Linkage analysis of the visible mutations Sel and Xan of Bombyx mori (Lepidoptera: Bombycidae) using SSR markers

Xuexia Miao,Marian R Goldsmith,Cheng Lu,Fangyin Dai,Yongping Huang,Muwang Li

doi:10.14411/eje.2007.081

Abstract

Wild type silkworm larvae have opaque white skin, whereas the mutants Sel (Sepialumazine) and Xan (Xanthous) are yellow-skinned. Previous genetic analysis indicated that Sel and Xan are on established linkage groups 24 (0.0) and 27 (0.0), respectively. However, in constructing a molecular linkage map using simple sequence repeat (SSR) loci, we found that the two mutations were linked. To confirm this finding, we developed a set of SSR markers and used them to score reciprocal backcross populations. Taking advantage of the lack of crossing-over in female silkworms, we found that the progeny of backcrosses between F1 females and males of the parental strains (BC1F) of the two visible mutations had the same inheritance patterns linked to the same SSR markers. This indicated that the two visible mutations belonged to the same chromosome. To confirm this finding, we tested for independent assortment by crossing Sel and Xan marker strains with each other to obtain F1 and F2 populations. Absence of the expected wild type class among 5000 F2 progeny indicated that the two visible mutations were located on the same linkage group. We carried out recombination analysis for each mutation by scoring 190 progeny of backcrosses between F1 males and parental females (BC1M) and constructed a linkage map for each strain. The results indicated that the Sel gene was 12 cM from SSR marker S2404, and the Xan gene was 7.03 cM from SSR marker S2407. To construct a combined SSR map and to avoid having to discriminate the two similar dominant mutations in heterozygotes, we carried out recombination analysis by scoring recessive wild type segregants of F2 populations for each mutation. The results showed that the Sel and Xan genes were 13 cM and 13.7 cM from the S2404 marker, respectively, consistent with the possibility that they are alleles of the same locus, which we provisionally assigned to SSR linkage group 24. We also used the F2 recessive populations to construct two linkage groups for the Sel and Xan genes.

Highlights

The silkworm, Bombyx mori L., is one of the best characterized insects genetically
Among 500 clones we found 300 robust simple sequence repeat (SSR) loci that could amplify single bands in K07 and K14 which could be used for mapping relative to Dazao, a wild type strain used to construct a first generation genome-wide SSR linkage map (Miao et al, 2005)
To find which SSR markers were linked to Sel or Xan, we screened the genotypes of 20 BC1F individuals using Dazao as a standard wild type to determine which SSR markers (e.g., M) showed a heterozygous (M1M2) or homozygous (M2M2) pattern among the yellow-skinned (Sel+Sel or Xan+Xan) or normal skin (+Sel+Sel or +Xan +Xan) individuals, respectively

Summary

Introduction

At least 400 visible and biochemical mutations have been identified and more than 200 of them have been placed on linkage maps (Doira, 1992; Goldsmith, 1995). These silkworm mutants are invaluable biological resources for basic and applied science. Yellow-skinned mutants are important for linkage analysis as these genes are the only available markers in a few established linkage groups (ELGs). Sel larvae are reported to accumulate sepialumazine (7,8-dihydro -6-lactyllumazine) as a by product of pteridine metabolism, accompanied by high levels of sepiapterin deaminase activity (Mazda, 1980); the Xan mutation, possibly introgressed from the silkworm’s wild ancestor, B. mandarina, has not been characterized biochemically. Sel and Xan are usually selected as visible markers for linkage groups 24 and 27, respectively (Fujii et al, 1998)

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Conclusion