Polymorphic EST–SSR markers and their mode of inheritance in Fenneropenaeus chinensis

Abstract

229 SSRs (simple sequence repeats) were identified among 10,443 ESTs (expressed sequence tags) of Chinese shrimp (Fenneropenaeus chinensis). The average density of SSRs was one SSR per 19.1 kb of EST sequence screened. The dinucleotide repeats appeared to be the most abundant SSRs detected. Nine EST–SSR markers were detected polymorphisms of the thirty SSR primer pairs derived from F. chinensis ESTs. The number of alleles per locus ranged from 5 to 15, with an average of 9.1 alleles per locus. The observed heterozygosity of nine loci ranged from 0.47 to 0.87. These loci were used successfully for pedigree analysis in three families of Fenneropenaeus chinensis. Two of the nine microsatellite loci showed the existence of null alleles. Assuming the existence of null alleles at Fc07 and Fc14 loci, the allelic inheritance mode of the EST–SSR DNA markers (Fc04, Fc06, Fc07, Fc10, Fc14, Fc18, Fc22, Fc24, and Fc27) was consistent with Mendelian segregation.

Introduction

Chinese shrimp (Fenneropenaeus chinensis) is one of the most popular cultured species in China. As for many marine species, the Chinese shrimp culture industry still relies largely on wild broodstock. With the depletion of the wild prawn broodstrock and outbreaks of viral and bacterial disease, genetic improvement programs are urgently needed for the sustainability of the industry. One of the difficulties in implementing breeding programs for aquacultural stocks is maintaining pedigree information (Norris et al., 2000). Microsatellite markers have been proposed as a tool for pedigree identification of mass-reared aquacultural populations (Garcia de Leon et al., 1998, Volckaert and Hellemans, 1999).

Microsatellite markers (also known as simple sequence repeats or SSRs) are short tandem nucleotide repeats in nuclear DNA, which exhibit high polymorphism and are inherited co-dominantly in a Mendelian fashion. They are sensitive and useful for studying genetic variation, especially in closely related populations (Wright and Bentzen, 1994). Microsatellites have been used as genetic markers in aquatic animals such as European sea bass (Garcia de Leon et al., 1995), Atlantic salmon (Slettan et al., 1993) and European flat oyster (Naciri et al., 1995). They also have been widely used as genetic markers for analysis of the genetic diversity of wild and cultured penaeid species and the population structure in Litopenaeus vannamei (Wolfus et al., 1997), Penaeus japonicus (Moore et al., 1999), Penaeus stylirostris (Bierne et al., 2000) and Penaeus monodon (Tassanakajon et al., 1998, Xu et al., 1999, Brooker et al., 2000, Pongsomboon et al., 2000). Recently, parentage and kinship determination using microsatellite marker have been reported in several penaeid species, e.g., P. monodon (Suwit et al., 2003) and P. japonicus (Sugaya et al., 2002, Jerry et al., 2004). However, few SSR markers for F. chinensis have been reported. The 31 microsatellites of the Chinese shrimp have been isolated through PCR screening of small size fractionated libraries of P. chinensis by our laboratory (Xu et al., 2001).

In recently years, significant information on Expressed Sequence Tags (ESTs) from F. chinensis has accumulated with 10,443 cDNA sequences established by our laboratory (Xiang et al., 2002). ESTs are being put to a variety of uses, including the development of molecular markers such as EST–SSRs and EST–SNPs (Holton et al., 2002, Kantety et al., 2002, Somers et al., 2003). The EST databases provide a valuable resource for the development of SSR markers, which are time and cost saving. EST–SSRs as rich resources of type I markers associated with known function genes can be more useful for comparative gene mapping (Liu et al., 1999), and also facilitate physical mapping.

In this paper, we report the characterization and cross-species transferability of SSRs derived from F. chinensis ESTs, describe the inheritance mode of these microsatellite loci in the three F. chinensis families, and examine the feasibility of these markers for detecting pedigrees in Chinese shrimp.