Construction Of Genetic Map And Evolutionary Analysis Of Repetitive Elements In Zhikong Scallop (Chlamys Farreri)

1. Construction of AFLP linkage map for C. farreriThe linkage maps of C. farreri were constructed with AFLP markers generated from 21 primer combinations. The polymorphism level of F1 progeny is 62.94%. Of the 783 polymorphic markers detected, 317 segregated in 1:1 ratios and 190 in 3:1 ratios, whereas the other 276 showed segregation distortion. Linkage analysis used the program MAPMAKER/EXP at LOD=2.5 andθ=0.30. The male map containing 94 markers in 19 linkage groups, was 1511.4 cM in total length and 66.56% in genome coverage. The female map containing 97 markers in 20 linkage groups, was 1610.2 cM in total length and 66.05% in genome coverage. The distribution of AFLP markers is relatively even in chromosomes of male and female map.2. Dynamics of concerted evolution of rDNA family in natural population of C. farrerirDNA family, as one of tandemly repeated families, exhibits the general pattern of concerted evolution. In order to study the homogenization process of rDNA family in the population of Zhikong scallop, this study focused on the insertion/deletion (indel) polymorphism of the internal transcribed spacer (ITS) sequence. Under the optimized condition, ITS heteroduplexes caused by indels could be well identified in denaturing high-performance liquid chromatography (DHPLC) analysis. Using this technique, ITS indel polymorphism in 40 individuals from natural population was investigated. Surprisingly, only 7.5 percent of individuals were homogeneous in ITS constitution, while the others (92.5%) were heterozygous. Based on peak types appeared in DHPLC analysis, seven representative individuals were chosen and ITS polymorphism within an individual was then investigated using sequencing method. Further, ITS indel polymorphism within single sperm was investigated in more individuals. Based on these results, a new model was proposed for concerted evolution of rDNA family in natural population of C. farreri. Different from previous models, 2-peak individuals with the highest proportion (62.5%) in natural population can be seen as being in a steady and balanced state maintained by rapid intrachromosomal recombination. 1-peak individuals can be seen as being in the state of full homogenization. They occupy the low proportion (7.5%) in natural population, which suggests that interchromosomal recombination is slower than intrachromosomal recombination. 3-peak individuals can be seen as being in the state of homogenizing a variant which may be generated by mutation or interchromosomal recombination. They occupy relatively high proportion in natural population, which implies that the rate of interchromosomal recombination may be not rare.3. Rapid concerted evolution via biased gene conversion occurs in the rDNA family of a hybrid scallop (Chlamys farreri♀×Argopecten irradians♂) at the early developmental stageThis study also focused on the variation of the parental ITSs in a hybrid scallop (Chlamys farreri♀×Argopecten irradians♂) at the early developmental stage. The main results obtained in this study are: (i) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that the quantity of paternal ITS in the hybrid had decreased gradually accompanying the development of the hybrid and even nearly disappeared at the 14th day after fertilization; (ii) fluorescence in situ hybridization (FISH) analysis in the hybrid revealed that maternal ITS had been found in the paternal ITS-bearing chromosomes, while paternal ITS had not been found in the maternal ITS-bearing chromosome; (iii) with PCR-RFLP technique, six recombinant variants were found in the hybrid and identification of short recombination regions implied that site-specific recombination may be involved in the biased gene conversion. Based on these results, it was concluded that rapid concerted evolution via biased gene conversion had occurred in the rDNA family of the hybrid at the early developmental stage.4. Analysis of the secondary structure of ITS1 in Pectinidae: implications for phylogenetic reconstruction and structural evolutionIt is at present difficult to accurately position gaps in sequence alignment and to determine substructural homology in structure alignment when reconstructing phylogenies based on highly divergent sequences. Therefore, this study developed a new strategy for inferring phylogenies based on highly divergent sequences. In this new strategy, the whole secondary structure presented as a string in bracket notation is used as phylogenetic characters to infer phylogenetic relationships. It is no longer necessary to decompose the secondary structure into homologous substructural components. In this study, reliable phylogenetic relationships of eight species in Pectinidae were inferred from the structure alignment, but not from sequence alignment, even with the aid of structural information. The results suggest that this new strategy should be useful for inferring phylogenetic relationships based on highly divergent sequences. Moreover, the structural evolution of ITS1 in Pectinidae was also investigated. The whole ITS1 structure could be divided into 4 structural domains. Compensatory changes were found in all 4 structural domains. Structural motifs in these domains were identified further. These motifs, especially those in D2 and D3, may have important functions in the maturation of rRNAs.5. Cloning and characterization of two novel elements (CFG1 and PYG1) of Mag lineage of Ty3/Gypsy retrotransposons from Zhikong scallop (Chlamys farreri) and Japanese scallop (Patinopecten yessoensis)Two novel elements (CFG1 and PYG1) which belong to Mag lineage of Ty3/Gypsy retrotransposons were cloned from Zhikong scallop (Chlamys farreri) and Japanese scallop (Patinopecten yessoensis). The total length of CFG1 element is 4826 bp, including 5'-LTR (192 bp), entire ORF (4047 bp) and 3'-LTR (189 bp). The entire ORFs of both CFG1 and PYG1 elements are composed of 1348 aa and do not have any frameshifts. Their closest relative is Jule element from the poeciliid fish (Xiphophorus maculatus). On average, the diploid genome of C. farreri contains approximate 84 copies of CFG1 elements. For CFG1, PYG1 and other elements of Mag lineage of Ty3/Gypsy group, this study summarized their major features and compared their 3D structures of RT domains. The mRNA expression of CFG1 element in larvae increases gradually before gastrulae stage and decreases gradually after gastrulae stage, whereas expressions in adductor muscle and digestive gland are relatively lower than those in other tissues of adults. Overall, the mRNA expression of CFG1 element in the early larvae is significantly higher than those in adult tissues. The promoter and partial GAG domain of CFG1 element are unmethylated, however, the partial RT domain is highly methylated. Therefore, it seems that CFG1 expression may be controlled by post-transcriptional suppression rather than methylation.