油用向日葵芽苗期抗旱性鉴定及SSR分子标记的研究
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摘要
油葵芽苗期抗旱性强弱,直接关系到出苗率和幼苗的长势,所以油葵芽苗期抗旱性鉴定及遗传改良研究对油葵的农业生产具有非常重要的现实意义。近年来,分子标记技术的发展为加快作物育种进程提供了强有力的武器,但应用于油葵的分子标记研究起步较晚。本研究利用11个油葵育种材料和K55×K58构建的分离群体,对油葵抗旱性鉴定方法、抗旱性遗传和SSR分子标记等进行了研究。主要研究结果如下:
1、利用室内发芽试验和室外盆栽试验对11个抗旱性不同的油葵育种材料的相对发芽指数、萌发抗旱指数和株高等14项抗旱性相关性状进行了测定,并运用灰色关联度理论,以种子萌发抗旱指数与幼苗干旱成活率的乘积作为参考数列X0,分析各指标与芽苗期抗旱性的关系,从中筛选出关联度较大的相对发芽率、相对发芽指数、芽相对生长量、根相对生长量、相对电导率、相对含水量等8个指标。然后通过隶属函数法对抗旱性进行综合分析,定量地评价11个油葵育种材料芽苗期的抗旱性。选出抗旱性差异明显的两个品种K55、K58作为构建作图群体的杂交亲本。
2、F2:3群体及F1各抗旱相关性状的表型分析表明:F2:3群体根生长量和根冠比性状的变异系数较高,表现出较大的变幅;丙二醛、芽生长量和发芽率性状的变异系数较低,表现出较小的变幅。除丙二醛性状外, F2:3群体各性状的平均表现均低于双亲及杂种F1。K55×K58杂交组合的亲本、F1、F2丙二醛生理指标鉴定及遗传分析结果表明:K58为强抗旱性品种,K55为弱抗旱性品种,F1为高抗,F2抗、感分离比例符合3:1,结合亲本在各抗旱性状的表现、遗传力大小及χ2测验结果初步认为模拟干旱胁迫下丙二醛含量是由1~2对主效基因控制的性状,属于简单遗传的质量性状或主效基因控制的数量性状,受环境影响小。
3、在DNA含量、退火温度和循环次数上分别设置几个水平,采用单一因素的方法,建立了一套适合油葵抗旱的SSR体系。其中2×Taq Master Mix 12.5μl终浓度1×,DNA模板1μl终浓度<500ng,上下游引物均为0.5μl,终浓度0.1-1.0μM,剩余用Nuclease-Free Waters补足25μl。利用该体系进行扩增,所得谱带清晰、稳定、非特异性带少。
4、60对油葵SSR特异引物对杂交组合K55×K58的亲本、抗感基因池进行扩增,结果表明:两亲本之间,有7对引物表现明显而稳定的多态性,多态性频率为13.5%;其中引物ORS510、ORS317在抗感基因池间表现稳定的多态性。
5、采用MAPMAKER数据分析软件对(K55×K58)F2单株SSR扩增结果和F2:3群体干旱胁迫下丙二醛含量进行统计分析,表明ORS510、ORS317是与干旱胁迫下丙二醛基因相关的连锁位点,它们与干旱胁迫下丙二醛基因的遗传距离分别为6.8cM、9.7cM。
Drought resistance of sunflower shoots period, strong or weak, is directly related to the emergence and growth of seedlings, so it is a very important practical significance for the agricultural production of sunflower to identify the drought resistance of sunflower shoots period and research on genetic improvement of sunflower. Recently, the development of molecular markers which speeds up the process of crop breeding provides a powerful weapon. However, the application of molecular markers on the sunflower research began rather late, thus it affects the sunflower breeding for drought resistance process. Based on 11 sunflower breeding materials and K55×K58 hybrid combinations, this paper carries on a preliminary study of the identification methods of sunflower drought-resistance, inheritance of resistance, SSR molecular markers and so on. The main findings are as follows:
1. Research on the 11 different drought-resistance of sunflower breeding material through the germination tests indoor and outdoor pot tests to identify 14 indicators related to drought resistance such as determination of the germination index, germination index and plant height. Analysis the relationship of above indicators and drought resistance at seedling stage by use of gray relational grade theory, regard the product of drought index in seed germination and drought seedling survival as Gray Relational Analysis series reference X0, and select eight larger correlative indicators, such as relative germination rate, relative germination index, shoot relative growth rate, root relative growth, relative conductivity, relative water content and so on. Finally, use the membership function to analysis the drought resistance and quantitatively evaluate the drought in the 11 sunflower shoots’period. Select two lager drought separated species K55 and K58 for future experiments.
2. The relevant traits’performance of the F2: 3 population and the F1 show that the F2: 3 groups of root and shoot growth traits have the higher coefficient of variation and a larger amplitude, MDA, bud growth and germination rate have the lower coefficient of variation of traits and a smaller variation, and the average performance of the characters of F2: 3 groups were lower than F1 hybrids and their parents. Through the analysis of K55×K58 hybrid combinations of parents, F1, F2 field identification and genetics, the results show that: K58 has drought resistance, K55 has emotional variety, F1 has the high resistance, F2 has resistant and susceptible separation in line with the 3:1 ratio, therefore the resistance of K55 is controlled by1 ~ 2 pairs of major genes, drought resistance is dominant, F2 has dominant performance.
3. Establish an anti-drought SSR system used the Single-factor method for sunflower by setting on several levels in the DNA contents and annealing temperature. 2×Taq Master Mix 12.5μl final concentration 1×, DNA template 1μl final concentration <500ng, upstream and downstream primer 0.5μl final concentration of 0.1-1.0μM, the Nuclease-Free Waters complements 25μl.Amplified by using the system, bands are clear and stable, less non-specific.
4. Use 60 pairs of SSR primers to amplify genetic parents and anti-sense gene pool of K55×K58 hybrid combinations, the results show that: Between the two parents, seven pairs of primers have clear and stable performance polymorphism, which is scattered throughout the 17 linkage groups, the polymorphism frequency is 13.5%, however, two different pairs of primers exist in anti-influenza gene pool which is related with the sunflower drought-resistance gene.
5. Depend on data analysis software MAPMAKER (K55×K58) to statistically analysis F2 single SSR amplification results , it shows that ORS510, ORS 317 are under drought stress MDA chain gene-related sites, and genetic distance is the 6.8cM, 9.7cM.
1、利用室内发芽试验和室外盆栽试验对11个抗旱性不同的油葵育种材料的相对发芽指数、萌发抗旱指数和株高等14项抗旱性相关性状进行了测定,并运用灰色关联度理论,以种子萌发抗旱指数与幼苗干旱成活率的乘积作为参考数列X0,分析各指标与芽苗期抗旱性的关系,从中筛选出关联度较大的相对发芽率、相对发芽指数、芽相对生长量、根相对生长量、相对电导率、相对含水量等8个指标。然后通过隶属函数法对抗旱性进行综合分析,定量地评价11个油葵育种材料芽苗期的抗旱性。选出抗旱性差异明显的两个品种K55、K58作为构建作图群体的杂交亲本。
2、F2:3群体及F1各抗旱相关性状的表型分析表明:F2:3群体根生长量和根冠比性状的变异系数较高,表现出较大的变幅;丙二醛、芽生长量和发芽率性状的变异系数较低,表现出较小的变幅。除丙二醛性状外, F2:3群体各性状的平均表现均低于双亲及杂种F1。K55×K58杂交组合的亲本、F1、F2丙二醛生理指标鉴定及遗传分析结果表明:K58为强抗旱性品种,K55为弱抗旱性品种,F1为高抗,F2抗、感分离比例符合3:1,结合亲本在各抗旱性状的表现、遗传力大小及χ2测验结果初步认为模拟干旱胁迫下丙二醛含量是由1~2对主效基因控制的性状,属于简单遗传的质量性状或主效基因控制的数量性状,受环境影响小。
3、在DNA含量、退火温度和循环次数上分别设置几个水平,采用单一因素的方法,建立了一套适合油葵抗旱的SSR体系。其中2×Taq Master Mix 12.5μl终浓度1×,DNA模板1μl终浓度<500ng,上下游引物均为0.5μl,终浓度0.1-1.0μM,剩余用Nuclease-Free Waters补足25μl。利用该体系进行扩增,所得谱带清晰、稳定、非特异性带少。
4、60对油葵SSR特异引物对杂交组合K55×K58的亲本、抗感基因池进行扩增,结果表明:两亲本之间,有7对引物表现明显而稳定的多态性,多态性频率为13.5%;其中引物ORS510、ORS317在抗感基因池间表现稳定的多态性。
5、采用MAPMAKER数据分析软件对(K55×K58)F2单株SSR扩增结果和F2:3群体干旱胁迫下丙二醛含量进行统计分析,表明ORS510、ORS317是与干旱胁迫下丙二醛基因相关的连锁位点,它们与干旱胁迫下丙二醛基因的遗传距离分别为6.8cM、9.7cM。
Drought resistance of sunflower shoots period, strong or weak, is directly related to the emergence and growth of seedlings, so it is a very important practical significance for the agricultural production of sunflower to identify the drought resistance of sunflower shoots period and research on genetic improvement of sunflower. Recently, the development of molecular markers which speeds up the process of crop breeding provides a powerful weapon. However, the application of molecular markers on the sunflower research began rather late, thus it affects the sunflower breeding for drought resistance process. Based on 11 sunflower breeding materials and K55×K58 hybrid combinations, this paper carries on a preliminary study of the identification methods of sunflower drought-resistance, inheritance of resistance, SSR molecular markers and so on. The main findings are as follows:
1. Research on the 11 different drought-resistance of sunflower breeding material through the germination tests indoor and outdoor pot tests to identify 14 indicators related to drought resistance such as determination of the germination index, germination index and plant height. Analysis the relationship of above indicators and drought resistance at seedling stage by use of gray relational grade theory, regard the product of drought index in seed germination and drought seedling survival as Gray Relational Analysis series reference X0, and select eight larger correlative indicators, such as relative germination rate, relative germination index, shoot relative growth rate, root relative growth, relative conductivity, relative water content and so on. Finally, use the membership function to analysis the drought resistance and quantitatively evaluate the drought in the 11 sunflower shoots’period. Select two lager drought separated species K55 and K58 for future experiments.
2. The relevant traits’performance of the F2: 3 population and the F1 show that the F2: 3 groups of root and shoot growth traits have the higher coefficient of variation and a larger amplitude, MDA, bud growth and germination rate have the lower coefficient of variation of traits and a smaller variation, and the average performance of the characters of F2: 3 groups were lower than F1 hybrids and their parents. Through the analysis of K55×K58 hybrid combinations of parents, F1, F2 field identification and genetics, the results show that: K58 has drought resistance, K55 has emotional variety, F1 has the high resistance, F2 has resistant and susceptible separation in line with the 3:1 ratio, therefore the resistance of K55 is controlled by1 ~ 2 pairs of major genes, drought resistance is dominant, F2 has dominant performance.
3. Establish an anti-drought SSR system used the Single-factor method for sunflower by setting on several levels in the DNA contents and annealing temperature. 2×Taq Master Mix 12.5μl final concentration 1×, DNA template 1μl final concentration <500ng, upstream and downstream primer 0.5μl final concentration of 0.1-1.0μM, the Nuclease-Free Waters complements 25μl.Amplified by using the system, bands are clear and stable, less non-specific.
4. Use 60 pairs of SSR primers to amplify genetic parents and anti-sense gene pool of K55×K58 hybrid combinations, the results show that: Between the two parents, seven pairs of primers have clear and stable performance polymorphism, which is scattered throughout the 17 linkage groups, the polymorphism frequency is 13.5%, however, two different pairs of primers exist in anti-influenza gene pool which is related with the sunflower drought-resistance gene.
5. Depend on data analysis software MAPMAKER (K55×K58) to statistically analysis F2 single SSR amplification results , it shows that ORS510, ORS 317 are under drought stress MDA chain gene-related sites, and genetic distance is the 6.8cM, 9.7cM.
引文
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