Study On Molecular Marker Analysis Between Color-different Individuals Of The Blue Swimming Crab (Portunus Trituberculatus)

There have been some crab individuals with purple carapaces and appendages newly discovered in the wild captured blue swimming crab Portunus trituberculatus in recent years, while the normal carapace color of the blue swimming crab is tea-green. Given the morphological characters of purple crabs identical with that of the tea-green ones except the body color, it is expected that the purple crabs and the tea-green ones were from the same species. In order to identify the phylogenetic relationships between them at the molecular level, we amplified and compared two conserved mitochondrial gene fragments of purple and tea-green crabs through PCR-SSCP and DNA sequencing analyses. The results showed that there was no distinct variation of DNA bands of both the COI and 16srRNA genes detected in polyacrylamide gels through SSCP analysis between purple and tea-green crab samples, and the following sequence analysis of color-different crab individuals presented 99.87% and 99.88% nucleotide sequence identity of the COI and 16srRNA genes respectively. These indicated that there was no species or subspecies differentiation between purple and tea-green crabs, meaning that they belonged to the same species, Portunus trituberculatus. And some crabs with other carapace colors were found in their natural habitats. It was expected that the carapace color may be regulated by multiple genes located on the chromosomes.Then the phylogenetic relationships and genetic differences were determined between purple and tea-green crab individuals of P. trituberculatus by RAPD technique. 40 color-different crab individuals were evaluated using 80 RAPD primers. 30 primers were selected using bulk segregation analysis (BSA) and RAPD-PCR analysis. The results showed that the ratio of polymorphic loci was 61.29%, Nei's gene diversity was 0.3756, Shannon's information index was 0.5545 among purple crab individuals; and 62.44% the ratio of polymorphic loci, 0.3451 Nei's gene diversity, 0.5099 Shannon's information index among tea-green crab individuals. It was clear that the genetic diversity between purple and tea-green crabs was similar, which indicated close phylogenetic relationships between the purple and tea-green crabs. In addition, two primers (SBS A09 and SBS G16) got specific DNA bands by comparing forty individuals of color-different crab individuals through RAPD-PCR analysis and the following electrophoresis. One specific DNA band (750~1000 bp) was discovered using the primer SBS A09, with 22.22% and 75.00% polymorphism within purple and tea-green crab individuals respectively; and one DNA band (1000~2000 bp) from the primer SBS G16 showed 35.00% and 75.00% polymorphism within purple and tea-green crab individuals respectively. And the SSR primer MIH-SSR also got specific DNA bands within color-different crab individuals. It indicated that the body color transmision was affected by the heredity of some quantitative characters. These indicated close phylogenetic relationships between purple and tea-green crabs.