Molecular Detection Of Geographic Populations And Interspecies Genetic Relationships Of Several Important Bean Weevils (Coleoptera Bruchidae)

The species of Bruchidae have economic significance in stored-product pests. Most of them feed on and damage the seeds of legume by means of boring seeds at the phase of larva. The four major detrimental bruchid species widely distributed in China are Bruchus rufimanus Behoman, B. pisorum (Linnaeus), Callosobruchus chinensis and C. maculatus (Fabricius). Thereinto, C. maculatus (Fabricius) is a kind of pest to agricultural plant quarantined and an A 3 quarantine pest in China. So, it is of great significance to study these pests.The sequences of mitochondrial DNA (mtDNA) cytochrome c oxidase I gene (COI), Cytochrome b gene (Cytb), and the second internal transcribed spacer of nuclear ribosomal DNA (rDNA ITS2) of different geo-populations of the four bruchid species were sequenced. Combined with the correlative COI and Cytb gene sequences of bean weevils downloaded from the GenBank, they were used to analyse the phylogenetic relationship among different populations and species. The main results were as follows:1. Through the study, we screened out a good method for extracting the genomic DNA of the large-scale bruchid species under the laboratory conditions: the CTAB method. Three methods of preserving samples for extracting genomic DNA by CTAB method, including preserving samples in alcohol soaking, drying samples in oven and drying samples in shades, were compared. The results showed that the first two methods had better effects in extracting genomic DNA by CTAB method while the last method had not.2. The sequence analysis of several bruchid species showed the content of Cytb and COI gene has the bias of A+T for 69.8% and 67.9% respectively, and was most pronounced at the third sites of codons for 85.0% and 85.2% respectively. Among the nucleotides substitution of Cytb and COI gene, transition is obviously more than transversion. Transversion has the bias of A/T, and always happen at the 3rd sites of codons. The high content of A+T at the 3rd sites of codons is correlated with the variance of amion acid. As a typical protein-coding gene, the second sites were least variable while the first sites were more variable and the third sites were the most variable. The topological structure of the molecular phylogenetic trees of Cytb and COI gene constructed by two methods were basically identical. The species sampled divided into two clades and the species in the same genus clustered together in both trees by the two methods.3. Analysis of the haplotype and its distribution, based on the mtDNA Cytb and COI gene, demonstrated low genetic variation among the geo-populations of the four bruchids for few exclusive haplotypes existed. The analysis of gene flow for both gene sequences showed that both gene flow and genetic differences existed among the geo-populations sampled. Thereinto, we observed very few base variations among the geo-populations of C. chinensis. The differences of the values of Nm between COI and Cytb gene of B. pisorum demonstrated that the molecular evolutionary rate of Cytb gene is faster than that of COI gene. COI and Cytb gene are good molecular markers for studying the geo-populations of B. rufimanus, B. pisorum, and C. maculatus.4. Analysis of rDNA ITS2 gene of different geo-populations of the four bruchids showed that very few variations existed in the geo-populations of C. chinensis and C. maculatus. For our delight, we detected the relations between molecular variations and geographical distribution of the populations of B. rufimanus. So we considered rDNA ITS2 a good molecular marker for studying the geo-populations of B. rufimanus.