0534858AI).EuroPean Patent Office,Paris.
Zheng-Song Peng , Petr Martinek , Kazumasa Kosuge , Tsutomu Kuboyama , Nobuyoshi Watanabe.2008.Genetic mapping of a mutant gene producing three pistils per floret in common wheat.J Appl Genet.49(2):135-139
Zietkiewicz E , Rafalski A , Labuda D. 1994.Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification. Genomics,20:176-183