Xiang H, Li M, Yang F, Guo Q, Zhan S, Lin H, Miao X, Huang Y
Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Center of Insect Science, The Chinese Academy of Sciences, Shanghai, P.R. China.
Heredity (Edinb). 2008 May;100(5):533-40. doi: 10.1038/hdy.2008.10. Epub 2008 Mar 26.
The silkworm homeotic mutant E(kp) has a pair of rudimentary abdominal legs, called prolegs, in its A2 segment. This phenotype is caused by a single dominant mutation at the E(kp)-1 locus, which was previously mapped to chromosome 6. To explore the possible association of Hox genes with proleg development in the silkworm, a map-based cloning strategy was used to isolate the E(kp)-1 locus. Five E(kp)-1-linked simple sequence repeat markers on chromosome 6 were used to generate a low-resolution map with a total genetic distance of 39.5 cM. Four additional cleaved amplified polymorphic sequence markers were developed based on the initial map. The closest marker to E(kp)-1 was at a genetic distance of 2.7 cM. A high-resolution genetic map was constructed using nine BC1 segregating populations consisting of 2396 individuals. Recombination suppression was observed in the vicinity of E(kp)-1. Four molecular markers were tightly linked to E(kp)-1, and three were clustered with it. These markers were used to screen a BAC library. A single bacterial artificial chromosome (BAC) clone spanning the E(kp)-1 locus was identified, and E(kp)-1 was delimited to a region less than 220 kb long that included the Hox gene abdominal-A and a non-coding locus, iab-4. These results provide essential information for the isolation of this locus, which may shed light on the mechanism of proleg development in the silkworm and possibly in Lepidoptera.
家蚕同源异型突变体E(kp)在其A2节段有一对退化的腹足，称为原足。这种表型是由E(kp)-1位点的单个显性突变引起的，该位点先前已定位到6号染色体上。为了探究Hox基因与家蚕原足发育之间可能的关联，采用了基于图谱的克隆策略来分离E(kp)-1位点。利用6号染色体上5个与E(kp)-1连锁的简单序列重复标记构建了一个总遗传距离为39.5 cM的低分辨率图谱。基于初始图谱又开发了另外4个酶切扩增多态性序列标记。与E(kp)-1最接近的标记的遗传距离为2.7 cM。使用由2396个个体组成的9个BC1分离群体构建了一个高分辨率遗传图谱。在E(kp)-1附近观察到重组抑制现象。4个分子标记与E(kp)-1紧密连锁，其中3个与之聚集在一起。这些标记用于筛选BAC文库。鉴定出一个跨越E(kp)-1位点的细菌人工染色体(BAC)克隆，并将E(kp)-1限定在一个长度小于220 kb的区域，该区域包括Hox基因腹-A和一个非编码位点iab-4。这些结果为该位点的分离提供了重要信息，可能有助于揭示家蚕以及可能在鳞翅目中原足发育的机制。
