CAJ | 학술논문

【目的】红松已开发的分子标记缺乏共显性的遗传标记，尚不能满足分子标记辅助育种的需要。利用转录组数据开发 SSR标记目前仍然是较为经济高效的 DNA分子标记开发策略，本研究利用高通量测序技术开发红松 EST-SSR标记，掌握其在转录组序列中的分布类型及特征，为红松的 SSR多样性分析及变异分析提供基础。【方法】利用 SSR检索程序从红松转录组41476条 Unigenes中筛选得到1757个 SSR 位点，并对 SSR 位点的数量、分布特征进行统计分析。设计合成101对 SSR引物，采用琼脂糖电泳初步检查和聚丙烯酰胺凝胶电泳分离检测方法确定引物的多态性，并收集扩增产物，送测序验证，最终开发出16对 SSR引物。合成6对荧光引物，采用荧光标记技术对来自黑龙江省鹤岗、林口、铁力、苇河4个种子园的53份自由授粉子代进行遗传多样性分析。【结果】基于转录组序列开发出的 EST-SSR的分布频率( SSR 的个数与总 Unigene 的数量比)为4.24%。单、二、三核苷酸重复单元分别占总SSR的46.90%，17.12%和34.66%； SSR重复单元的重复次数分布在5～24次之间。参试的101对引物中有21对引物扩增可检测出多态性位点，占引物总数的20.8%；重测序验证，有16对引物能够扩增出目标序列。6对荧光引物共检测出18个等位基因，多态性信息量(PIC)为0.0363～0.6674，平均为0.325。【结论】红松属于基因组序列比较庞大的裸子植物，本研究可扩增出多态性位点的引物重复单元以二、三核苷酸重复为主。所研究红松种子园子代属于中等多态性水平。本研究印证了利用红松转录组数据开发 SSR标记的可行性，同时采用荧光标记技术检测红松自由授粉子代材料，为红松种质资源的多样性水平分析及变异水平的研究提供基础。
【목적】홍송이개발적분자표기결핍공현성적유전표기，상불능만족분자표기보조육충적수요。이용전록조수거개발 SSR표기목전잉연시교위경제고효적 DNA분자표기개발책략，본연구이용고통량측서기술개발홍송 EST-SSR표기，장악기재전록조서렬중적분포류형급특정，위홍송적 SSR다양성분석급변이분석제공기출。【방법】이용 SSR검색정서종홍송전록조41476조 Unigenes중사선득도1757개 SSR 위점，병대 SSR 위점적수량、분포특정진행통계분석。설계합성101대 SSR인물，채용경지당전영초보검사화취병희선알응효전영분리검측방법학정인물적다태성，병수집확증산물，송측서험증，최종개발출16대 SSR인물。합성6대형광인물，채용형광표기기술대래자흑룡강성학강、림구、철력、위하4개충자완적53빈자유수분자대진행유전다양성분석。【결과】기우전록조서렬개발출적 EST-SSR적분포빈솔( SSR 적개수여총 Unigene 적수량비)위4.24%。단、이、삼핵감산중복단원분별점총SSR적46.90%，17.12%화34.66%； SSR중복단원적중복차수분포재5～24차지간。삼시적101대인물중유21대인물확증가검측출다태성위점，점인물총수적20.8%；중측서험증，유16대인물능구확증출목표서렬。6대형광인물공검측출18개등위기인，다태성신식량(PIC)위0.0363～0.6674，평균위0.325。【결론】홍송속우기인조서렬비교방대적라자식물，본연구가확증출다태성위점적인물중복단원이이、삼핵감산중복위주。소연구홍송충자완자대속우중등다태성수평。본연구인증료이용홍송전록조수거개발 SSR표기적가행성，동시채용형광표기기술검측홍송자유수분자대재료，위홍송충질자원적다양성수평분석급변이수평적연구제공기출。
Objective]In Korean pine ( Pinus koraiensis ) breeding programs,lack of co-dominant genetic markers constrained the development of molecular marker assisted breeding. At present,development of SSR markers based on transcriptome data is still an economic and efficient development strategy of DNA molecular markers. In this study,we used high-throughput sequencing technology to develop EST-SSR markers for Korean pine. Distribution patterns of the markers in the transcriptome sequences and their characteristics were analyzed,in order to provide a basis for analysis of SSR diversity and mutation of Korean pine. [Method]A total of 1 757 SSR sites were identified from 41 476 unigenes in Korean pine transcriptome by using SSR searching program. Statistical analyses were conducted for number,distribution and characteristics of the SSR loci. And 101 pairs of SSR primers were designed and synthesized. Agarose electrophoresis was used for initial check and polyacrylamide gel electrophoresis for separation and detection of the polymorphisms of primers. Then amplification products were collected and sequenced for validation. Finally,16 pairs of SSR primers and 6 pairs of fluorescence primers were identified. To study the genetic variation,53 samples of open-pollinated progeny were collected from four seed orchards respectively in Hegang,Linkou,Tieli and Weihe. [Result]The distribution frequency of EST-SSRs ( ratio of the number of SSRs to the total number of unigenes) was 4. 24%,based on the transcriptome sequences. Mononucleotide dinucleotide and trinucleotide repeats were 46. 90% ,17. 12% and 34. 66% of total SSR, respectively. The SSR repeat number of SSR repeat units was between 5 and 24. Twenty-one pairs of primers showed polymorphism among 101 pairs of primer,which accounting for 20. 8% of the total number of primer pairs. By sequencing validation,16 pairs of primers amplified the target sequence. Eighteen alleles were tested from 6 pairs of fluorescence primers. Polymorphic information content ( PIC) was 0. 036 3 - 0. 667 4 and the mean was 0. 325 0. [Conclusion] Korean pine has a relatively large genome in gymnosperms. The amplified primers of the polymorphism loci were mainly dinucleotide and trinucleotide repeats. The progeny of the Korean pine seed orchard revealed a medium level of polymorphism. This study demonstrated that using Korean pine transcriptome data to develop SSR markers was feasible. The technique of using fluorescent markers to analyze the progeny materials provided a basis for studies of genetic diversity and variation of the Korean pine germplasm resources.