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香菇分子遗传连锁图谱的构建

摘   要

采用香菇（Lentinula edodes）两个栽培菌株L205和武香1号各自的孢子单核体L₆和W₂₆进行杂交（L₆×W₂₆）、出菇和孢子单核体制备实验，获得了148个孢子单核体的遗传分离群体，通过杂交配对观察锁状联合的有无、核迁移实验和OWE-SOJ实验，准确鉴定了此分离群体中个菌株的交配型。

通过SSR、SRAP、TRAP和一个信息素受体基因片段对得到的香菇遗传分离群体进行分子扩增分析：从77对SSR引物中筛选得到2对具有多态性条带的引物；在56对SRAP引物组合中，25对引物组合表现出较高的多态性，多态性条带在4~17条之间，共扩增得到225个多态性条带，平均每对引物组合扩增得到9条条带；通过对36个与香菇子实体发育相关基因的分析，设计TRAP固定引物，与随机引物组合，共得到43对TRAP引物组合，TRAP分析总计扩增得到474个多态性条带，平均每对引物产生11.1个多态性条带。SRAP和TRAP都表现出较高的扩增效率，有利于高密度遗传连锁图谱的构建；根据本实验室前期所得到的香菇交配型B因子信息素受体基因片段，进行特异性引物的设计，在分离群体中进行扩增实验，扩增结果具有多态性，扩增片段大小为371bp，与理论设计片段一致。

702条多态性条带（2个SSR标记、一个信息素受体基因片段、225个SRAP标记位点、474个TRAP标记）和两个交配型位点，运用Joinmap作图软件进行数据分析，构建了一个包含有581个标记和位点，11个连锁群的香菇高密度分子遗传连锁图谱。图谱总长度为963.2 cM，连锁群的大小在21cM~151 cM之间，平均大小为87.6cM；标记间的距离在0~20.45 cM之间，标记间的平均距离为1.70 cM；两个交配型因子MAT-A、MAT-B分别定位在连锁群LG 3和LG 10上，大小分别为111cM和39cM；与MAT-A位点邻近的两个标记zip1-4-295和exg2-3-180距离MAT-A位点分别为6.6 cM和7.3 cM；与MAT-B位点邻近的两个标记zip1-4-390和exp1-4-190与MAT-B位点的距离分别为2.48 cM和6.55 cM；信息素受体基因片段“ste-1-371”（STE 3）定位在LG 10连锁群上，但并未与MAT-B位点紧密连锁，两标记间的遗传距离为6.93 cM，两标记间不连锁的原因可能与基因转座或者基因插入等有关；本实验所构建的香菇高密度分子遗传连锁图谱在10cM、5 cM和2 cM以内一个标记的覆盖度分别为99.9%、98.9%和83.7%。

平均图距为1.70 cM的香菇遗传连锁图谱是迄今为止的香菇遗传连锁图谱研究中图谱密度最高的，在5 cM遗传距离内一个标记的覆盖度高达98.9%，这与采用了高扩增效率的SRAP和TRAP标记直接相关，为将来的图位克隆、QTL数量性状定位、分子遗传育种等奠定了良好的基础。 

关键词：香菇，分子遗传连锁图谱，SSR，SRAP，TRAP，信息素受体基因，交配型因子

Abstract

In this study, the single spores L₆ and W₂₆, derived from two genetic-differentiated cultivated mushroom strains L205 and WuXiang NO.1 respectively, used as parents, the hybrid L₆×W₂₆ was cultivated from which the spore monokaryons were obtained. Their mating-types were identified accurately by the clamp connection appearing or not during hybrid pairing, nuclear transfer experiments and SOJ-OWE experiments. we constructed a genetic segregation population with 148 spores monokaryons as map population of Lentinula edodes.

    SSR, SRAP, TRAP, and a pheromone receptor gene were used to analyse the map population for the construction of a molecular genetic linkage map in Lentinula edodes. Two polymorphic primers were screened from 77 tested SSR primers. 25 out of 56 SRAP primer combinations were selected which had high polymorphic, PCR amplification results showed that the number of polymorphic bands was from 4 to 17 and the total polymorphic bands was 225, on tne average of 9 bands amplified by each primer combination. Through the analysis of 36 genes that associated with the fruit body development in Lentinula edodes, fixed primers for TRAP were designed, and 43 TRAP primer combinations were obtained. PCR amplification results showed that total of 474 polymorphic bands was obtained, on the average of 11.1 bands amplified by each TRAP primer combination. SRAP and TRAP molecular markers have shown high amplification efficiency, which was very beneficial to constructing high-density molecular genetic linkage map. According to the previous study about the Mat-B factor gene segments which also belonged to a pheromone receptor in our laboratory, we designed a specific primer and fortunately, a 371bp polymorphic DNA bands was obtained in our genetic segregation population, which aslo consistent with the theoretical size.

In molecule experiments, we got total of 702 polymorphic bands (two SSR markers, one pheromone receptor gene, 225 SRAP and 474 TRAP markers ), and two mating type sites（MAT-A and MAT-B, total of 706 polymorphic bands and sites were analysed by JoinMap^R 3.0 software for graphical representation of the genetic linkage map. JoinMap^R 3.0 generated a framework map consisting of 581 molecular marker and two mating type sties which distributed over 11 linkage groups. The 11 linkage groups was 963.2 cM with an average distance of 1.70 cM between two markers, the groups ranged in size from 21 cM ~151 cM, and the largest distance between markers was 20.45 cM. Two mating type factors were linked in LG 3 and LG 10 groups, which was 111 cM and 39 cM respectively. Two markers zip1-4-295 and exg2-3-180 were closely linkaged to the MAT-A stie to which the distance was 6.6 cM and 7.3 cM respectively (LG 3), two markers zip1-4-390 and exp1-4-190 were closely linkaged to MAT-B stie to which the distance was 2.48 cM and 6.55 cM respectively (LG 10). Pheromone receptor gene fragment “ste-1-371”（STE3） was linked in LG 10 too, but not closely with the MAT-B locus, the distance between two locus was 6.93 cM, which may be due to some transposition or gene insertion in B mating type system. The coverage of markers within 10 cM, 5 cM and 2 cM were 99.9%, 98.9% and 83.7% respectively.

In our experiments, we constructed a high-density molecular genetic linkage map in Lentinula edodes. With the shortesr average distance 1.70 cM in recent research. The coverage of markers within 5 cM was 98.9%, which owed to the high amplification efficiency SRAP and TRAP molecular markers, this also lay a good foundation for future positional cloning, quantitative trait position, and other modern molecular genetic breeding in Lentinula edodes.

Key words：Lentinula edodes， Molecular Genetic Linkage Map， SSR，SRAP，TRAP，Pheromone Receptor Gene，Mating-Type Factor

香菇分子遗传连锁图谱的构建.pdf