狗牙根种质资源遗传多样性分析及评价
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摘要
狗牙根(Cynodon dactylon)是禾本科狗牙根属的多年生植物,是世界著名的暖季型草坪草之一,同时也是优质牧草。
鉴于狗牙根在分布范围和生长习性上的多样性,本研究在全面调查国内外野生狗牙根种群生态分布的基础上,从所收集的材料中选出475份(473份野生狗牙根和2份应用广泛的栽培品种'Tifway'和'Tifgreen')作为试验材料,从形态学水平及分子水平研究其遗传多样性；并分别采用草坪综合评价体系及概略养分法对其坪用价值及营养价值进行评价,旨在探明狗牙根种源的遗传关系,筛选具有优良性状及品质的草坪和饲草新品系,为狗牙根品种选育和开发利用奠定基础。主要研究结果如下：
(1)对475份种源的17个外部性状进行分析表明：狗牙根形态特征存在广泛变异。17个形态性状变异系数均大于10%,在形态水平上证实了不同狗牙根之间差异较大。对狗牙根的10个主要形态指标进行聚类分析,可将475份材料明显分为2大类群：即低矮型和高大型。
(2)经过比较多种方法对狗牙根基因组DNA的提取效果,最终获得了以健康嫩叶提取纯度高及完整性好的狗牙根基因组DNA的提取方法,即改良CTAB法。
(3)利用单因子试验及正交设计试验优化了狗牙根SRAP-PCR反应体系,即25μL反应体系中,含1.5 mmol/L Mg2+、0.25 mmol/L dNTPs、0.4μmol/L引物、1.5U Taq酶和80ng模板DNA。
利用该体系筛选的15对多态性高且重复性好的SRAP引物组合对475份种质进行扩增,共扩增出500条谱带,且均是多态性条带,平均每对引物组合扩增带数为33.33条,从而在分子水平上证实了不同狗牙根之间差异较大,种质间的遗传相似系数为0.554-0.964,平均值为0.723。聚类分析可将475份种质分为3大类。
(4)利用正交设计试验优化了狗牙根ISSR-PCR反应体系,即25μL反应体系中含2.5 mmol/L Mg2、0.25 mmol/L dNTPs、0.2μmol/L引物、1.0U Taq DNA聚合酶和40ng模板DNA。
利用该体系筛选的14条多态性高且重复性好的ISSR引物对所有种质进行扩增,共扩增出266条谱带,且均是多态性带,平均每条引物扩增带数是19条,能将475份狗牙根种质区分开,种质之间的遗传相似系数为0.549-0.981,平均值为0.761。聚类分析可将475份种质分为4大类。
(5)筛选出坪用价值较高的材料50份,其中接近或优于世界著名品种'Tifway'和'Tifgreen'的有14份。
(6)筛选出高营养饲用品系32份,可以以之为基础材料,开展饲用型牧草新品种的选育。
Cynodon dactylon (Common bermudagrass), the most important member of the gramineous genus Cynodon, is a world-famous warm-season turfgrass and high quality pasture.
In view of its diversity in distribution area and growth habits, after a whole investigation of ecological distribution areas and growth habits of bermudagrass populations, a total of 475 bermudagrass accessions including 473 wild accessions and 2 commercial cultivars ('Tifway' and Tifgreen') were selected as experimental materials in this study. In view of their abundant variation and genetic diversity among bermudagrass accessions, systematic studies on different levels had been made on morphological marker and molecular marker in order to supplying the theoretical base of bermudagrass genetic improvement. Meantime, turf and nutrition characteristics of bermudagrass accessions were evaluated through turf quality comprehensive assessment system and nutrition value assessment system. The purpose was to select excellent accessions. The information from this study will be beneficial for the breeding of bermudagrass. The main results were as follows:
(1) Seventeen morphological characteristics of 475 bermudagrass accessions were observed and analyzed. Five main results were received as:abundant morphological variation existed in bermudagrass accessions and the coefficients of variation were over 10%. Based on 10 morphological characteristics,475 bermudagrass accessions were clustered into two morphological types, designated as low type and high type.
(2) Comparing of lots of DNA extraction method, the improved protocol based on CTAB for isolating total genomic DNA from fresh and healthy leaves of bermudagrass was found.
(3) The optimal SRAP-PCR amplification system of C. dactylon was established by employing single factor tests and orthogonal design approaches using young leaves, which came out total 25μL reaction system containing 1.5 mmol/L Mg2+,0.25 mmol/L dNTP,0.4μmol/L primer,1.5 U Taq DNA polymerase, and 80 ng DNA.
475 bermudagrass accessions were studied for the analysis of genetic diversity by the optimum SRAP-PCR system. The results indicated that 475 accessions could be separated by 15 primer combinations, and the 15 primer combinations amplified 500 reproducible fragments with an average of 33.33 and all scorable bands were polymorphic in nature and none of the primer combinations used produced monomorphic bands indicating a high level of genetic variation in this grass. GSC among the 475 accessions ranged from 0.554 to 0.964 with an average of 0.723. The accessions were clustered into three major groups by unweighted pair-group method with arithmetic averages.
(4) The concentration of Mg2+, dNTPs, Taq DNA polymerase, primers, and DNA which affected the ISSR-PCR reactions was optimized by orthogonal design. Results showed that the optimum system in 25μL reaction mixture was as follows,2.5 mmol/L Mg2+,0.25 mmol/L dNTPs,0.2μmol/L primer,1.0 U Taq DNA polymerase, and 40 ng DNA.
The fourteen selected primers amplified a total of 266 scorable fragments among 475 accessions with an average of 19, and the size of the fragments varied from 200 to 3000 bp. The percentage of polymorphic bands was 100%, meaning that the bands were present in at least one accession but were not observed in others. GSC among the 475 accessions ranged from 0.549 to 0.981. The accessions were clustered into four major groups by unweighted pair-group method with arithmetic averages.
(5) 50 accessions which had excellent turf characteristics were screened out.14 accessions were more excellent than'Tifway' and'Tifgreen'among them.
(6) 32 accessions which had high nutritional quality were screened out.
鉴于狗牙根在分布范围和生长习性上的多样性,本研究在全面调查国内外野生狗牙根种群生态分布的基础上,从所收集的材料中选出475份(473份野生狗牙根和2份应用广泛的栽培品种'Tifway'和'Tifgreen')作为试验材料,从形态学水平及分子水平研究其遗传多样性；并分别采用草坪综合评价体系及概略养分法对其坪用价值及营养价值进行评价,旨在探明狗牙根种源的遗传关系,筛选具有优良性状及品质的草坪和饲草新品系,为狗牙根品种选育和开发利用奠定基础。主要研究结果如下：
(1)对475份种源的17个外部性状进行分析表明：狗牙根形态特征存在广泛变异。17个形态性状变异系数均大于10%,在形态水平上证实了不同狗牙根之间差异较大。对狗牙根的10个主要形态指标进行聚类分析,可将475份材料明显分为2大类群：即低矮型和高大型。
(2)经过比较多种方法对狗牙根基因组DNA的提取效果,最终获得了以健康嫩叶提取纯度高及完整性好的狗牙根基因组DNA的提取方法,即改良CTAB法。
(3)利用单因子试验及正交设计试验优化了狗牙根SRAP-PCR反应体系,即25μL反应体系中,含1.5 mmol/L Mg2+、0.25 mmol/L dNTPs、0.4μmol/L引物、1.5U Taq酶和80ng模板DNA。
利用该体系筛选的15对多态性高且重复性好的SRAP引物组合对475份种质进行扩增,共扩增出500条谱带,且均是多态性条带,平均每对引物组合扩增带数为33.33条,从而在分子水平上证实了不同狗牙根之间差异较大,种质间的遗传相似系数为0.554-0.964,平均值为0.723。聚类分析可将475份种质分为3大类。
(4)利用正交设计试验优化了狗牙根ISSR-PCR反应体系,即25μL反应体系中含2.5 mmol/L Mg2、0.25 mmol/L dNTPs、0.2μmol/L引物、1.0U Taq DNA聚合酶和40ng模板DNA。
利用该体系筛选的14条多态性高且重复性好的ISSR引物对所有种质进行扩增,共扩增出266条谱带,且均是多态性带,平均每条引物扩增带数是19条,能将475份狗牙根种质区分开,种质之间的遗传相似系数为0.549-0.981,平均值为0.761。聚类分析可将475份种质分为4大类。
(5)筛选出坪用价值较高的材料50份,其中接近或优于世界著名品种'Tifway'和'Tifgreen'的有14份。
(6)筛选出高营养饲用品系32份,可以以之为基础材料,开展饲用型牧草新品种的选育。
Cynodon dactylon (Common bermudagrass), the most important member of the gramineous genus Cynodon, is a world-famous warm-season turfgrass and high quality pasture.
In view of its diversity in distribution area and growth habits, after a whole investigation of ecological distribution areas and growth habits of bermudagrass populations, a total of 475 bermudagrass accessions including 473 wild accessions and 2 commercial cultivars ('Tifway' and Tifgreen') were selected as experimental materials in this study. In view of their abundant variation and genetic diversity among bermudagrass accessions, systematic studies on different levels had been made on morphological marker and molecular marker in order to supplying the theoretical base of bermudagrass genetic improvement. Meantime, turf and nutrition characteristics of bermudagrass accessions were evaluated through turf quality comprehensive assessment system and nutrition value assessment system. The purpose was to select excellent accessions. The information from this study will be beneficial for the breeding of bermudagrass. The main results were as follows:
(1) Seventeen morphological characteristics of 475 bermudagrass accessions were observed and analyzed. Five main results were received as:abundant morphological variation existed in bermudagrass accessions and the coefficients of variation were over 10%. Based on 10 morphological characteristics,475 bermudagrass accessions were clustered into two morphological types, designated as low type and high type.
(2) Comparing of lots of DNA extraction method, the improved protocol based on CTAB for isolating total genomic DNA from fresh and healthy leaves of bermudagrass was found.
(3) The optimal SRAP-PCR amplification system of C. dactylon was established by employing single factor tests and orthogonal design approaches using young leaves, which came out total 25μL reaction system containing 1.5 mmol/L Mg2+,0.25 mmol/L dNTP,0.4μmol/L primer,1.5 U Taq DNA polymerase, and 80 ng DNA.
475 bermudagrass accessions were studied for the analysis of genetic diversity by the optimum SRAP-PCR system. The results indicated that 475 accessions could be separated by 15 primer combinations, and the 15 primer combinations amplified 500 reproducible fragments with an average of 33.33 and all scorable bands were polymorphic in nature and none of the primer combinations used produced monomorphic bands indicating a high level of genetic variation in this grass. GSC among the 475 accessions ranged from 0.554 to 0.964 with an average of 0.723. The accessions were clustered into three major groups by unweighted pair-group method with arithmetic averages.
(4) The concentration of Mg2+, dNTPs, Taq DNA polymerase, primers, and DNA which affected the ISSR-PCR reactions was optimized by orthogonal design. Results showed that the optimum system in 25μL reaction mixture was as follows,2.5 mmol/L Mg2+,0.25 mmol/L dNTPs,0.2μmol/L primer,1.0 U Taq DNA polymerase, and 40 ng DNA.
The fourteen selected primers amplified a total of 266 scorable fragments among 475 accessions with an average of 19, and the size of the fragments varied from 200 to 3000 bp. The percentage of polymorphic bands was 100%, meaning that the bands were present in at least one accession but were not observed in others. GSC among the 475 accessions ranged from 0.549 to 0.981. The accessions were clustered into four major groups by unweighted pair-group method with arithmetic averages.
(5) 50 accessions which had excellent turf characteristics were screened out.14 accessions were more excellent than'Tifway' and'Tifgreen'among them.
(6) 32 accessions which had high nutritional quality were screened out.
引文
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