Development And Application Of Molecular Markers In Pacific Oyster (Crassostrea Gigas)

1. Development and characterization of 68 EST-SSR markers in the Pacific Oyster, Crassostrea gigasA set of 68 new EST-SSRs for C. gigas were developed from ESTs database. The average numbers of alleles was 4.9 alleles per locus, observed heterozygosities varied from 0.0333 to 0.9667, while expected heterozygosities varied from 0.0655 to 0.9149. Twenty-two of the loci deviated significantly from HWE after a Bonferroni correction. Significant linkage disequilibrium was detected between two pairwise combinations of four loci (CgF114 and CgF147, CgL40 and CgL51) after correction. Fifteen ESTs had homology to known genes and predicted proteins from other organisms. Cross-species amplification was examined in two other Crassostrea species, including C. angulata and C. ariakensis. Fifty primer sets amplified successfully at least one species, with 40 individuals of C. angulata and 45 individuals of C. ariakensis. Mendelian segregations were tested in a full-sib family, 36 loci were monomorphic (AA×AA genotype), the remaining 32 polymorphic markers were segregated and 11 loci showed unexpected progeny phenotypes that were best explained by null alleles (16.2%), thus 32.4% of the 68 loci significantly deviating from HWE might result from the existence of null alleles. The frequency of null alleles is lower compared to genomic SSRs in the C. gigas. Although some of the EST-SSRs have low levels of polymorphism, the 68 new SSRs should be useful for genome mapping and population genetics studies.2. Characterization of 20 single nucleotide polymorphism markers in the Pacific oyster, C. gigasWe report the first set of polymorphic SNP markers by tetra-primer ARMS-PCR method. A total of 20 SNP markers were identified from EST database, and sequences containing SNPs were annotated using BLAST software, and sequence homology was accepted based on a cut-off E-value of 1.0×10-6. All loci had 2 alleles and the minor allele frequency ranged from 0.033 to 0.467 in WH, and from 0.033 to 0.367 in LYG Polymorphism was evaluated in the wild Weihai population (WH) and cultured Lianyungang population (LYG). The observed and expected heterozygosities in WH ranged from 0.067 to 0.800 and from 0.064 to 0.500, while those in LYG ranged from 0.067 to 0.733 and from 0.064 to 0.464. LD was not found, but significant deviation from HWE was observed at the same two loci in both populations after Bonferroni correction (P<0.05). The development of polymorphic SNP markers will be useful for mapping studies, population genetics and marker-assisted breeding in C. gigas.3. Discovery and evaluation of 12 exon-primed intron-crossing (EPIC) PCR markers for the Pacific oyster (C.gigas)Using the gene-sequence alignment between Crassostrea gigas cDNAs from public databases and the genomic sequences of Ciona intestinalis and Danio rerio homologs, we developed exon-primed, intron-crossing polymerase chain reaction (EPIC-PCR) markers of C. gigas. Among the satisfactory amplified systems,12 showed length polymorphism with 3-13 alleles with an average of 7.3 alleles per locus. The observed and expected heterozygosities ranged from 0.067 to 0.633 and from 0.098 to 0.853, respectively. Six of the 12 EPIC-PCR markers significantly deviated from the HWE after correction for multiple tests, and all of them showed heterozygote deficiency (P<0.05/12). Sequencing of multiple individuals across two randomly selected loci (Cglns and CgEF) showed that allele length polymorphisms were usually from random insertion/deletion events. The transferability of these EPIC markers was tested on four other Crassostrea species including C. hongkongensis, C. ariakensis, C. angulata and C. sikamea, nine loci gave successful amplifications in the species. One controlled cross demonstrated Mendelian inheritance patterns for these EPIC markers and absence of null alleles. The results obtained in this study indicated that the EPIC markers developed from C. gigas genes may be uniquely useful in population genetic studies, phylogenetic analysis, and in anchoring linkage maps across Crassostrea.4. Comparative assessment of genomic SSR, EST-SSR, and SNP markers for evaluation of genetic diversity and conservation of genetic resources using wild, and cultivated Pacific oysters (C.gigas)A set of 8 genomic SSRs,10 EST-SSRs and 10 EST-derived SNP markers were analyzed on 2 wild and 4 cultured populations to access their applicability for population genetics. Genomic SSR markers were found the most polymorphic with an average PIC value of 0.8725, higher than EST-SSRs (0.7020) and SNP (0.4482). The observed and expected heterozygosities in genomic SSR ranged from 0.280 to 0.880 and from 0.762 to 0.933, while those in EST-SSR ranged from 0.200 to 0.900 and from 0.580 to 0.849, in SNP ranged from 0.067 to 0.833 and from 0.066 to 0.500, respectively. The average Fis value in genomic SSR, EST-SSR and SNP was 0.280, 0.314,0.133, respectively. Phylogenetic trees were constructed the using NJ method by the 3 types of markers according to the genetic distances (Dc) between populations. Relevance exists among the 3 NJ trees although there were differences in branching pattern. The effective marker index (EMI) was recorded highest (0.6544) for genomic SSR markers and lowest (0.4482) for the SNP markers while the EST-SSR markers had an intermediate EMI (0.5265). The present study suggests that the genomic SSR as the best class of markers for population genetics diversity analysis, the EST-SSR markers may be more suitable for phylogenetic and evolution studies, SNP markers may play an important role in high density linkage map construction.5. Molecular phylogenetics of six oyster species based on EST-SSR markersTaking the advantage of the conservation in ESTs from sequencing cDNA libraries,10 EST-SSR markers derived from C. gigas were used for phylogenetic analysis in six commercial and non-commercial oyster species including C. sikamea, C. nippona, C. angulata, C. ariakensis, C. hongkongensis and C. gigas. The average numbers of alleles were 4.1 in C. sikamea,3.5 in C. nippona,4.3 in C. angulata,4.0 in C. ariakensis,4.3 in C. hongkongensis and 5.0 in C. gigas.21.6% of the observed heterozygosities were higher than expected heterozygosities. Cg154 and CgL40 conformed tq HWE in all the 6 oyster species. Allele distribution at all 10 EST-SSR loci in each oyster species demonstrated that most of the alleles (27.5-64.25%) were in moderate frequencies (0.1-0.5). Fst value ranged from 0.016 to 0.450, the lowest Fst was between C. ariakensis and C. hongkongensis. Cluster analysis using both UPGMA and NJ method according to chord distance Dc. NJ trees partitioned the 6 oyster species into two main groups, one contained C. ariakensis and C. hongkongensis, the rest of the oysters belonged to the other group. In contrast, UPMGA tree had 3 groups, the C. gigas group, the C. ariakensis and C. hongkongensis group, and the rest 3 species conbined into the third groups. We noticed that the two pairs, C. ariakensis and C. hongkongensis, C. sikamea and C. nippona had closely related, whether they were in UPMGA or NJ trees.