灵宝大枣遗传多样性的表型与ISSR分析
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摘要
灵宝大枣(Ziziphus jujuba‘Lingbaodazao’)果实个大,糖分含量高,营养丰富,是枣(Ziziphus jujuba Mill.)的一个优良地方品种。灵宝大枣有栽培历史悠久,在长期的栽培过程中,由于气候、土壤等自然条件和人类生产活动的影响,其形态特征、生物学特征和品质特征均发生了较大的变异,对灵宝大枣生产的标准化和商品化产生了不利的影响,但同时也为从中选育优良品系奠定了良好的基础。本文采用传统形态学标记方法并结合技术成熟稳定、经济适用且多态性检测水平高的ISSR标记方法,对灵宝大枣群体的遗传多样性、遗传学结构及遗传学关系进行了研究,收集了一些优良单株并进行了嫁接实验,为灵宝大枣新品种选育、种质资源保护提供理论依据。主要结果如下:
1.对70份灵宝大枣材料28个表型性状的变异系数分析结果显示:灵宝大枣14个植株性状的平均变异系数为17.36%,平均方差为9.3267;8个果实外观性状的平均变异系数为25.29%,平均方差为15.1270;6个果实主要内含物性状的平均变异系数为11.39%,平均方差为8.1293。表明在灵宝大枣表型总变异中果实的变异程度最高,植株变异程度居中,而内含物最为稳定,变异系数也最小。
2.表型性状的聚类分析结果表明:供试的70份材料可以分为3个群组,第一群组为大果型,为灵宝大枣的典型代表类型,第二群组为小果型和长果型,在灵宝大枣中种类中比较稀少。第三组群仅有1株,形态特征多方面异于其他单株,可能是灵宝大枣的变异单株或者同名异物单株。对灵宝大枣10个主要性状的相关性分析结果表明:果形主要受到果横径变异的影响最大;单果重和内含物含量的关系比较密切,内含物各性状之间的相关关系不明显。
3.改良后的SDS法提取的灵宝大枣基因组DNA,纯度较好平均OD260/280值为1.77;平均得率为93.7μg/g;完整性好。可以完全满足后期ISSR扩增的需要。
4.建立和适合灵宝大枣的ISSR-PCR反应体系:总体积20μL,其中含模板DNA80ng,引物浓度0.7μmol/L,2×Taq PCR MasterMix 10.0μl,用无菌去离子水补足20μl。扩增程序:94℃预变性5min,94℃变性30s,54℃(视引物而定)复性45s,72℃延伸75s,35个循环;72℃延伸10 min,4℃保存,筛选出多态性较好的12条ISSR引物用于扩增。
5.共选出扩增稳定、多态性较好的引物12个,对70份灵宝大枣基因组DNA进行了扩增,共扩增出105个位点,平均每引物扩增位点8.7个,其中多态位点91个,多态位点百分率86.67%,检查到的等位基因数na=1.8667,有效等位基因数ne= 1.5491,基因多样性h= 0.3183,香农信息指数I= 0.4725,说明70份灵宝大枣材料间存在着较高的遗传变异。
6.对灵宝大枣的群体遗传结构分析表明:灵宝大枣群体的基因多样度Ht为0.3193,群体内基因多样度Hs为0.2828,群体间基因多样度Dst为0.0365,基因分化系数Gst为0.1144,说明仅有11.44%的变异来源于居群之间,而主要的遗传变异来自群体内的个体之间,基因流Nm为3.8717>1,说明居群间的基因流动性较大,居群间的分化比较小。
7.基于ISSR数据的聚类分析同基于表型的聚类分析的结果有所差异,但基本保持一致;主坐标分析更加直观地揭示灵宝大枣各材料之间的亲缘关系;对遗传距离和地理关系的相关系分析表明,遗传距离和地理距离有着弱相关关系。
8.综合表型特征和分子生物学特征,从70株优良单株中挑选出具有代表性优良单株,用高接换头嫁接的方法,在灵宝和新郑进行异地对比试验和遗传稳定性测定,结果有待于经一步观察。
Ziziphus jujuba 'Lingbaodazao' which is a fine local variety with big fruit, high sugar, nutrient-rich, and has a long history cultivated, in the long-term cultivation process, because of climate, soil and other natural conditions and the impact of human activities, their morphological characteristics, biological characteristics and quality features of the natural are significant variation of Lingbao jujuba production standardization and commercialization of adverse effects, but also for the breeding of improved strains from which laid a favorable foundation. In this paper, the traditional morphological markers combined with technology is mature and stable economy with high levels of applicable and polymorphism of ISSR markers, the genetic diversity Lingbao jujuba, the relationship between genetic structure and genetic studies, collecting some fine individual and carrying out grafting experiments, the Lingbao jujuba breeding, germplasm conservation provide a theoretical basis. The main results are as follows:
1. Ziziphus jujuba 'Lingbaodazao' material on 70 of 28 phenotypic analysis showed that the coefficient of variation: 14 Ziziphus jujuba 'Lingbaodazao' plant traits, the average coefficient of variation was 17.36%, the average variance of 9.3267; 8 traits of the average fruit coefficient of variation was 25.29%, the average variance is 15.1270; 6 characters inclusions, the average coefficient of variation was 11.39%, the average variance is 8.1293. Shows that Ziziphus jujuba 'Lingbaodazao' fruit phenotypic variation of the total variance in the highest degree of plant variation center, while the inclusion of the most stable, the coefficient of variation is also minimal.
2. Phenotypic characteristics of cluster analysis showed that: 70 materials were divided into three groups, the first group for large fruit, as a typical representative of the type, and the second group of small fruit type and long-shaped fruit, these are rare species. The third group only one, different from many other plant morphology, might be a variety in the population or homonym. With the 10 main characters Ziziphus jujuba 'Lingbaodazao'correlation analysis showed that: fruit shaped largely by the impact of fruit diameter; fruit weight and content of inclusions close relationship, inclusion relationship between the characters is not obvious.
3. The improved SDS method is the best one with fewer steps, easy to handle and stable. The average OD260/280 1.77; the average yield was 93.7μg / gThe extraction is suitable acting as template DNA for ISSR-PCR amplification.
4. The optimal ISSR-PCR reaction system was determined.Each 20uL reaction system should contain 80ng template DNA , 0.7mmol/L primer , 10.0μL 2×Taq PCR MasterMix.The amplification program as follows:predenature at 94℃for 5min,followed by denature at 94℃for 45s,annealing at 54℃(depend on primer ) for 1 min,extend at 72℃for 90s,35 cycles,and then extend at 72℃for 8min, final store at 4℃for ever.
5. 12 stable polymorphisms primers were selected from 100 ISSR primes, 70 Ziziphus jujuba 'Lingbaodazao' on genomic DNA was amplified to a total out of 105 sites per primer locus 8.7 a, 91 polymorphic sites, the percentage of polymorphic loci was 86.67%, the number of alleles na = 1.8667, the effective number of alleles ne = 1.5491, gene diversity h = 0.3183, Shannon information index I = 0.4725 shows that between 70 Ziziphus jujuba 'Lingbaodazao' material there is a high genetic variation.
6. Population genetic structure analysis shows that: Ziziphus jujuba 'Lingbaodazao' group gene diversity Ht was 0.3193, gene diversity within populations Hs was 0.2828, gene diversity among populations Dst was 0.0365, coefficient of gene differentiation Gst was 0.1144, that indicating only 11.44% of the variation comes from the population between the main genetic variation among individuals within populations and gene flow Nm to 3.8717> 1, indicating that gene flow among populations is large,therefore,the differentiation between populations is relatively small.
7. Based on ISSR data of cluster analysis with cluster analysis based on phenotypic results were inconsistent, but basically indentical; principal coordinate analysis revealed Ziziphus jujuba 'Lingbaodazao' genetic relationship between the various materials for futher; on the genetic distance and the geographical relationship of the correlation analysis showed that genetic distance and geographic distance has a weak correlation.
8. Comprehensive phenotypic and molecular characteristics of an integrated morphological and molecular cluster, from 70 to select 6 fine individuals, with grafting methods for remote testing and species comparison determination of genetic stability, the results need to be observed further.
1.对70份灵宝大枣材料28个表型性状的变异系数分析结果显示:灵宝大枣14个植株性状的平均变异系数为17.36%,平均方差为9.3267;8个果实外观性状的平均变异系数为25.29%,平均方差为15.1270;6个果实主要内含物性状的平均变异系数为11.39%,平均方差为8.1293。表明在灵宝大枣表型总变异中果实的变异程度最高,植株变异程度居中,而内含物最为稳定,变异系数也最小。
2.表型性状的聚类分析结果表明:供试的70份材料可以分为3个群组,第一群组为大果型,为灵宝大枣的典型代表类型,第二群组为小果型和长果型,在灵宝大枣中种类中比较稀少。第三组群仅有1株,形态特征多方面异于其他单株,可能是灵宝大枣的变异单株或者同名异物单株。对灵宝大枣10个主要性状的相关性分析结果表明:果形主要受到果横径变异的影响最大;单果重和内含物含量的关系比较密切,内含物各性状之间的相关关系不明显。
3.改良后的SDS法提取的灵宝大枣基因组DNA,纯度较好平均OD260/280值为1.77;平均得率为93.7μg/g;完整性好。可以完全满足后期ISSR扩增的需要。
4.建立和适合灵宝大枣的ISSR-PCR反应体系:总体积20μL,其中含模板DNA80ng,引物浓度0.7μmol/L,2×Taq PCR MasterMix 10.0μl,用无菌去离子水补足20μl。扩增程序:94℃预变性5min,94℃变性30s,54℃(视引物而定)复性45s,72℃延伸75s,35个循环;72℃延伸10 min,4℃保存,筛选出多态性较好的12条ISSR引物用于扩增。
5.共选出扩增稳定、多态性较好的引物12个,对70份灵宝大枣基因组DNA进行了扩增,共扩增出105个位点,平均每引物扩增位点8.7个,其中多态位点91个,多态位点百分率86.67%,检查到的等位基因数na=1.8667,有效等位基因数ne= 1.5491,基因多样性h= 0.3183,香农信息指数I= 0.4725,说明70份灵宝大枣材料间存在着较高的遗传变异。
6.对灵宝大枣的群体遗传结构分析表明:灵宝大枣群体的基因多样度Ht为0.3193,群体内基因多样度Hs为0.2828,群体间基因多样度Dst为0.0365,基因分化系数Gst为0.1144,说明仅有11.44%的变异来源于居群之间,而主要的遗传变异来自群体内的个体之间,基因流Nm为3.8717>1,说明居群间的基因流动性较大,居群间的分化比较小。
7.基于ISSR数据的聚类分析同基于表型的聚类分析的结果有所差异,但基本保持一致;主坐标分析更加直观地揭示灵宝大枣各材料之间的亲缘关系;对遗传距离和地理关系的相关系分析表明,遗传距离和地理距离有着弱相关关系。
8.综合表型特征和分子生物学特征,从70株优良单株中挑选出具有代表性优良单株,用高接换头嫁接的方法,在灵宝和新郑进行异地对比试验和遗传稳定性测定,结果有待于经一步观察。
Ziziphus jujuba 'Lingbaodazao' which is a fine local variety with big fruit, high sugar, nutrient-rich, and has a long history cultivated, in the long-term cultivation process, because of climate, soil and other natural conditions and the impact of human activities, their morphological characteristics, biological characteristics and quality features of the natural are significant variation of Lingbao jujuba production standardization and commercialization of adverse effects, but also for the breeding of improved strains from which laid a favorable foundation. In this paper, the traditional morphological markers combined with technology is mature and stable economy with high levels of applicable and polymorphism of ISSR markers, the genetic diversity Lingbao jujuba, the relationship between genetic structure and genetic studies, collecting some fine individual and carrying out grafting experiments, the Lingbao jujuba breeding, germplasm conservation provide a theoretical basis. The main results are as follows:
1. Ziziphus jujuba 'Lingbaodazao' material on 70 of 28 phenotypic analysis showed that the coefficient of variation: 14 Ziziphus jujuba 'Lingbaodazao' plant traits, the average coefficient of variation was 17.36%, the average variance of 9.3267; 8 traits of the average fruit coefficient of variation was 25.29%, the average variance is 15.1270; 6 characters inclusions, the average coefficient of variation was 11.39%, the average variance is 8.1293. Shows that Ziziphus jujuba 'Lingbaodazao' fruit phenotypic variation of the total variance in the highest degree of plant variation center, while the inclusion of the most stable, the coefficient of variation is also minimal.
2. Phenotypic characteristics of cluster analysis showed that: 70 materials were divided into three groups, the first group for large fruit, as a typical representative of the type, and the second group of small fruit type and long-shaped fruit, these are rare species. The third group only one, different from many other plant morphology, might be a variety in the population or homonym. With the 10 main characters Ziziphus jujuba 'Lingbaodazao'correlation analysis showed that: fruit shaped largely by the impact of fruit diameter; fruit weight and content of inclusions close relationship, inclusion relationship between the characters is not obvious.
3. The improved SDS method is the best one with fewer steps, easy to handle and stable. The average OD260/280 1.77; the average yield was 93.7μg / gThe extraction is suitable acting as template DNA for ISSR-PCR amplification.
4. The optimal ISSR-PCR reaction system was determined.Each 20uL reaction system should contain 80ng template DNA , 0.7mmol/L primer , 10.0μL 2×Taq PCR MasterMix.The amplification program as follows:predenature at 94℃for 5min,followed by denature at 94℃for 45s,annealing at 54℃(depend on primer ) for 1 min,extend at 72℃for 90s,35 cycles,and then extend at 72℃for 8min, final store at 4℃for ever.
5. 12 stable polymorphisms primers were selected from 100 ISSR primes, 70 Ziziphus jujuba 'Lingbaodazao' on genomic DNA was amplified to a total out of 105 sites per primer locus 8.7 a, 91 polymorphic sites, the percentage of polymorphic loci was 86.67%, the number of alleles na = 1.8667, the effective number of alleles ne = 1.5491, gene diversity h = 0.3183, Shannon information index I = 0.4725 shows that between 70 Ziziphus jujuba 'Lingbaodazao' material there is a high genetic variation.
6. Population genetic structure analysis shows that: Ziziphus jujuba 'Lingbaodazao' group gene diversity Ht was 0.3193, gene diversity within populations Hs was 0.2828, gene diversity among populations Dst was 0.0365, coefficient of gene differentiation Gst was 0.1144, that indicating only 11.44% of the variation comes from the population between the main genetic variation among individuals within populations and gene flow Nm to 3.8717> 1, indicating that gene flow among populations is large,therefore,the differentiation between populations is relatively small.
7. Based on ISSR data of cluster analysis with cluster analysis based on phenotypic results were inconsistent, but basically indentical; principal coordinate analysis revealed Ziziphus jujuba 'Lingbaodazao' genetic relationship between the various materials for futher; on the genetic distance and the geographical relationship of the correlation analysis showed that genetic distance and geographic distance has a weak correlation.
8. Comprehensive phenotypic and molecular characteristics of an integrated morphological and molecular cluster, from 70 to select 6 fine individuals, with grafting methods for remote testing and species comparison determination of genetic stability, the results need to be observed further.
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