CAJ | 학술논문

【目的】探究国兰种质资源遗传多样性水平，并构建分子指纹图谱及分子身份证，为在分子水平上鉴定国兰种质提供技术支撑，也为国兰种质资源开发、保存利用及种质创新奠定基础。【方法】以收集于华南及邻近地区的139份国兰栽培品种和野生种质为材料，采用改良的CTAB法提取基因组DNA，由13条正向引物和16条反向引物随机组成208对SRAP引物，每对引物用品种‘宋梅’和‘大勋’扩增产物进行筛选；PCR扩增产物应用6%非变性聚丙烯酰胺凝胶电泳，电泳胶图进行人工读带，并计算多态性引物的总扩增条带数、多态性条带数和多态性条带比率。按照Botstein公式计算多态信息含量；用POPGENE32软件计算观测等位基因数、有效等位基因数、Nei’s基因多样性指数、Shannon’s信息指数，并进行遗传多样性分析。用NTSYS-pc2.10e软件UPGMA方法进行聚类分析，并计算遗传相似性系数。采用引物对组合的方法，构建139份国兰种质资源数字指纹图谱。【结果】从208对SRAP引物中共筛选出17对多态性好且重复性高的引物，对供试材料进行PCR扩增，共扩增出DNA条带489条，其中多态性谱带484条，多态性比率(PPB)为98.89%，观测等位基因数平均值为2.00，多态性信息含量平均值为0.94，每个位点的有效等位基因数平均值为1.49，Nei’s基因多样多样性指数平均值为0.30，Shannon信息指数平均值为0.45。UPGMA聚类分析表明，139份国兰种质资源的遗传相似系数变化范围为0.51—0.91。在相似系数为0.70时，可划分为6个类群。用SRAP多种引物组合方法可有效区分所有材料，并构建出139份国兰种质资源特异性分子身份证，置信概率达到99.99%。根据聚类结果可以将国兰种质分为4组：一为春兰组，由春兰种质单独构成；二为建墨兰组，主要由建兰和墨兰种质组成；三为寒蕙兰组，主要由寒兰、蕙兰和杂交系组成；四为剑莲兰组，由春剑和莲瓣兰种质组成。而四组之间剑莲兰组与寒蕙兰组亲缘关系最近，与建墨兰组次之，与春兰组亲缘关系较远。【结论】所试国兰种质具有较丰富的遗传多样性水平，基于17对SRAP引物组合所构建的139份国兰种质的分子身份识别体系具有唯一性和高效性，SRAP标记是在分子水平鉴定国兰种质的有效方法之一。【目的】探究國蘭種質資源遺傳多樣性水平，併構建分子指紋圖譜及分子身份證，為在分子水平上鑒定國蘭種質提供技術支撐，也為國蘭種質資源開髮、保存利用及種質創新奠定基礎。【方法】以收集于華南及鄰近地區的139份國蘭栽培品種和野生種質為材料，採用改良的CTAB法提取基因組DNA，由13條正嚮引物和16條反嚮引物隨機組成208對SRAP引物，每對引物用品種‘宋梅’和‘大勛’擴增產物進行篩選；PCR擴增產物應用6%非變性聚丙烯酰胺凝膠電泳，電泳膠圖進行人工讀帶，併計算多態性引物的總擴增條帶數、多態性條帶數和多態性條帶比率。按照Botstein公式計算多態信息含量；用POPGENE32軟件計算觀測等位基因數、有效等位基因數、Nei’s基因多樣性指數、Shannon’s信息指數，併進行遺傳多樣性分析。用NTSYS-pc2.10e軟件UPGMA方法進行聚類分析，併計算遺傳相似性繫數。採用引物對組閤的方法，構建139份國蘭種質資源數字指紋圖譜。【結果】從208對SRAP引物中共篩選齣17對多態性好且重複性高的引物，對供試材料進行PCR擴增，共擴增齣DNA條帶489條，其中多態性譜帶484條，多態性比率(PPB)為98.89%，觀測等位基因數平均值為2.00，多態性信息含量平均值為0.94，每箇位點的有效等位基因數平均值為1.49，Nei’s基因多樣多樣性指數平均值為0.30，Shannon信息指數平均值為0.45。UPGMA聚類分析錶明，139份國蘭種質資源的遺傳相似繫數變化範圍為0.51—0.91。在相似繫數為0.70時，可劃分為6箇類群。用SRAP多種引物組閤方法可有效區分所有材料，併構建齣139份國蘭種質資源特異性分子身份證，置信概率達到99.99%。根據聚類結果可以將國蘭種質分為4組：一為春蘭組，由春蘭種質單獨構成；二為建墨蘭組，主要由建蘭和墨蘭種質組成；三為寒蕙蘭組，主要由寒蘭、蕙蘭和雜交繫組成；四為劍蓮蘭組，由春劍和蓮瓣蘭種質組成。而四組之間劍蓮蘭組與寒蕙蘭組親緣關繫最近，與建墨蘭組次之，與春蘭組親緣關繫較遠。【結論】所試國蘭種質具有較豐富的遺傳多樣性水平，基于17對SRAP引物組閤所構建的139份國蘭種質的分子身份識彆體繫具有唯一性和高效性，SRAP標記是在分子水平鑒定國蘭種質的有效方法之一。
【목적】탐구국란충질자원유전다양성수평，병구건분자지문도보급분자신빈증，위재분자수평상감정국란충질제공기술지탱，야위국란충질자원개발、보존이용급충질창신전정기출。【방법】이수집우화남급린근지구적139빈국란재배품충화야생충질위재료，채용개량적CTAB법제취기인조DNA，유13조정향인물화16조반향인물수궤조성208대SRAP인물，매대인물용품충‘송매’화‘대훈’확증산물진행사선；PCR확증산물응용6%비변성취병희선알응효전영，전영효도진행인공독대，병계산다태성인물적총확증조대수、다태성조대수화다태성조대비솔。안조Botstein공식계산다태신식함량；용POPGENE32연건계산관측등위기인수、유효등위기인수、Nei’s기인다양성지수、Shannon’s신식지수，병진행유전다양성분석。용NTSYS-pc2.10e연건UPGMA방법진행취류분석，병계산유전상사성계수。채용인물대조합적방법，구건139빈국란충질자원수자지문도보。【결과】종208대SRAP인물중공사선출17대다태성호차중복성고적인물，대공시재료진행PCR확증，공확증출DNA조대489조，기중다태성보대484조，다태성비솔(PPB)위98.89%，관측등위기인수평균치위2.00，다태성신식함량평균치위0.94，매개위점적유효등위기인수평균치위1.49，Nei’s기인다양다양성지수평균치위0.30，Shannon신식지수평균치위0.45。UPGMA취류분석표명，139빈국란충질자원적유전상사계수변화범위위0.51—0.91。재상사계수위0.70시，가화분위6개류군。용SRAP다충인물조합방법가유효구분소유재료，병구건출139빈국란충질자원특이성분자신빈증，치신개솔체도99.99%。근거취류결과가이장국란충질분위4조：일위춘란조，유춘란충질단독구성；이위건묵란조，주요유건란화묵란충질조성；삼위한혜란조，주요유한란、혜란화잡교계조성；사위검련란조，유춘검화련판란충질조성。이사조지간검련란조여한혜란조친연관계최근，여건묵란조차지，여춘란조친연관계교원。【결론】소시국란충질구유교봉부적유전다양성수평，기우17대SRAP인물조합소구건적139빈국란충질적분자신빈식별체계구유유일성화고효성，SRAP표기시재분자수평감정국란충질적유효방법지일。
Objective] The genetic diversity of Chinese cymbidium germplasms was studied, meanwhile, the molecular fingerprinting and ID card were established in order to provide technique support for their identification at molecular level, and to lay the root for their exploitation, conservation, utilization and innovation in the future.[Method] 139 samples of Chinese cymbidium gemplasm containing cultivated and wild species were collected from south China and its neighborhood. Their genomic DNA was extracted by the modified method of CTAB. 208 pair SRAP primers was from random combinations with 13 pieces F-primer and 16 pieces R-primer, which each pair of SRAP primers was initially screened for amplification of a specific product from genomic DNA of two cultivars, one from ‘Songmei’ and the other from ‘Daxun’. The PCR amplification products were separated on 6% nondenaturing polyacrylamidegel electrophoresis. The electrophoresis pattern was artificially analyzed, and total bands, polymorphic bands and percentage of polymorphic bands(PPB) were counted. The polymorphism information content(PIC) were counted according to Botstein’s formula. The observed number of alleles (Na), effective number of alleles (Ne), Nei’s genetic diversity diversity index (H) , and Shannon’s information index (I) all were estimated by software POPGENE32. The UPGMA clustering analysis was performed by software NTSYS-pc2.10e, and attained the genetic similarity coefficient value. The method of primer combinations was used to construct a digital fingerprinting of 139 samples of Chinese cymbidium germplasm.[Result] 17 pair polymorphic primers were screened from 208 pair SRAP primers, and a total of 489 bands were amplified by these primers, of which 484 bands were polymorphic and PPB was 98.89%, observed number of alleles (Na), the effective number of alleles (Ne), Nei’s genetic diversity diversity index (H) , and Shannon’s information index (I) were respectively 2.00, 1.49, 0.30, 0.45. The genetic similar coefficient of 139 Chinese cymbidium ranged from 0.51 to 0.91, and the cluster analysis based on UPGMA showed that these germplasm can be divided into six major groups at similarity coefficient of 0.70. All of the samples could be effectively distinguished by different primer combinations. The specific molecular identity for 139 samples of Chinese cymbidium germplasm were established with 99.99% probability of confidence. However, Chinese cymbidium germplasm can be divided into four sections according to their genetic relationship, section one of which isC. goeringii germline, only consists of one species, section two mainly includesC. ensifoliumand C. sinense germline, section three consists ofC. faberi, C. kanranandhybrid species, section four includes C. longibractiumand C. lianpan.In terms of genetic relationship among them, section four is the nearest with section three, section two comes second, is farthest with section one.[Conclusion] A higher level of genetic diversity exists in Chinese cymbidium germplasm examined, it was unique and efficient that the molecular identity system of 139 samples was first established based on 17 pair primer combinations of SRAP. The SRAP technique can provide a powerful tool for Chinese cymbidium germplasm identification.