基于AFLP标记的农作物品种分子条码构建
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摘要
农作物种类多,品种十分丰富,而且随着农业生产的发展,育种成效愈来愈大,许多种类的遗传基础也越来越窄,在生产上应用的品种和传统种质资源的鉴定、甄别,一方面越来越重要,另一方面则越来越困难。因此,寻找简便、有效的鉴定、甄别、标识方法越来越重要。分子标记理论与方法的迅速发展,为其提供了有效的技术,但是由于其只是依赖少数特征标记进行区别,不能对大多数群体或个体相互间进行区别鉴定,难以形成一个稳定的、可供普遍使用的技术体系。在这里,我们将分子生物学技术与计算机技术结合起来,将商业上的条形码概念应用到生物鉴别上,提出了分子条码(molecular barcode)的概念,试图将现代信息技术和生物技术结合起来,为生物群体和个体鉴定、甄别、标识,品种审定、认定和鉴定,以及新品种权保护等提供了唯一标识和鉴定依据。鉴于十字花科(Cruciferae)蔬菜是浙江省栽培面积最大,总产量最高的一类经济作物,由于其品种数量巨大,加上新品种的遗传多样性越来越狭窄,使得在形态学上鉴定品种越来越困难。因此,本研究以搜集到的浙江省十字花科蔬菜主要栽培品种为例,利用其丰富的多样性,探讨采用AFLP标记构建分子条码的概念,探讨分子条码的构建、标准和可行性,以此技术为基础,建立一个农作物品种管理平台。其主要的研究结果如下:
(1)对普通白菜的19个形态学性状的观察,可以用于区分10个白菜品种;通过芥菜的12个形态学性状观察可以用区分4个芥菜品种;通过萝卜的20个形态性状的观察可以区分5个萝卜品种。结果表明,十字花科蔬菜具有丰富的多样性。当用17个质量性状做聚类分析是发现,‘蚕白'‘五月慢'没有别区分开,说明在大量品种鉴定时需要更多的标记。
(2)对AFLP标记实验体系进行优化的结果显示,在20μL酶切体系中,酶切DNA模板为250ng,预扩循环数为18的预扩结果最好。在每20μL选扩体系中,dNTPs的用量在4～8μmol·L~(-1)、Taq酶的量在0.5～0.8U、Buffer的量在2.0～2.5μL的范围内,选扩的效果没有明显区别。比较两种银染显色法的结果发现:用NaCO_3的慢速速显色法比用NaOH的快速显色法检测的多态性要多,经过筛选得到M5E2、M6E1、M3E2、M5E1、M6E2、M4E7、M1E2、M8E3多态性较高的引物,共扩增出251条带。利用AFLP标记聚类结果表明:在75个品种中,除‘矮抗青'和‘新选1号'没有被区分,其余的品种都被区分,说明十字花科蔬菜在DNA水平上有丰富的多样性,另外,研究结果进一步显示,大白菜和芜菁,大白菜和普通白菜的亲缘关系远。
(3)我们定义的分子条码是指采用信息技术对生物物种各分类阶元,以及农业上的品种,甚至变异的个体在分子水平上的多态性特征信息集合进行分析生成的条形码。在建立基于AFLP标记的农作物品种条码的标准的基础上,构建获得以PDF417编码技术编码的75个浙江省十字花科蔬菜主要栽培品种的分子条码。提出建立农作物品种分子条码管理平台。
There are many kinds of crops,and the cultivars are very rich,but with the development of agricultural technology,increasing the effectiveness of breeding,many genetic basis of crops become more and more narrow,identification of applied cultivars and traditional germplasm resources incarnates important Increasingly,on the other hand,more and more difficult.Therefore,looking for simple and effective identification methods will become important especially.Rapid development of Molecular markers theories and methods provides an effective technology,although because it only dependents on a few features to distinguish between tags,not a lot of groups or the individuals,so it is difficult to form a stable,available generally technical system.Here,we will combine the molecular biology and computer technology to make the business bar codes be applied to biometric identification.We proposed molecular barcode and trying to combine modern information technology and bio-technologies,providing a basis for the identification of the biological group and individuals,species identification,and protection of the new cultival rights.In view of Cruciferae vegetables in Zhejiang Province is the largest area of cultivation,the highest total output economic crops,but because of its huge number of cultivars,and new cultivars with narrow genetic diversity increasingly,which makes the identification of species on the morphology more and more difficult. Therefore,this study collected the Zhejiang Cruciferae vegetables main cultivars,using their rich diversity, to explore Construction of the concept of molecular bar code with AFLP marker,and study the construction, standards and feasibility of molecular bar code,On the basis of this technology,established a crop cultivars management platform.The main results are as follows:
(1)We identified 10 Chinese cabbage-pak-cho cultivars by 19 morphological characters;identified 4 mustards cultivars by 12 morphological characters;identified 5 radish cultivars by 20 morphological characters.The results indicated that there was rich genetic diversity in Cruciferae cultivars in Zhejiang province.
(2)By the research on AFLP system,the results showed that,in 20μL digestion system,the right DNA templet is 250 ng and the pre-amplification is 18 cycles:in 20μL amplification system,dNTPs at 4～8μmol·L~(-1)、Taq at 0.5～0.8U、Buffer at 2～2.5μL have no significant difference.Compared with two silver staining methods,showed that more diversity had been detected by quick Silver Staining than slow Silver Staining。By using AFLP technique then 251 bands were amplified by eight amplification primers(M5E2、M6E1、M3E2、M5E1、M6E2、M4E7、M1E2,M8E3).Tree dendrograms were constructed based on the AFLP marker data by system cluster analysis of WPGMA,75 cruciferae cultivars were identified except 'Aikanqing','Xinxuanlhao',The results indicated cruciferae vegetable have rich diversity on DNA level, on the side,the results showed that chinese cabbage-pe-tsai have far genetic relationship with turnip chinese cabbage-pak-choi.
(3)We posed a molecular barcode concept,and discussed its standar(?)an feasibility.Obtained the molecular barcode of the 75 cruciferae based on PDF417,established a platform for crops management information.
(1)对普通白菜的19个形态学性状的观察,可以用于区分10个白菜品种;通过芥菜的12个形态学性状观察可以用区分4个芥菜品种;通过萝卜的20个形态性状的观察可以区分5个萝卜品种。结果表明,十字花科蔬菜具有丰富的多样性。当用17个质量性状做聚类分析是发现,‘蚕白'‘五月慢'没有别区分开,说明在大量品种鉴定时需要更多的标记。
(2)对AFLP标记实验体系进行优化的结果显示,在20μL酶切体系中,酶切DNA模板为250ng,预扩循环数为18的预扩结果最好。在每20μL选扩体系中,dNTPs的用量在4～8μmol·L~(-1)、Taq酶的量在0.5～0.8U、Buffer的量在2.0～2.5μL的范围内,选扩的效果没有明显区别。比较两种银染显色法的结果发现:用NaCO_3的慢速速显色法比用NaOH的快速显色法检测的多态性要多,经过筛选得到M5E2、M6E1、M3E2、M5E1、M6E2、M4E7、M1E2、M8E3多态性较高的引物,共扩增出251条带。利用AFLP标记聚类结果表明:在75个品种中,除‘矮抗青'和‘新选1号'没有被区分,其余的品种都被区分,说明十字花科蔬菜在DNA水平上有丰富的多样性,另外,研究结果进一步显示,大白菜和芜菁,大白菜和普通白菜的亲缘关系远。
(3)我们定义的分子条码是指采用信息技术对生物物种各分类阶元,以及农业上的品种,甚至变异的个体在分子水平上的多态性特征信息集合进行分析生成的条形码。在建立基于AFLP标记的农作物品种条码的标准的基础上,构建获得以PDF417编码技术编码的75个浙江省十字花科蔬菜主要栽培品种的分子条码。提出建立农作物品种分子条码管理平台。
There are many kinds of crops,and the cultivars are very rich,but with the development of agricultural technology,increasing the effectiveness of breeding,many genetic basis of crops become more and more narrow,identification of applied cultivars and traditional germplasm resources incarnates important Increasingly,on the other hand,more and more difficult.Therefore,looking for simple and effective identification methods will become important especially.Rapid development of Molecular markers theories and methods provides an effective technology,although because it only dependents on a few features to distinguish between tags,not a lot of groups or the individuals,so it is difficult to form a stable,available generally technical system.Here,we will combine the molecular biology and computer technology to make the business bar codes be applied to biometric identification.We proposed molecular barcode and trying to combine modern information technology and bio-technologies,providing a basis for the identification of the biological group and individuals,species identification,and protection of the new cultival rights.In view of Cruciferae vegetables in Zhejiang Province is the largest area of cultivation,the highest total output economic crops,but because of its huge number of cultivars,and new cultivars with narrow genetic diversity increasingly,which makes the identification of species on the morphology more and more difficult. Therefore,this study collected the Zhejiang Cruciferae vegetables main cultivars,using their rich diversity, to explore Construction of the concept of molecular bar code with AFLP marker,and study the construction, standards and feasibility of molecular bar code,On the basis of this technology,established a crop cultivars management platform.The main results are as follows:
(1)We identified 10 Chinese cabbage-pak-cho cultivars by 19 morphological characters;identified 4 mustards cultivars by 12 morphological characters;identified 5 radish cultivars by 20 morphological characters.The results indicated that there was rich genetic diversity in Cruciferae cultivars in Zhejiang province.
(2)By the research on AFLP system,the results showed that,in 20μL digestion system,the right DNA templet is 250 ng and the pre-amplification is 18 cycles:in 20μL amplification system,dNTPs at 4～8μmol·L~(-1)、Taq at 0.5～0.8U、Buffer at 2～2.5μL have no significant difference.Compared with two silver staining methods,showed that more diversity had been detected by quick Silver Staining than slow Silver Staining。By using AFLP technique then 251 bands were amplified by eight amplification primers(M5E2、M6E1、M3E2、M5E1、M6E2、M4E7、M1E2,M8E3).Tree dendrograms were constructed based on the AFLP marker data by system cluster analysis of WPGMA,75 cruciferae cultivars were identified except 'Aikanqing','Xinxuanlhao',The results indicated cruciferae vegetable have rich diversity on DNA level, on the side,the results showed that chinese cabbage-pe-tsai have far genetic relationship with turnip chinese cabbage-pak-choi.
(3)We posed a molecular barcode concept,and discussed its standar(?)an feasibility.Obtained the molecular barcode of the 75 cruciferae based on PDF417,established a platform for crops management information.
引文
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