龙眼遗传多样性及亲缘关系的ISSR分析
摘要
龙眼起源于我国，是我国南方特产果树之一，种质资源十分丰富。但长期以来，对龙眼资源的分类和利用，都是采用林奈和恩格勒的形态特征分类法，往往有些种、品种和品系在外貌和形态特征上十分相似，但亲缘关系和遗传特性却相差巨大，使得龙眼的分类地位及亲缘关系一直存在着较多的争议，并给龙眼的新品种培育和资源利用带来困难。本试验以广西区域内广泛收集的60个龙眼品种为试材，应用ISSR分子标记技术，探讨龙眼的分类和品种之间亲缘关系的研究。试验结果表明：
1、通过对SDS、CTAB和改良CTAB法三种基因组DNA提取方法的比较，从纯度、产率等方面考虑，认为改良CTAB法较为适合龙眼基因组DNA的提取。采用该方法提取的龙眼基因组DNA，经电泳检测，主带清晰，无降解，分光光度计检测，A_(260)／A_(280)的值在1.81-1.84之间，符合ISSR反应的要求。
2、通过对Tag DNA聚合酶，dNTP，模板DNA，Primer不同浓度的优化试验及对PCR反应程序的筛选，确定了合适的反应体系和反应程序，从而建立了龙眼的ISSR分析技术体系。在20μl反应体系中，模板DNA 75ng，Primer 5μmol，rTaq polymerase 1 U，dNTP 4 mmol，Tris-HCl(pH 8.3)200μmol。反应程序为：预变性，94℃5min；变性，94′C 1min；退火1min；延伸，72℃2min；40个循环，72℃延伸10min，4℃保存。
3、利用筛选得到的12条ISSR引物对全部60个龙眼品种进行PCR扩增，共扩增产生119条总带，其中共有带52条，多态性条带67条，多态性比率为56.30％。扩增片段长度分布在100bp—2000bp之间，平均每个引物能扩出10条带。
4、运用NTSYS软件，采用Dice相似系数分析ISSR反应结果，得到了60个品种间的亲缘关系树状图。以0.80作为相似系数的分界点，60个样品通过基因组ISSR分成2大类：龙荔单独为一类，另一类在相似系数0.85的水平将其余的59个龙眼材料分为7个品种群：品种群Ⅰ：东壁、未知实生2；品种群Ⅱ：立冬本；品种群Ⅲ：乌龙岭、九叶乌、佳圆；品种群Ⅳ：灵龙、早白露；品种群Ⅴ：车站、桂香、香蜜、小广眼等12个品种(系)；品种群Ⅵ：大乌圆、大广眼、储良、古山二号、石硖等27个个品种(系)；品种群Ⅶ：青山水柜、洪武本、草灰本、处暑本、桂明等12个品种(系)。
5、利用ISSR技术可以对基因组的所有序列直接分析，不受环境的影响，在分子水平上利用ISSR技术对60个供试龙眼品种(系)进行遗传多样性和亲缘关系鉴定，结果非常可靠，是龙眼种质资源研究中快速、可靠、有效的新技术，能为杂交育种亲本组配提供有价值的分子水平上的信息。
Longan (Dimocarpus longan Lour.) is originated in south China, which is one of thespecial local fruits, and its variety resource is very abundant. The utilization andclassification of longan germplasm resources were adopted the botany classificationsystem of linnaean or Engler for a long time. There are arguements on the geneticrelationship and classification among the native varieties of Longan for many years,which had made grate trouble in breeding new variety and utilizating resource, as somespecies, cuitivars or strains are much similar in the appearance and morphologicalcharacters, while their relationships of heredity and relative were very different.Therefor, in this study we analyzed the classification and genetic relationship among 60cultivars of longan by ISSR-PCR. The main result of this research as follows:
1. Three DNA extraction methods of CTAB, SDS and modified CTAB were studied.The results showed that the modified CTAB method was the best to extract longangenomic DNA based on the purity and yield of DNA. The products (bands) were clearand degradation in gel electrophoresis. The values of A_(260)/A_(280) were between 1.81-1.84detected by U/V spectrophotometer. This method could get good purity DNA and wasqualified to apply to ISSR—PCR.
2. The different PCR program and the ingredients in ISSR reaction system such asTag DNA Polymerase, dNTP, DNA and primers were screened by differentconcentration. Suitable reaction system and program of the ISSR analysis for longangenomic DNA was setted. The following was the 20μL reaction system with DNAtemplate 75ng, Primer 5μmol, rTaq polymerase 1 U, dNTP 4 mmol, Tris-HCl (pH 8.3) 200μmol. The program was that predenature in 94℃for 5min, denature in 94℃for1min, renature in 36℃for 1min, elongated in 72℃for 2min, holding in 72℃for 10min,cycling number in 40, and final products kept at 4℃.
3. 12 ISSR primers which screened from 100 ISSR primers were used for theAmplification reaction to 60 longan material by PCR. A total of 119 bands were obtainedby amplification of the polymorphic primers, among which 52 bands were commonbands, and 67 polymorphic bands were found to be polymorphic. The percentage ofpolymorphic bands was 56.30%. Every primer could amplify averagely 10 bands, andthe size of band amplified was between 100bp and 3000bp.
4. The dendrogram obtained by the UPGMA clustering method. 60 longan cultivarscould be clustered into two groups on the dendrogram. Of there, the first group has onlya variety, that is longli [Dimocarpus confinis (How et Ho) H. S. Lour]. The other wereclustered into a big group, which could be divided into seven sub-groups, the firstsubgroup consisted of two cuitivars such as Dongbi and NO. 2 of unknown seeding, thesecond subgroup consisted of Lidongben cuitivar, the third subgroup consisted of threecultivars, such as Wulongling, Jiuyewu and Jiayuan, the fourthly subgroup consisted oftwo cultivars, such as Linglong and Zhaobailu, the fifth subgroup consisted of twelvecultivars, such as Chezhan, Guixiang and Xiangmi et. al, the sixth subgroup consisted oftwenty-seven cultivars, such as Dawuyuan, Daguangyan and Chuliang et. al, the seventhsubgroup consisted of twelve cultivars, such as Qingshanshuigui, Hongwuben andCaohuiben et. al.
5. All DNA sequences can be directly analysed by ISSR method, which is no failedunder the influence of environment condition. The results are very reliable that thegenetic diversity and relationship of 60 Longan cultivars are analysed by ISSR methodon the mmolecular level. It is a new technology, which is very effective, simple andreliable in studing on the longan germplasm resources. And it could provide informationof value in parent combination at Molecular Level on the hybridization breeding.
1、通过对SDS、CTAB和改良CTAB法三种基因组DNA提取方法的比较，从纯度、产率等方面考虑，认为改良CTAB法较为适合龙眼基因组DNA的提取。采用该方法提取的龙眼基因组DNA，经电泳检测，主带清晰，无降解，分光光度计检测，A_(260)／A_(280)的值在1.81-1.84之间，符合ISSR反应的要求。
2、通过对Tag DNA聚合酶，dNTP，模板DNA，Primer不同浓度的优化试验及对PCR反应程序的筛选，确定了合适的反应体系和反应程序，从而建立了龙眼的ISSR分析技术体系。在20μl反应体系中，模板DNA 75ng，Primer 5μmol，rTaq polymerase 1 U，dNTP 4 mmol，Tris-HCl(pH 8.3)200μmol。反应程序为：预变性，94℃5min；变性，94′C 1min；退火1min；延伸，72℃2min；40个循环，72℃延伸10min，4℃保存。
3、利用筛选得到的12条ISSR引物对全部60个龙眼品种进行PCR扩增，共扩增产生119条总带，其中共有带52条，多态性条带67条，多态性比率为56.30％。扩增片段长度分布在100bp—2000bp之间，平均每个引物能扩出10条带。
4、运用NTSYS软件，采用Dice相似系数分析ISSR反应结果，得到了60个品种间的亲缘关系树状图。以0.80作为相似系数的分界点，60个样品通过基因组ISSR分成2大类：龙荔单独为一类，另一类在相似系数0.85的水平将其余的59个龙眼材料分为7个品种群：品种群Ⅰ：东壁、未知实生2；品种群Ⅱ：立冬本；品种群Ⅲ：乌龙岭、九叶乌、佳圆；品种群Ⅳ：灵龙、早白露；品种群Ⅴ：车站、桂香、香蜜、小广眼等12个品种(系)；品种群Ⅵ：大乌圆、大广眼、储良、古山二号、石硖等27个个品种(系)；品种群Ⅶ：青山水柜、洪武本、草灰本、处暑本、桂明等12个品种(系)。
5、利用ISSR技术可以对基因组的所有序列直接分析，不受环境的影响，在分子水平上利用ISSR技术对60个供试龙眼品种(系)进行遗传多样性和亲缘关系鉴定，结果非常可靠，是龙眼种质资源研究中快速、可靠、有效的新技术，能为杂交育种亲本组配提供有价值的分子水平上的信息。
Longan (Dimocarpus longan Lour.) is originated in south China, which is one of thespecial local fruits, and its variety resource is very abundant. The utilization andclassification of longan germplasm resources were adopted the botany classificationsystem of linnaean or Engler for a long time. There are arguements on the geneticrelationship and classification among the native varieties of Longan for many years,which had made grate trouble in breeding new variety and utilizating resource, as somespecies, cuitivars or strains are much similar in the appearance and morphologicalcharacters, while their relationships of heredity and relative were very different.Therefor, in this study we analyzed the classification and genetic relationship among 60cultivars of longan by ISSR-PCR. The main result of this research as follows:
1. Three DNA extraction methods of CTAB, SDS and modified CTAB were studied.The results showed that the modified CTAB method was the best to extract longangenomic DNA based on the purity and yield of DNA. The products (bands) were clearand degradation in gel electrophoresis. The values of A_(260)/A_(280) were between 1.81-1.84detected by U/V spectrophotometer. This method could get good purity DNA and wasqualified to apply to ISSR—PCR.
2. The different PCR program and the ingredients in ISSR reaction system such asTag DNA Polymerase, dNTP, DNA and primers were screened by differentconcentration. Suitable reaction system and program of the ISSR analysis for longangenomic DNA was setted. The following was the 20μL reaction system with DNAtemplate 75ng, Primer 5μmol, rTaq polymerase 1 U, dNTP 4 mmol, Tris-HCl (pH 8.3) 200μmol. The program was that predenature in 94℃for 5min, denature in 94℃for1min, renature in 36℃for 1min, elongated in 72℃for 2min, holding in 72℃for 10min,cycling number in 40, and final products kept at 4℃.
3. 12 ISSR primers which screened from 100 ISSR primers were used for theAmplification reaction to 60 longan material by PCR. A total of 119 bands were obtainedby amplification of the polymorphic primers, among which 52 bands were commonbands, and 67 polymorphic bands were found to be polymorphic. The percentage ofpolymorphic bands was 56.30%. Every primer could amplify averagely 10 bands, andthe size of band amplified was between 100bp and 3000bp.
4. The dendrogram obtained by the UPGMA clustering method. 60 longan cultivarscould be clustered into two groups on the dendrogram. Of there, the first group has onlya variety, that is longli [Dimocarpus confinis (How et Ho) H. S. Lour]. The other wereclustered into a big group, which could be divided into seven sub-groups, the firstsubgroup consisted of two cuitivars such as Dongbi and NO. 2 of unknown seeding, thesecond subgroup consisted of Lidongben cuitivar, the third subgroup consisted of threecultivars, such as Wulongling, Jiuyewu and Jiayuan, the fourthly subgroup consisted oftwo cultivars, such as Linglong and Zhaobailu, the fifth subgroup consisted of twelvecultivars, such as Chezhan, Guixiang and Xiangmi et. al, the sixth subgroup consisted oftwenty-seven cultivars, such as Dawuyuan, Daguangyan and Chuliang et. al, the seventhsubgroup consisted of twelve cultivars, such as Qingshanshuigui, Hongwuben andCaohuiben et. al.
5. All DNA sequences can be directly analysed by ISSR method, which is no failedunder the influence of environment condition. The results are very reliable that thegenetic diversity and relationship of 60 Longan cultivars are analysed by ISSR methodon the mmolecular level. It is a new technology, which is very effective, simple andreliable in studing on the longan germplasm resources. And it could provide informationof value in parent combination at Molecular Level on the hybridization breeding.
引文
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