RAMP在黑木耳品种鉴定中的应用研究
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摘要
黑木耳[Auricularia auricula(L.ex Hook.)Underwood]是我国最先栽培的食用菌之一。我国黑木耳产量在全世界食用菌产量中居第七位,主要出口日本,东南亚,西欧和北美等一些国家,在我国食用菌出口中占有重要地位。随着“培育者权利”保护意识的增加,世界各国都开始制定种苗保护制度,加强本国种质资源的保护。在世界品种资源保护兴起的大环境中,我国也应该加强种质资源的保护力度。为了能够更好的对食用菌种质资源进行保护,开发一种可靠、稳定的食用菌鉴定方法是关键。本论文对吉林省生产中广泛应用的22个黑木耳品种进行鉴定研究,旨在探索黑木耳遗传变异的规律和适合黑木耳品种鉴定的方法、分析生产中菌种名称混乱的原因、为相关研究提供参考依据。本论文的研究分为三个部分:
首先,对能够提取高质量黑木耳基因组DNA的方法进行了探索。由于黑木耳菌丝中含有大量的胶状物质,在DNA提取时常常和DNA结合在一起形成粘稠状悬浮物,影响DNA沉淀。本文对DNA提取时常用的SDS法进行了改进,并与CTAB法、试剂盒法、改进前的SDS法进行比较,发现改进后的方法在提取黑木耳基因组DNA时能够很好的去除菌丝体中的多糖和胶状物质。对这几种方法提取的DNA进行经紫外扫描分析,证明改进后的方法显著的提高了DNA的质量。
其次,对黑木耳的遗传稳定性进行了研究。食用菌的菌丝在生产与保存过程中很容易产生菌种退化现象,菌种退化是由于菌丝体的遗传物质发生变异而造成的。为了证明这些潜在的变异是否已经达到了品种级别,本文将同一个黑木耳品种进行连续继代培养12代,然后利用RAMP方法比较第2代和第12代之间的区别。结果发现黑木耳在传代过程中具有较高的稳定性。常规的操作引起的机械损伤并不能导致黑木耳菌株产生品种水平上的变异。
最后,利用RAMP(随机扩增微卫星多态性)标记对22个在吉林省黑木耳生产中广泛应用的品种进行了鉴定研究。本文对40对引物进行了筛选,发现其中有21对引物可以区分不同的黑木耳品种,在这21对引物中有9对呈现出了很好的多态性,其中有3对结果比较接近而且有很好的重复性和多态性,可以作为黑木耳品种鉴定的有效引物。通过对22个黑木耳品种比较分析,发现其中存在同种异名现象。尤其是在菌农自己繁育的品种中,这种现象更为明显。
通过本文的研究筛选出了适合对黑木耳品种进行鉴定的RAMP标记引物,通过对鉴定结果的分析发现吉林省黑木耳生产中应用的品种存在同种异名现象。并进一步证明了黑木耳品种具有较高的传代稳定性,因此生产中产生的同种异名现象更可能是人为因素造成的。本文通过对常用DNA提取方法—SDS法进行改进,提高了黑木耳基因组DNA的得率和质量。
Auricularia auricular is a kind of edible fungi which firstly cultivated in China. The production of Auricularia auricular is the seventh compared to the world production of fungi. Auricularia auricular, which stand a significant place for the export of edible fungi mainly export to Japan, Southeast Asia, Western Europe, North America and so on. With the increase of "breeder authority" consciousness, many countries begin to legislate some institution to protect germplasm resource. During the protective current of germplasm resource, our country should enhance the protection. The key point of identical strains is to exploiting a reliable and stabile method. The research chooses 22 production strains of Auricularia auricular which is widespread at production in Jilin province to identify. The purpose is to exploit the rule of hereditary, suitable method of identifying the strains of Auricularia auricular and the reasons of different strains have the same name. This study can offer some base on correlate research. There are three parts in this thesis.
First, exploit a method of extracting high quality genome DNA from Auricularia auricular. Because of much jell in mycelium, which combined with DNA when extracting DNA interfere the precipitation of DNA. The study improved a commonly used SDS method and compared with CTAB method, kit and unimproved SDS method. The results indicated that the improved method could well remove polysaccharide and jell in the mycelium when extracting DNA. Through ultraviolet scanning DNA, we found that the improved method obviously elevate the quality of DNA.
Second, study the hereditary stability of Auricularia auricular. The mycelium of edible fungi can easily generate degeneration when applying and conserving. The phenomenon of mycelium degeneration is possible due to the variation of hereditary substance. In order to certify this potential variation whether or not generating different strains, this study use the same strain of Auricularia auricular to continual cultivated for 12 generation, than analysis the distinction between the second generation and the twelve generation with RAMP method. The result showed that inheritance of Auricularia auricular is very stability and the conventional machinery damage will not lead to the variation on strain level.
Last, using the method of RAMP to identify 22 strains of Auricularia auricular which is widespread produced in Jilin province. This research analysis 40 pair primers, 21 pair primers of which can identify strains of Auricularia auricular, 9 pair primers of the 21 pair primers show well polymorphism, 3 pair primers show well repeatability and polymorphism which can be used for utility primers on strain identify. We can find the phenomenon that same strain have different name through the analysis of the 22 strains, especially in the breeding by farmers.
In this research, we find that the primers suited for identifying different strain of Auricularia auricular with RAMP method and find some identical strains which widespread at production in Jilin province have different names. We further proved that Auricularia auricular do not occur variation at strain level. So the phenomenon of "identical-strain-different-name" is caused by human. We improved the DNA isolation methods that purify the quality of DNA significantly.
首先,对能够提取高质量黑木耳基因组DNA的方法进行了探索。由于黑木耳菌丝中含有大量的胶状物质,在DNA提取时常常和DNA结合在一起形成粘稠状悬浮物,影响DNA沉淀。本文对DNA提取时常用的SDS法进行了改进,并与CTAB法、试剂盒法、改进前的SDS法进行比较,发现改进后的方法在提取黑木耳基因组DNA时能够很好的去除菌丝体中的多糖和胶状物质。对这几种方法提取的DNA进行经紫外扫描分析,证明改进后的方法显著的提高了DNA的质量。
其次,对黑木耳的遗传稳定性进行了研究。食用菌的菌丝在生产与保存过程中很容易产生菌种退化现象,菌种退化是由于菌丝体的遗传物质发生变异而造成的。为了证明这些潜在的变异是否已经达到了品种级别,本文将同一个黑木耳品种进行连续继代培养12代,然后利用RAMP方法比较第2代和第12代之间的区别。结果发现黑木耳在传代过程中具有较高的稳定性。常规的操作引起的机械损伤并不能导致黑木耳菌株产生品种水平上的变异。
最后,利用RAMP(随机扩增微卫星多态性)标记对22个在吉林省黑木耳生产中广泛应用的品种进行了鉴定研究。本文对40对引物进行了筛选,发现其中有21对引物可以区分不同的黑木耳品种,在这21对引物中有9对呈现出了很好的多态性,其中有3对结果比较接近而且有很好的重复性和多态性,可以作为黑木耳品种鉴定的有效引物。通过对22个黑木耳品种比较分析,发现其中存在同种异名现象。尤其是在菌农自己繁育的品种中,这种现象更为明显。
通过本文的研究筛选出了适合对黑木耳品种进行鉴定的RAMP标记引物,通过对鉴定结果的分析发现吉林省黑木耳生产中应用的品种存在同种异名现象。并进一步证明了黑木耳品种具有较高的传代稳定性,因此生产中产生的同种异名现象更可能是人为因素造成的。本文通过对常用DNA提取方法—SDS法进行改进,提高了黑木耳基因组DNA的得率和质量。
Auricularia auricular is a kind of edible fungi which firstly cultivated in China. The production of Auricularia auricular is the seventh compared to the world production of fungi. Auricularia auricular, which stand a significant place for the export of edible fungi mainly export to Japan, Southeast Asia, Western Europe, North America and so on. With the increase of "breeder authority" consciousness, many countries begin to legislate some institution to protect germplasm resource. During the protective current of germplasm resource, our country should enhance the protection. The key point of identical strains is to exploiting a reliable and stabile method. The research chooses 22 production strains of Auricularia auricular which is widespread at production in Jilin province to identify. The purpose is to exploit the rule of hereditary, suitable method of identifying the strains of Auricularia auricular and the reasons of different strains have the same name. This study can offer some base on correlate research. There are three parts in this thesis.
First, exploit a method of extracting high quality genome DNA from Auricularia auricular. Because of much jell in mycelium, which combined with DNA when extracting DNA interfere the precipitation of DNA. The study improved a commonly used SDS method and compared with CTAB method, kit and unimproved SDS method. The results indicated that the improved method could well remove polysaccharide and jell in the mycelium when extracting DNA. Through ultraviolet scanning DNA, we found that the improved method obviously elevate the quality of DNA.
Second, study the hereditary stability of Auricularia auricular. The mycelium of edible fungi can easily generate degeneration when applying and conserving. The phenomenon of mycelium degeneration is possible due to the variation of hereditary substance. In order to certify this potential variation whether or not generating different strains, this study use the same strain of Auricularia auricular to continual cultivated for 12 generation, than analysis the distinction between the second generation and the twelve generation with RAMP method. The result showed that inheritance of Auricularia auricular is very stability and the conventional machinery damage will not lead to the variation on strain level.
Last, using the method of RAMP to identify 22 strains of Auricularia auricular which is widespread produced in Jilin province. This research analysis 40 pair primers, 21 pair primers of which can identify strains of Auricularia auricular, 9 pair primers of the 21 pair primers show well polymorphism, 3 pair primers show well repeatability and polymorphism which can be used for utility primers on strain identify. We can find the phenomenon that same strain have different name through the analysis of the 22 strains, especially in the breeding by farmers.
In this research, we find that the primers suited for identifying different strain of Auricularia auricular with RAMP method and find some identical strains which widespread at production in Jilin province have different names. We further proved that Auricularia auricular do not occur variation at strain level. So the phenomenon of "identical-strain-different-name" is caused by human. We improved the DNA isolation methods that purify the quality of DNA significantly.
引文
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