The Construction Of A Genetic Linkage Map Of Scylla Paramamosain Based On Microsatellite And AFLP Markers

Scylla paramamosain (S.paramamosain), an importantly commercial species ofcoastal marine mud crab, widely distributed in the southeastern coastal regions ofChina. Due to its economic and nutritional value, the mud crab S.paramamosainincreasingly becomes a significant fishery resource and the scale of aquacutureprogresively increases in Chnia. In the meantime, biotechnology has been used toenhance the quality and quantity of the S.paramamosain, particularly the genomicinformation analysis technique and molecular marker have been used to researchgenetic diversity and assisted breeding. This paper focused on developingpolymorphic microsatellite loci, the genetic diversity of G1family of the mud craband genetic linkage map of S.paramamosain to provide theoretical basis for artificialbreeding and cultivation. The detailed works and results are as follows:1. Based on454data of transcriptome high-throughput sequencing ofS.paramamosain,90pairs of primers were designed by Primer Premier5.0and35polymorphic microsatellite loci were proved to be polymorphic. Meanwhile, therewere197alleles detected in a population of32Qinglan S.paramamosains and thenumber of alleles per locus ranged from2to12with a average value of5.6. Inaddition, the PIC, observed heterozygosity (Ho) and expected heterozygosity(He)varied from0.201to0.890,0.125to1.000and0.490to0.915with an average valueof0.651,0.704and0.736per locus, respectively. Finally, there was only one locusdeviated from Hardy-Weinberg equilibrium in term of Bonferroni correction, whichindicated that these loci was reliable and credible to analyse the genetic diversity ofS.paramamosain.2. It was investigated that the segregating pattern of AFLP (amplified fragmentlength polymorphism) and the genetic diversity of G1family of the mud crab(S.paramamosain) by32selective primer combinations. A total of1128loci were produced, of which376loci were polymorphic (11.8%), and752loci weremonomorphic. Among376segregating loci,229loci (60.9%) segregated inMendelian ratio while147loci (39.1%) segregated in distortion estimated byChi-square test (P=0.05)．There were259(68.9%) and117(31.1%) loci segregatingin the ratios of1:1and3:1, respectively. The number of loci segregated in Mendelianratios were155(59.8%) for1:1and74(63.3%) for3:1, respectively. Effectivenumber of alleles, Shannon genetic diversity index and Nei’s gene diversity indexwere between1.13to1.73, between0.10to0.56, and between0.07to0.40,respectively. This study will be helpful for constructing a genetic linkage map andmolecular marker-assisted selection in this important crab species.3. An initial genetic linkage map of S.paramamosain was constructed based onSSR and AFLP markers. The linkage map totally contained189markers that groupedin57linkage groups, including33SSR markers and156AFLP markers. The numberof markers per group ranged from2to8covering1208.6cM with an averageresolution of15.5cM. And the length of LGs varied from6.3cM to140.3cM. Inaddition, the genome lengths were3821.1cM calculated by Gel and3907cM byGe2,respectively. Therefore, the estimated average genome length (Ge) was3864cM witha coverage of30.1%. Besides, the genome coverage of linkage map of theS.paramamosain tongue was approximately at53.1%in term of the ratio between Goaand Ge. According to the above data, It is useful for quantify trait loci, molecularmarker-assisted breeding and construct middle or high density linkage map of themud crab (S.paramamosain).