家蚕AFLP分子标记连锁图的构建

【作者】 孙德斌；

【导师】 鲁成； 朱勇；

【作者基本信息】 西南农业大学 ， 特种经济动物饲养， 2001， 硕士

【摘要】 家蚕是一种重要的经济昆虫，在我国的国民经济中具有非常重要的地位，家蚕分子连锁图谱的构建对促进家蚕分子生物学的研究具有重要意义。 扩增片段长度多态性（Amplified Fragment Length Polymorphism，AFLP）标记技术是一种新型的分子标记技术，兼具RAPD标记的多态性检测率高和RFLP标记稳定性好的特点，目前已广泛应用于多种生物的连锁图谱的构建和数量性状位点（QuantitativeTrait Locus， QTL）定位，具有良好的应用前景。 本文使用AFLP标记技术构建了一张高密度的家蚕遗传图谱。主要结果如下： （1）24组AFLP引物，共扩增出652个多态性位点，每组引物扩增出27.2条多态性带。本实验中可检测的片段大小在100-1000之间。 （2）297个有效位点中的197个构成了33个连锁群，占有效位点的66.33％，占总多态性位点的30.22％，还有100个位点不连锁，不能进入连锁图中，。 （3）33个连锁群中，最大的连锁群为第4连锁群，共有46个标记，最小的连锁群只有2个标记；最长的连锁群为890cM，最小的连锁群为1.1cM，位点间的最大图距为53.8cM，最小图距为0.55cM。 （4）33个连锁群覆盖基因组的总长度为2692.4cM，每条连锁群的平均长度为81.6cM，家蚕有28条染色体，本实验所构建的连锁群总数为33个，说明我们所构建的连锁群中有些是相互连锁的，只是目前还缺乏特定的标记将其整合在一起。 （5）在33个连锁群中平均图距小于10cM的连锁群有23个，图距在10-20cM之间的连锁群有8个，大于20cM的连锁群有2个，位点间的平均距离为9.1cM，每个连锁群上的位点数平均为6个。 （6）在197个构成连锁群的位点中，有91个位点间的图距小于10cM，占连锁位点的16.2％；有64个位点间的距离小于20cM大于10cM，二者共占197个位点的78.7％。 （7）分析表明，连锁群的长度与标记数目呈正相关，即当一个连锁群上的标记数目较多时，其长度也相应地增长；同时标记中有部分成簇的现象，估计是因为位点之间联系较紧密， 在育种过程中，借助高密度的分于连锁图，根据实际需要，追踪分子标记并结合其控制的表型性状进行选择，减少盲目性，达到提高育种效率的目的，加快育种进程。目前，分子标记育种在动植物中应用己得到了广泛重视，在西瓜、油菜等物种中己建立了分于标记辅助选择（MAS）的模型。家蚕中己定位的质量性状基因和数量性状基囚都较少，家蚕已构建的连锁图谱的密度也较低，主要是框架图的建立，本文所构建的是一张密度较高的分于连锁图，为后一步的 QTL基因定位和分子标记辅助选择（MAS）奠定基础。更多还原

【Abstract】 The silkworm. Born byv mon.. one of the important economic insects, plays an important role in our country. Constructing the linkage map of silkworm, Bombyx Mon., will lead to much great progress of molecular biology of Bombyx mon. Amplified Fragment Length Polymorphism, i.e. AFLP, is a novel molecular marker technique. having both the high polymorphism of RAPD and the reliability of RFLP. It has a good applied prospect considering the fact that it has been used to construct the linkage map and to map the quantitative trait locus(QTL) widely. AFLP was used to construct a high dense linkage map of Bombyx mon. in the paper. The following are the results: First, total 652 polymorphic loci were amplified using 24 primer combinations. On average, 27.2 polymorphic bands are amplified by one primer combination. The detected fragments are from I OObp to 1 000bp. Second. 33 linkage groups were constructed using 197 polymorphic loci, which was part of the 297 efficient polymorphic loci. The other doesn抰 link each other by our calculation and cant be used to construct the linkage map. The 197 loci account about 66.33% of the 297 efficient loci and 30.22% of the total polymorphic loci. Third. iii the 33 linkage groups. the biggest one is No.4. which has 46 markers: ~lhe smallest has only two markers. The longest linkage group is 89OcM,while the shortest is VII 1/ 1.lcM.The maximum distance between markers is 53.8cM, and te minimum is 0.55cM. Fourth, 33 linkage groups cover about 2692.4cM of the genomics of Bombyx mon. Average length of each linkage group is 81.6cM. In the paper 33 linkage groups are constructed, however Bombyx man, has only 28 chromosomes.It reflects that some of the linkage groups are linked together.But we can not link them together for the absence of the specific markers. Fifth, of the 33 linkage groups.23 has an average distance less than lOcM,and 8 is no less than 10cM and no more than 20cM. Two of them has an average distance more than 2OcN4. Average distance between loci is 9. lcM.And there are 6 markers in one linkage group on average. Sixth, there are 91 loci which map distance is less than lOcMgccounting 46.2% of the 197 loci that enter the linkage map; And the map distance of 64 loci is from 10cM to 20cM. The two account about 78.7% of the 197 loci. The last one, the results show that the length of linkage groi~~ has a positive correlation with the number of markers. That is to say, if there are more markers in a linkage group, the length it has will be longer. Meanwhile there are partial clustering markers. The reason would be that the markers are too close to have recombination. During the process of breeding, via the high dense molecular linkage map and considering the practical needs, ~ can follow the molecular markers and select the phenotype it controlls to reduce the blindness and to improve the breeding efficiency and to accelerate the breeding. Now breeding via marker-assisited selec~on (MAS) has caught the attention. Models of MAS have been constructed in the Brassica napus and watermelon, etc. The qualitative trait locus and quantitative trait locus mapped in 8 cm byx mon. have not so much. The linkage maps of Bombyx man, are mainly the construation of the scaffold. What w更多还原