野生狗牙根分子遗传多样性及优异种质筛选
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摘要
狗牙根(Cynodon dactylon L.Pers.),属于禾本科(Gramineae)狗牙根属(Cynodon)。广泛分布于东西半球热带、亚热带甚至温带地区。在欧亚大陆、印尼、马来西亚和印度等地均有分布。在我国,狗牙根主要分布于黄河流域及其以南地区,此外新疆、吉林、青海、甘肃、河北等地均有分布。狗牙根植株低矮,繁殖能力强、抗旱、耐践踏、质地纤细、色泽好,是优良的暖季性草坪草,被广泛地用于运动场草坪、观赏草坪、机场草坪、水土保持、护坡护堤等诸多领域。试验以栽培品种为对照,从DNA分子标记等方面进行系统的遗传多样性研究和优异种质筛选。主要结果如下：
1.利用SRAP分子标记技术,对采自中国西南五省区的44份野生普通狗牙根(C. dactylon)及8份非洲狗牙根(C. transvalensis)材料进行遗传多样性研究。结果表明：18对引物组合共得到扩增总条带236条,多态性条带数206条,平均每对引物扩增出11.4条带,多态性位点百分率为87.29%,材料间的遗传相似系数范围在0.569到0.929之间,平均GS值为0.723。聚类分析结果表明,52份材料可聚为5类；基于Shannon多样性指数估算了8个狗牙根生态地理类群内和类群间的遗传分化,类群内的遗传变异占总变异的63.81%,类群间的遗传变异占总变异的36.19%,表明这些种源材料遗传差异较大,各生态地理类群间的遗传分化与其所处的生态地理环境具有一定的相关性。
2.以采自中国西南五省区的55份野生普通狗牙根(C. dactylon)和8份非洲狗牙根(C transvalensis)材料,利用SSR标记技术进行群体遗传结构的研究。结果表明,在6个地理类群中：(1)18对引物组合共获得353条清晰可辨的总条带,多态性条带数267条,平均每对引物扩增出19.61条带,多态性位点百分率为75.10%,材料间的遗传相似系数(GS)范围在0.687到0.904之间,平均GS值为0.788,变幅为0.217,说明供试材料具有比较丰富的遗传多样性。(2) UPGMA聚类分析结果表明,GS=0.81时,63份材料可聚为7类,大部分来自相同或相似生态地理环境的材料聚为一类,表明供试材料的聚类和其生态地理环境间有一定的相关性。(3)基于Shannon多样性指数估算了7个狗牙根生态地理类群内和类群间的遗传分化,类群内的遗传变异占总变异的66.57%,类群间的遗传变异占总变异的33.43%。(4)对各生态地理类群基于Nei氏无偏估计的遗传一致度的聚类分析表明,各生态地理类群间的遗传分化与其所处的生态地理环境具有一定的相关性。上述结果为保护和利用野生狗牙根资源提供了科学依据。
3.利用AFLP标记对采自中国西南五省区的51份野生普通狗牙根(C. dactylon)和8份非洲狗牙根(C. transvalensis)为材料进行遗传多样性分析。获得结果如下：11对引物共扩增出670个条带,其中多态性条带有663条,多态性条带比率为98.74%,材料间的遗传相似系数(GS)范围在0.64～0.96之间,平均GS值为0.78；基于Shannon多样性指数估算了7个狗牙根生态地理类群内和类群间的遗传分化,类群内的遗传变异占总变异的51.68%,类群间的遗传变异占总变异的48.32%；聚类分析和主成分分析显示,可将供试材料分成5大类,分类结果与材料的地理分布大致相符,呈现出一定的地域性分布规律。由此可见,丰富的地理生态条件造就了狗牙根资源丰富的遗传多样性和明显的地域性分布规律。对制定科学的资源保护策略、促进其开发利用以及新品种的选育具有重要的意义。
4.本研究采用了SRAP、SSR、AFLP三种分子标记分析了中国西南区野生普通狗牙根及采自非洲的野生狗牙根种质的遗传多样性及地理分化。三种分子标记都揭示了供试种质间具有丰富的遗传多样性并存在一定的地理分化。相关性结果表明了三种标记之间具有显著的相关性,但相关系数存在差异,其中SRAP和SSR的相关性较高。
5.在遗传多样性研究基础上,对初步筛选表现较好的五个野生狗牙根品系以“南京”和‘Tifway"作为对照品种,分别在雅安、眉山、成都、重庆和黔南五个试验点进行了周期3年的区域性试验研究,观测指标有草坪密度、质地、均一性、色泽、成坪密度以及抗性等。经试验观察以及Excel 2007和SPSS12.0软件对试验数据进行的分析表明,材料Sau9926的绿色期长、成坪速度较快、密度较高、抗逆性强,具有良好的坪用特性。在五个试验点的平均绿色期天数为295d,成坪天数为45d左右,密度为200枝/100cm2左右。从五份狗牙根新品系及两份对照品种的质地、色泽、密度、抗逆性、成坪速度和绿色期等方面综合分析,Sau9926的综合表现最佳,优于两个对照品种。
Cynodon dactylon L.Pers.belongs to Cynodon L.C.Rich.,Gramineae.It distributed in tropical, subtropical and even temperate regions of eastern and western hemispheres widely, and it also distributed in Eurasia, Indonesia, Malaysia and India and other places. In China, C.dactylon mainly distributed in the south of the Yellow River, and also distributed in Xinjiang, Jilin, Qinghai, Gansu, Hebei and other places. C. dactylon own low plants, strong breeding ability, high drought resistance, forbear trampling, delicate texture, good color, it is an excellent warm-season turf grass,which is widely used as playground lawn, ornamental turf, airport lawn, soil and water conservation, slope protection berm and other areas. The cultivars were selected as control varieties in the experiment.The systematic study of the genetic diversity and excellent germplasm filtration were from the DNA molecular markers and field test. The main results were as follows:
1. Sequence-related amplified polymorphism (SRAP) markers were used to detect the genetic diversity of 44 wild accessions of C.dactylon collected from Sichuan, Chongqing,Yunnan, Guizhou and Tibet in China and 8 wild accessions of C.dactylon collected from Africa.The results showed that Eighteen primer pairs produced 236 polymorphic bands, among which 206 bands were found to be polymorphic.averaged 11.4 bands per primer pair. The percentage of polymorphic bands in average was 87.29%.The Nei's genetic similarity coefficient of the tested accessions ranged from 0.569 to 0.929, and the average Nei's coefficient was0.723.The 52 accessions were classified into five major groups:Ⅰ,Ⅱ,Ⅲ,ⅣandⅤby cluster analysis using UPGMA. Genetic differentiation between and within five eco-geographical groups of C. dactylon was estimated by Shannon's diversity index, which showed that 63.81% genetic variance existed within group, and 36.19% genetic variance was among groups. which showed that these materials have great differences in heredity, and own significant relationship with the origin regions of accessions.
2. The population's genetic structure of 63 agrestal accessions of C.dactylon collected from Sichuan, Chongqing, Guizhou, Yunnan, Tibet and Africa were analyzed using simple sequence repeat (SSR) molecular markers. The results showed that, In the six populations: (1) Eighteen primer pairs produced 353 polymorphic bands, among which 267 bands were found to be polymorphic.averaged 19.61 bands per primer pair. The percentage of polymorphic bands in average was 75.10%.The Nei's genetic similarity coefficient of the tested accessions ranged from 0.687 to 0.904, and the average Nei's coefficient was 0.788,the range was 0.217. It showed that the test materials have ample genetic diversity. (2) Analysis of cluster showed that all the accessions could be divided into 5 groups when the genetic similarity coefficient is 0.81. accessions from the same area were almost classified into the same group associated with their geographical distributions. (3) Genetic differentiation between and within seven eco-geographical groups of C. dactylon was estimated by Shannon's diversity index, which showed that 66.57% genetic variance existed within group, and33.43% genetic variance was among groups. (4) Analysis of cluster in every eco-geographical groups by Shannon's diversity index showed that they own significant relationship with the origin regions of accessions. The results provided a scientific basis for the conservation and use of the wild resources of Cynodon dactylon.
3. Amplified fragment length polymorphism (AFLP) molecular markers were applied to detect the genetic variation of 59 agrestal accessions of C.dactylon collected from Africa, Sichuan, Chongqing, Yunnan, Guizhou and Tibet in China. The following results were obtained,A total of 670 bands were amplified by eleven AFLP primer pairs from C.dactylon genetic DNA, among which 663 (98.74%) bands were found to be polymorphic.The AFLP-based genetic similarity values among 59 C.dactylon accessions ranged from 0.64 to 0.96,and the average Nei's coefficient was 0.78. Genetic differentiation between and within seven eco-geographical groups of C. dactylon was estimated by Shannon's diversity index, which showed that 51.68% genetic variance existed within group, and48.32% genetic variance was among groups.Analysis of cluster and principal component analysis showed that all the accessions could be distinguished by AFLP markers and divided into 5 groups, accessions from the same area were almost classified into the same group associated with their geographical distributions.Therefore, complex geographical ecological environment is important factor to the genetic diversity and geographical distribution of C.dactylon.It will own great significance in constituting the scientific protected strategy,promoting its exploitation and utilization, and breeding new varieties.
4. Sequence-related amplified polymorphism (SRAP), simple sequence repeat (SSR) and Amplified fragment length polymorphism (AFLP) molecular markers were applied to detect the genetic diversity and geographical differentiation of wild accessions of C.dactylon collected from Southwest China and Africa. The three molecular markers all showed the ample genetic diversity and geographical differentiation of the tested Germplasm. In this paper, three kinds of molecular markers'correlation were analysed. The result indicated that three molecular markers' correlation was remarkable.but the correlation coefficients were different, and the correlation between SRAP and SSR was higher.
5. The adaptability of five new wild accessions of C.dactylon were evaluated comprehensively in five regions of southwest China for 3 years. The introduction variety Tifway and national variety Nanjing were selected as control varieties. The trial sites were located at Ya'an, Meishan and Chengdu in Sichuan, Rongchang in Chongqing and Dushan in Guizhou. Comprehensively analysising density, texture, homogeneity, color, mature time, resistance and so on. The test observations and Excel 2007, SPSS 12.0 software analysis showed that, Sau9926 has long green period, fast grow speed,high density,strong resistance, and it is own good turf characteristics. In the five pilot sites, the average number of the green period is 295d, the mature time is about 45d, and the density is about 200/100 cm2. Comprehensively analysising texture, color, density, resistance, mature time and green period of five new Bermudagrass lines and two control Varieties, overall performance of Sau9926 is best, which is better than two control Varieties.
1.利用SRAP分子标记技术,对采自中国西南五省区的44份野生普通狗牙根(C. dactylon)及8份非洲狗牙根(C. transvalensis)材料进行遗传多样性研究。结果表明：18对引物组合共得到扩增总条带236条,多态性条带数206条,平均每对引物扩增出11.4条带,多态性位点百分率为87.29%,材料间的遗传相似系数范围在0.569到0.929之间,平均GS值为0.723。聚类分析结果表明,52份材料可聚为5类；基于Shannon多样性指数估算了8个狗牙根生态地理类群内和类群间的遗传分化,类群内的遗传变异占总变异的63.81%,类群间的遗传变异占总变异的36.19%,表明这些种源材料遗传差异较大,各生态地理类群间的遗传分化与其所处的生态地理环境具有一定的相关性。
2.以采自中国西南五省区的55份野生普通狗牙根(C. dactylon)和8份非洲狗牙根(C transvalensis)材料,利用SSR标记技术进行群体遗传结构的研究。结果表明,在6个地理类群中：(1)18对引物组合共获得353条清晰可辨的总条带,多态性条带数267条,平均每对引物扩增出19.61条带,多态性位点百分率为75.10%,材料间的遗传相似系数(GS)范围在0.687到0.904之间,平均GS值为0.788,变幅为0.217,说明供试材料具有比较丰富的遗传多样性。(2) UPGMA聚类分析结果表明,GS=0.81时,63份材料可聚为7类,大部分来自相同或相似生态地理环境的材料聚为一类,表明供试材料的聚类和其生态地理环境间有一定的相关性。(3)基于Shannon多样性指数估算了7个狗牙根生态地理类群内和类群间的遗传分化,类群内的遗传变异占总变异的66.57%,类群间的遗传变异占总变异的33.43%。(4)对各生态地理类群基于Nei氏无偏估计的遗传一致度的聚类分析表明,各生态地理类群间的遗传分化与其所处的生态地理环境具有一定的相关性。上述结果为保护和利用野生狗牙根资源提供了科学依据。
3.利用AFLP标记对采自中国西南五省区的51份野生普通狗牙根(C. dactylon)和8份非洲狗牙根(C. transvalensis)为材料进行遗传多样性分析。获得结果如下：11对引物共扩增出670个条带,其中多态性条带有663条,多态性条带比率为98.74%,材料间的遗传相似系数(GS)范围在0.64～0.96之间,平均GS值为0.78；基于Shannon多样性指数估算了7个狗牙根生态地理类群内和类群间的遗传分化,类群内的遗传变异占总变异的51.68%,类群间的遗传变异占总变异的48.32%；聚类分析和主成分分析显示,可将供试材料分成5大类,分类结果与材料的地理分布大致相符,呈现出一定的地域性分布规律。由此可见,丰富的地理生态条件造就了狗牙根资源丰富的遗传多样性和明显的地域性分布规律。对制定科学的资源保护策略、促进其开发利用以及新品种的选育具有重要的意义。
4.本研究采用了SRAP、SSR、AFLP三种分子标记分析了中国西南区野生普通狗牙根及采自非洲的野生狗牙根种质的遗传多样性及地理分化。三种分子标记都揭示了供试种质间具有丰富的遗传多样性并存在一定的地理分化。相关性结果表明了三种标记之间具有显著的相关性,但相关系数存在差异,其中SRAP和SSR的相关性较高。
5.在遗传多样性研究基础上,对初步筛选表现较好的五个野生狗牙根品系以“南京”和‘Tifway"作为对照品种,分别在雅安、眉山、成都、重庆和黔南五个试验点进行了周期3年的区域性试验研究,观测指标有草坪密度、质地、均一性、色泽、成坪密度以及抗性等。经试验观察以及Excel 2007和SPSS12.0软件对试验数据进行的分析表明,材料Sau9926的绿色期长、成坪速度较快、密度较高、抗逆性强,具有良好的坪用特性。在五个试验点的平均绿色期天数为295d,成坪天数为45d左右,密度为200枝/100cm2左右。从五份狗牙根新品系及两份对照品种的质地、色泽、密度、抗逆性、成坪速度和绿色期等方面综合分析,Sau9926的综合表现最佳,优于两个对照品种。
Cynodon dactylon L.Pers.belongs to Cynodon L.C.Rich.,Gramineae.It distributed in tropical, subtropical and even temperate regions of eastern and western hemispheres widely, and it also distributed in Eurasia, Indonesia, Malaysia and India and other places. In China, C.dactylon mainly distributed in the south of the Yellow River, and also distributed in Xinjiang, Jilin, Qinghai, Gansu, Hebei and other places. C. dactylon own low plants, strong breeding ability, high drought resistance, forbear trampling, delicate texture, good color, it is an excellent warm-season turf grass,which is widely used as playground lawn, ornamental turf, airport lawn, soil and water conservation, slope protection berm and other areas. The cultivars were selected as control varieties in the experiment.The systematic study of the genetic diversity and excellent germplasm filtration were from the DNA molecular markers and field test. The main results were as follows:
1. Sequence-related amplified polymorphism (SRAP) markers were used to detect the genetic diversity of 44 wild accessions of C.dactylon collected from Sichuan, Chongqing,Yunnan, Guizhou and Tibet in China and 8 wild accessions of C.dactylon collected from Africa.The results showed that Eighteen primer pairs produced 236 polymorphic bands, among which 206 bands were found to be polymorphic.averaged 11.4 bands per primer pair. The percentage of polymorphic bands in average was 87.29%.The Nei's genetic similarity coefficient of the tested accessions ranged from 0.569 to 0.929, and the average Nei's coefficient was0.723.The 52 accessions were classified into five major groups:Ⅰ,Ⅱ,Ⅲ,ⅣandⅤby cluster analysis using UPGMA. Genetic differentiation between and within five eco-geographical groups of C. dactylon was estimated by Shannon's diversity index, which showed that 63.81% genetic variance existed within group, and 36.19% genetic variance was among groups. which showed that these materials have great differences in heredity, and own significant relationship with the origin regions of accessions.
2. The population's genetic structure of 63 agrestal accessions of C.dactylon collected from Sichuan, Chongqing, Guizhou, Yunnan, Tibet and Africa were analyzed using simple sequence repeat (SSR) molecular markers. The results showed that, In the six populations: (1) Eighteen primer pairs produced 353 polymorphic bands, among which 267 bands were found to be polymorphic.averaged 19.61 bands per primer pair. The percentage of polymorphic bands in average was 75.10%.The Nei's genetic similarity coefficient of the tested accessions ranged from 0.687 to 0.904, and the average Nei's coefficient was 0.788,the range was 0.217. It showed that the test materials have ample genetic diversity. (2) Analysis of cluster showed that all the accessions could be divided into 5 groups when the genetic similarity coefficient is 0.81. accessions from the same area were almost classified into the same group associated with their geographical distributions. (3) Genetic differentiation between and within seven eco-geographical groups of C. dactylon was estimated by Shannon's diversity index, which showed that 66.57% genetic variance existed within group, and33.43% genetic variance was among groups. (4) Analysis of cluster in every eco-geographical groups by Shannon's diversity index showed that they own significant relationship with the origin regions of accessions. The results provided a scientific basis for the conservation and use of the wild resources of Cynodon dactylon.
3. Amplified fragment length polymorphism (AFLP) molecular markers were applied to detect the genetic variation of 59 agrestal accessions of C.dactylon collected from Africa, Sichuan, Chongqing, Yunnan, Guizhou and Tibet in China. The following results were obtained,A total of 670 bands were amplified by eleven AFLP primer pairs from C.dactylon genetic DNA, among which 663 (98.74%) bands were found to be polymorphic.The AFLP-based genetic similarity values among 59 C.dactylon accessions ranged from 0.64 to 0.96,and the average Nei's coefficient was 0.78. Genetic differentiation between and within seven eco-geographical groups of C. dactylon was estimated by Shannon's diversity index, which showed that 51.68% genetic variance existed within group, and48.32% genetic variance was among groups.Analysis of cluster and principal component analysis showed that all the accessions could be distinguished by AFLP markers and divided into 5 groups, accessions from the same area were almost classified into the same group associated with their geographical distributions.Therefore, complex geographical ecological environment is important factor to the genetic diversity and geographical distribution of C.dactylon.It will own great significance in constituting the scientific protected strategy,promoting its exploitation and utilization, and breeding new varieties.
4. Sequence-related amplified polymorphism (SRAP), simple sequence repeat (SSR) and Amplified fragment length polymorphism (AFLP) molecular markers were applied to detect the genetic diversity and geographical differentiation of wild accessions of C.dactylon collected from Southwest China and Africa. The three molecular markers all showed the ample genetic diversity and geographical differentiation of the tested Germplasm. In this paper, three kinds of molecular markers'correlation were analysed. The result indicated that three molecular markers' correlation was remarkable.but the correlation coefficients were different, and the correlation between SRAP and SSR was higher.
5. The adaptability of five new wild accessions of C.dactylon were evaluated comprehensively in five regions of southwest China for 3 years. The introduction variety Tifway and national variety Nanjing were selected as control varieties. The trial sites were located at Ya'an, Meishan and Chengdu in Sichuan, Rongchang in Chongqing and Dushan in Guizhou. Comprehensively analysising density, texture, homogeneity, color, mature time, resistance and so on. The test observations and Excel 2007, SPSS 12.0 software analysis showed that, Sau9926 has long green period, fast grow speed,high density,strong resistance, and it is own good turf characteristics. In the five pilot sites, the average number of the green period is 295d, the mature time is about 45d, and the density is about 200/100 cm2. Comprehensively analysising texture, color, density, resistance, mature time and green period of five new Bermudagrass lines and two control Varieties, overall performance of Sau9926 is best, which is better than two control Varieties.
引文
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