甜菜栽培品种的DNA指纹图谱构建及遗传多样性分析
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摘要
品种的真实性和纯度是种子检测最重要的指标之一，而研究快速、准确、简便的ＤＮＡ分子标记技术鉴定甜菜种子和将来新品种授权均具有重要的意义。本研究首次利用SSR分子标记对我国生产上应用的甜菜品种进行指纹图谱的构建，同时对影响品种纯度和真实性鉴定的各种因素进行系统的分析，建立了一套快速、高效的品种纯度和真实性鉴定方法，使快速鉴定甜菜品种及不同实验室的交流成为可能。主要结果如下：
1.建立了快速高效提取甜菜基因组DNA的方法。
本研究使用甜菜干种子或者叶片经真空冷冻干燥机处理1-2天后的干粉为原料，使用96孔PCR板代替单个离心管，利用碱裂解法对两种样品进行DNA的提取，该方法无需使用液氮，提取上百份DNA仅需要20分钟,提取的DNA能够作为SSR-PCR的模板。
2．筛选出29对可用于指纹图谱构建的SSR核心引物。
利用8%非变性聚丙烯酰胺凝胶电泳对101对甜菜SSR扩增产物进行检测，共找到29对带型清晰、重复性好、多态性高、易于识别的引物，每对引物平均扩增出2～11个等位基因。扩增的片段大小在90～500bp之间，这29对引物可以作为甜菜品种纯度和真实性鉴定的核心引物。
3．建立了甜菜多重SSR-PCR扩增体系。
在单一SSR～PCR的基础上，甜菜2～3重SSR-PCR的体系为：每增加1重SSR，仅仅增加相应引物的量以及减少相应去离子水的量，其余成分不变。甜菜4～5重SSR-PCR要在单一PCR的基础上增加0.5倍的DNTPs以及相应引物的量，同时根据个别引物扩增效率的不同，相应减少个别引物的用量，此体系对大多数4重和5重PCR有效。多重PCR扩增体系的检测效率是单引物PCR的2～5倍。
4.有7对引物可单独进行品种鉴定，利用其中3对引物构建了46个甜菜品种的SSR指纹图谱。
聚类分析结果表明，在遗传距离0.20处，所有的品种被分为一类，在遗传距离0.16处，46个品种被分为4个类群，聚类分析结果与材料的来源有很好的一致性，基本上是同一公司或者同一国别的品种聚在了一起，从分子水平上说明了甜菜品种之间的遗传基础狭窄。另外，有7对引物SB04、S7、S6、BVV45、SB13、S13和S8单独使用时能够鉴别不同的品种，利用其中的3对引物SB04、S6和S7构建的指纹图谱就能区别所有的品种。
5.建立了一套快速鉴定甜菜品种纯度和真实性的方法。
该方法使用96孔PCR板，利用碱裂解法提取干粉DNA；使用GV及λ DNA检测提取样品DNA的浓度和质量； PCR反应体系为10μL；PCR反应程序将循环中的变性和退火温度均改为15s,延伸为30s，35个循环；PCR产物的分离使用8%非变性聚丙烯酰胺凝胶电泳；最后使用快速银染法对电泳后的聚丙烯酰胺凝胶进行检测；同时我们将模板DNA、引物稀释到工作浓度后和DNTPs以及PCR Buffer一起放入到冰箱保鲜层中，使用时不需要融化，可直接吸取，3个月内均能正常使用。利用以上的方法最终确立了一套简单、快速、节约、污染小的甜菜品种真实性和纯度的鉴定体系,促进了该技术的大规模使用，利用优化后的程序，一个成熟的实验室操作人员只需一天就可以完成192份样品的检测。
One of the most major targets of seed quality testing is the authenticity and Purity of cultivar.Theimportant significance to research rapidly,simple and accurate method of molecular maker with beetvarieties identified during seed quality testing and granting the new variety authorigation.Thefingerprint was built for the sugarbeet varieties applied in the sugar beet production in our country.SSRmolecular marker used in this research first time,and various factors affecting the variety purity andauthenticity identification were analyzed to establish a set of quick and efficient method for the varietyidentifying, which would make it possible to interchange between different laboratories.The majorresults obtained as follows:
1. An improved method of rapid,high effective DNA extraction was set up.
In this study, Used dry beet seeds or leaves which treated1～2days with frozen and dried in thevacuum freezing machine and extract DNA,the PCR plates of96were used to instead of a singlecentrifugal tube,Alkali decomposition method was used for DNA extraction,and in stead of liquidnitrogen.one person can extract about100DNA samples in20minutes,DNA extracted to be used astemplate of SSR-PCR.
2.29primers were screened as the core primer of SSR to apply in fingerprint construction
Using8%non denaturing polyacrylamide gel to test101pairs of sugar beet SSR amplificationproduct, each experiment repeated once at least,29pairs of primers were found which with clear bands,good repeatability, high polymorphism and easy to identify, each pair of primers amplified out2-11alleles average, there were159alleles all together，including116polymorphic alleles,and the ratio ofpolymorphism was72.95%.The fragment sizes vaired from90-500bp.The29primers can be used ascore primers for sugar beet varieties purity and authenticity identification.
3.Multiplex SSR-PCR amplification system of beet was established
Based on the single-pair of primer SSR-PCR, Duplex or triplex SSR-PCR system for Beet is thatevery increase one plex of SSR, and the amount of the appropriate primers increases only and thecorresponding amount of deionized water reduced, and the remaining ingredients unchanged. Four orfive plex SSR-PCR of beet is on the basis of the single PCR to increase the amount of0.5times DNTPsand the corresponding primer, at the same time which is according to the different individual primers inamplification efficiency, a corresponding reduction of the individual primer in mutiplex PCRamplification system, In this system,most four or five plex PCR is effective. the detection efficiency is2～5times than the single-primer PCR.
4. Seven pairs of primer can be used to identify different varieties independently, The DNA fingerprintsof46beet varieties were constructed by three primers。
UPGMA cluster analysis of genetic distance showed that all of the varieties were clustered in onegroup at the genetic distance of0.20, In genetic distance0.16,46varieties were divided into four groups,Cluster analysis and the source of the material has fine consistency, varieties come from the samecompany or the same country basically to join together,it is indicated that the genetic basis of beetvarieties was narrow. In addition, there are seven pairs of primers SB04, S7, S6, BVV45、SB13、S13、 S8, which can be able to identify the different varieties independently, the primer of SB04, S6and S7will made distinguishi for all varieties.
5. A rapid method to identify beet varieties purity and authenticity was established
A96-well PCR plate was used which instead of a single PCR tube, alkaline lysis method was usedto extract form dry seeds (or dry powder) DNA; the GV (Goldview) and λ DNA were used to test DNAconcentration and quality; PCR reaction system is10μL; PCR reaction program cycle denaturation andannealing temperature were converted to the15s, and extends for30s,35cycles; the high resolution of8%non-denaturing polyacrylamide gel was used to separate the PCR product; finally, A rapid silverstaining was used to detect polyacrylamide gel. At the same time，we put DNA templates, primers,DNTPs and PCR Buffer into the refrigerator-fresh-layer, Don't need to melt when using, all of themcan normally use within3months.In this way,we established a set of simple, fast, low costing and littlepollution technical system of the beet varieties authenticity and purity identification system, which is topromote the application of the technology in large-scale,A mature operator of laboratory can detect192samples by one day.
1.建立了快速高效提取甜菜基因组DNA的方法。
本研究使用甜菜干种子或者叶片经真空冷冻干燥机处理1-2天后的干粉为原料，使用96孔PCR板代替单个离心管，利用碱裂解法对两种样品进行DNA的提取，该方法无需使用液氮，提取上百份DNA仅需要20分钟,提取的DNA能够作为SSR-PCR的模板。
2．筛选出29对可用于指纹图谱构建的SSR核心引物。
利用8%非变性聚丙烯酰胺凝胶电泳对101对甜菜SSR扩增产物进行检测，共找到29对带型清晰、重复性好、多态性高、易于识别的引物，每对引物平均扩增出2～11个等位基因。扩增的片段大小在90～500bp之间，这29对引物可以作为甜菜品种纯度和真实性鉴定的核心引物。
3．建立了甜菜多重SSR-PCR扩增体系。
在单一SSR～PCR的基础上，甜菜2～3重SSR-PCR的体系为：每增加1重SSR，仅仅增加相应引物的量以及减少相应去离子水的量，其余成分不变。甜菜4～5重SSR-PCR要在单一PCR的基础上增加0.5倍的DNTPs以及相应引物的量，同时根据个别引物扩增效率的不同，相应减少个别引物的用量，此体系对大多数4重和5重PCR有效。多重PCR扩增体系的检测效率是单引物PCR的2～5倍。
4.有7对引物可单独进行品种鉴定，利用其中3对引物构建了46个甜菜品种的SSR指纹图谱。
聚类分析结果表明，在遗传距离0.20处，所有的品种被分为一类，在遗传距离0.16处，46个品种被分为4个类群，聚类分析结果与材料的来源有很好的一致性，基本上是同一公司或者同一国别的品种聚在了一起，从分子水平上说明了甜菜品种之间的遗传基础狭窄。另外，有7对引物SB04、S7、S6、BVV45、SB13、S13和S8单独使用时能够鉴别不同的品种，利用其中的3对引物SB04、S6和S7构建的指纹图谱就能区别所有的品种。
5.建立了一套快速鉴定甜菜品种纯度和真实性的方法。
该方法使用96孔PCR板，利用碱裂解法提取干粉DNA；使用GV及λ DNA检测提取样品DNA的浓度和质量； PCR反应体系为10μL；PCR反应程序将循环中的变性和退火温度均改为15s,延伸为30s，35个循环；PCR产物的分离使用8%非变性聚丙烯酰胺凝胶电泳；最后使用快速银染法对电泳后的聚丙烯酰胺凝胶进行检测；同时我们将模板DNA、引物稀释到工作浓度后和DNTPs以及PCR Buffer一起放入到冰箱保鲜层中，使用时不需要融化，可直接吸取，3个月内均能正常使用。利用以上的方法最终确立了一套简单、快速、节约、污染小的甜菜品种真实性和纯度的鉴定体系,促进了该技术的大规模使用，利用优化后的程序，一个成熟的实验室操作人员只需一天就可以完成192份样品的检测。
One of the most major targets of seed quality testing is the authenticity and Purity of cultivar.Theimportant significance to research rapidly,simple and accurate method of molecular maker with beetvarieties identified during seed quality testing and granting the new variety authorigation.Thefingerprint was built for the sugarbeet varieties applied in the sugar beet production in our country.SSRmolecular marker used in this research first time,and various factors affecting the variety purity andauthenticity identification were analyzed to establish a set of quick and efficient method for the varietyidentifying, which would make it possible to interchange between different laboratories.The majorresults obtained as follows:
1. An improved method of rapid,high effective DNA extraction was set up.
In this study, Used dry beet seeds or leaves which treated1～2days with frozen and dried in thevacuum freezing machine and extract DNA,the PCR plates of96were used to instead of a singlecentrifugal tube,Alkali decomposition method was used for DNA extraction,and in stead of liquidnitrogen.one person can extract about100DNA samples in20minutes,DNA extracted to be used astemplate of SSR-PCR.
2.29primers were screened as the core primer of SSR to apply in fingerprint construction
Using8%non denaturing polyacrylamide gel to test101pairs of sugar beet SSR amplificationproduct, each experiment repeated once at least,29pairs of primers were found which with clear bands,good repeatability, high polymorphism and easy to identify, each pair of primers amplified out2-11alleles average, there were159alleles all together，including116polymorphic alleles,and the ratio ofpolymorphism was72.95%.The fragment sizes vaired from90-500bp.The29primers can be used ascore primers for sugar beet varieties purity and authenticity identification.
3.Multiplex SSR-PCR amplification system of beet was established
Based on the single-pair of primer SSR-PCR, Duplex or triplex SSR-PCR system for Beet is thatevery increase one plex of SSR, and the amount of the appropriate primers increases only and thecorresponding amount of deionized water reduced, and the remaining ingredients unchanged. Four orfive plex SSR-PCR of beet is on the basis of the single PCR to increase the amount of0.5times DNTPsand the corresponding primer, at the same time which is according to the different individual primers inamplification efficiency, a corresponding reduction of the individual primer in mutiplex PCRamplification system, In this system,most four or five plex PCR is effective. the detection efficiency is2～5times than the single-primer PCR.
4. Seven pairs of primer can be used to identify different varieties independently, The DNA fingerprintsof46beet varieties were constructed by three primers。
UPGMA cluster analysis of genetic distance showed that all of the varieties were clustered in onegroup at the genetic distance of0.20, In genetic distance0.16,46varieties were divided into four groups,Cluster analysis and the source of the material has fine consistency, varieties come from the samecompany or the same country basically to join together,it is indicated that the genetic basis of beetvarieties was narrow. In addition, there are seven pairs of primers SB04, S7, S6, BVV45、SB13、S13、 S8, which can be able to identify the different varieties independently, the primer of SB04, S6and S7will made distinguishi for all varieties.
5. A rapid method to identify beet varieties purity and authenticity was established
A96-well PCR plate was used which instead of a single PCR tube, alkaline lysis method was usedto extract form dry seeds (or dry powder) DNA; the GV (Goldview) and λ DNA were used to test DNAconcentration and quality; PCR reaction system is10μL; PCR reaction program cycle denaturation andannealing temperature were converted to the15s, and extends for30s,35cycles; the high resolution of8%non-denaturing polyacrylamide gel was used to separate the PCR product; finally, A rapid silverstaining was used to detect polyacrylamide gel. At the same time，we put DNA templates, primers,DNTPs and PCR Buffer into the refrigerator-fresh-layer, Don't need to melt when using, all of themcan normally use within3months.In this way,we established a set of simple, fast, low costing and littlepollution technical system of the beet varieties authenticity and purity identification system, which is topromote the application of the technology in large-scale,A mature operator of laboratory can detect192samples by one day.
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