Project	Silkworm Genome Analysis Survey and Analysis Microsatellites Construction of Genetic and Physical Maps Z-chromosome mapping
Investigators PhD Scholars Technical Staff	Sunil Archak A Sobhan Babu, M Muthulakshmi
Funding Agency	Department of Biotechnology, Govt of India, New Delhi

Genetic analysis using molecular markers

B. mori has been used as a model system for formal genetic studies since the discovery of Mendelian inheritance at the turn of the century because of its large size, ease of rearing in the laboratory and economic importance. The well-developed genetics of this species includes more than 400 mutations, which have been mapped to 28 linkage groups or chromosomes. In addition, the existence of hundreds of geographic races and genetically improved strains used for silk production which differ not only in Mendelian traits but also in quantitative traits such as body size, feeding duration, thermal tolerance and disease resistance. These traits remain to be subjected to systematic analysis using modern genetic tools.

Molecular markers have several advantages over the traditional phenotypic markers that are available in silkworm genetic stocks. They are unaffected by environment, detectable in all stages of development and ubiquitous in number covering the entire genome. Development of molecular markers is important in silkworm for construction of linkage map, fingerprinting of strains for breeding, and marker-assisted selection. Many techniques have been employed in silkworm to reveal the genome-wide polymorphism. These are Restriction Fragment Length Polymorphisms (RFLP), Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR) and Inter-Simple Sequence repeats (ISSR) anchored PCR. Three criteria define ideal molecular markers: (i) they should be highly polymorphic, (ii) they should be easy to identify and type, and (iii) they should be amenable to automation.

Microsatellite based genetic markers, which are distributed across genomes of most of the eukaryotes, fulfill these criteria. Microsatellites also known as Simple Sequence Repeats (SSRs) are short stretches of DNA which consist of an array of simple tandemly repeated mono, di-, tri-, tetra-, penta or hexanucleotide repeats such as (A)_n, (CA)_n, (GA)_n, (GTA)_n, (ATT)_n, (GATA)_n, (ATTTT)_n, (ACGTCG)_n. They are ubiquitous in prokaryotic and eukaryotic genomes and are randomly distributed, both in protein coding and non-coding regions. A unique oligonucleotide on each side of the repeat region is chosen for the production of a primer pair for each of the microsatellite loci. PCR products of different lengths can be amplified using primers flanking the variable microsatellite region. Allelic variations among individuals are based mostly on differences in the number of tandem repeats in a microsatellite array providing a ready source of polymorphism. Thus, the only way in which alleles can be distinguished is by measuring the total length of the microsatellite allele. This is most readily accomplished through PCR amplification of microsatellite itself along with a short stretch of defined flanking sequences on both sides with designed primer pairs for each locus. Details are given in protocols. Cost effectiveness of the assay is achieved by combining two or more loci for simultaneous analysis through multiplex PCR. By using the fluorescent-labelled primers and automated sequencers, the entire process of microsatellite analysis can be automated. Due to these advantages, microsatellites have been used to construct linkage maps. In addition to their clear utility for practical applications, information about the distribution and variability of microsatellite sequences in the genome of a given species can help elucidate its genetic history from the standpoint of evolution and artificial selection. We undertook to survey, characterise, catalogue and analyse the microsatellite motifs that are distributed in the silkworm genome.

Survey and Analysis of Microsatellites in the Silkworm, Bombyx mori

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori(Prasad et al. 2005). We mined microsatellites of o315 bases of mononucleotide repeats and o35 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2b B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.

Microsatellite mapping initiative

Lepidoptera has received little attention in terms of genetics and genetic maps despite immense consequences in terms of economy and ecology. In B. mori, all the linkage maps except the RFLP map are based on dominant markers.

In B. mori, achiasmatic oogenesis results in absolute linkage in females. In males, linkage depends upon the crossover events occurring during spermatogenesis. This biphasic linkage behavior typical to lepidopterans aids mapping by sequential approach. F₁ individuals from a cross NB₄D₂ X Nistari were backcrossed to Nistari (the recurrent parent) to generate the mapping population. In the first step, F₁ females were backcrossed with Nistari males [backcross (BC)-I] to identify the linkage groups. In a second backcross, F₁ males were mated with Nistari females, generating recombinant progeny (BC-II) to obtain the order of the markers within each linkage group. Forty individuals from BC-I and 60 individuals from BC-II were genotyped along with the parents and F₁. The data matrix was input into MAPMAKER version 3.0. Two-point linkage was determined at LOD=3.0 and multipoint analysis based on Kosambi function yielded the order of the markers in each linkage group.

Genetic linkage map

A total of 46 polymorphic SSR markers were detected in the study. Among them, 10 markers exhibited null alleles in the male parent, Nistari, and segregated as dominant markers. Four markers showed Z-specific inheritance, which was confirmed by homology search in Z chr-BACs. Four markers exhibited monomorphism among BC progeny and were thus excluded. We carried out ² analyses to carry forward only those markers fitting the expected 1:1 ratio in the BC-II population. Five of the markers showed segregation distortion at P=0.05. Finally, 37 markers were employed for the construction of the SSR linkage map. Eight linkage groups including one Z-group were separated (Figure 4). They ranged from 58.7 cM (group 2) to 6.6 cM (group 5). Eight markers remained unlinked. This effort is a part of our ongoing program to develop a dense genetic linkage map of silkworm using fluorescent inter-simple sequence repeat (FISSR) markers and microsatellite markers. These mapped codominant markers will be useful in anchoring a genetic map on a physical map.

Z-chromosome Mapping

In the silkworm, female is the heterogametic (ZW) sex and the male is homogametic (ZZ). The female heterogamety is a typical situation in the insect order Lepidoptera. Although the W chromosome in silkworm is strongly female determining, no W-linked gene for a morphological character has been found on it. The Z chromosome carries important traits of economic value as well as genes for various phenotypic traits, but, based on its relative size, only 2% of molecular information based on its relative size is known. Studies conducted so far indicate that the Z-linked genes are not dosage compensated. In the present study, we constructed a genetic map of RAPD, SSR and FISSR markers for the Z chromosome using a backcross mapping population. Sixteen Z-linked markers were identified, characterized, and mapped using od, a recessive trait for translucent skin, as an anchor marker, yielding a total recombination map of 334.5 cM. The linkage distances obtained suggested that the markers were distributed throughout the Z chromosome. We also identified four RAPD and four SSR markers that were linked to W chromosome. The proposed mapping approach should be useful to identify and map sex-linked traits in the silkworm.

Future line of work

Public release of silkworm sequences from Japanese and Chinese researchers has facilitated the anchoring of genetic markers onto the actual chromosomal locations as the next logical step. In our lab, efforts are on for identifying the contigs/scaffolds carrying the mapped loci as well as genes. This contributes to the identification of additional markers to generate a high-density genetic map as well as to position the known genes on the linkage groups.