Development Of Genomic Microsatellite DNA Markers And Construction Of Genetic Linkage Map Of Guppy (Poecilia Reticulata) Based On AFLP And SSR Markers

In order to establish an efficient method to clone microsatellite DNA sequences and use microsatellite DNA and AFLP markers to construct genetic linkage maps of guppy (Poecilia reticulata), two parts of experiments were done: (1) isolation microsatellite DNA by magnetic beads enrichment; (2) construction of guppy (P. reticulata) genetic linkage maps using microsatellite DNA and AFLP markers and pseudo-testcross mapping strategy. The main contents were as follow:A genomic DNA library enriched with AC/TG-microsatellite DNA was constructed using DNA extracted from a guppy individual using FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) method. A biotinylated (AC)20 oligonucleotide was used as probe. DNA fragments containing microsatellite DNA were ligated with pMD18-T vector and electroporated into E. coli JM109.In total, about 450 white colonies were screened, each colony was subjected to 3 individual PCR reactions. In the first reaction, universal forward and reverse sequencing primers (M13-47 and M13-48) were used. In the second reaction, the universal forward primer was used in combination with a (CA)20 oligonucleotide and in the third one the universal reverse primer was used in combination with a (CA)20 oligonucleotide. In the white colonies screened, 287 clones are positive. After sequencing, 262 contained microsatellite repeat sequences. Besides the motif of (CA/GT) contained in the oligoprobe, we also found GCTTT,AAAAG,ATGG,TGCG,TATG,GTAT,CAAG,CACG,CGCA,TCC,TAA,AGG,ACA,AAC,GAA,GAG,CAA,CT,GA,A/T repeats. In addition, 1 (CACACAC) minisatellite clone was obtained. According to Weber (1990),the microsatellite sequences could be categorized structurally as follows: perfect (27.05%), imperfect (61.57%), and compound (11.39%).One hundred and eighty-nine microsatellite DNA primers of guppy (P.reticulata) were developed. Fifty-one of the markers detected the diversity in a guppy population. The number of alleles each locus ranged from 2 to16. Observed and expected heterozygosities varied from 0.10 to 0.63 and from 0.23 to 0.77, respectively. Four loci (G95, G278, G307and G401) displayed significant deviations from the expectations of Hardy–Weinberg equalifiation ( P<0.05 ). There was no null allele detected at any of the loci.Cross-species amplification was investigated in 3 closely related species, Poecilia latipinna, Xiphophorus hellerii and Xiphophorus maculates. The cases of successful cross-species amplification were higher in P. latipinna than in two species of genus Xiphophorus.Genetic linkage maps of guppy (P.reticulata) were constructed using microsatellite DNA and AFLP markers and pseudo-testcross mapping strategy. Of 221 microsatellite DNA markers, 101 segregated at 1:1 or 1:1:1:1 ratios. In addition, 336 AFLP markers segregated also in F1 progenies at 1:1 ratio, which were produced using 91 primer combinations.Two linkage maps were constructed: one for male parent and the other for female parent. The female parent map consisted of 219 markers, of which 135 (97 AFLP and 38 microsatellite DNA) were assigned to 22 linkage groups, at least four markers each. The 135 markers covered 1267.7 cM in length with an average interval of 11.2 cM,. The length of the individual linkage group varied from 19.6 to 202.3 cM. The remaining 46 markers distributed as two triplets and eighteen doublets. The male parent map consisted of 227 markers, of which 172 (122 AFLP, 49 microsatellite DNA and the sex determinant) were assigned to 20 linkage group. The 172 markers covered 1771.2 cM in length with an average interval of 11.7 cM. The length of the linkage groups ranged from 31.5 to 175.6 cM, the number of markers per group varied from 4 to 14. The remaining 16 distributed as four triplets and two doublets. On the basis of the expected genome lengths, genome coverage of female and male maps was 72.5% and 79.3%, respectively. When considering all the triplets and doublets, the map coverage increased to 84.0% for the female and 84.5% for the male.The sex of the mapping progenies was treated as a marker for linkage analysis in the female and male (Li et al., 2003). Of 61 progenies, 30 were female and 31 were male. The sex marker was the only phenotypic trait placed on the map. In the male parent map, sex was mapped onto linkage group 2, which contained 13 markers including four microsatellite DNA and nine AFLP. Four microsatellite DNA markers separated from the sex locus at the distance of 114.3 cM, 23.3 cM, 12.1cM and 9.2 cM, respectively.The present study has shown that enrichment by magnetic beads is a simple and efficient method for rapid isolation of guppy genome microsatellite DNA and the (AC) -enriched library created in this study will be useful resource for developing additional genomic microsatellite DNA markers for guppy in the future. Moreover, the construction of guppy linkage maps will be important for genetic studies of economically important fish.