Identificación, caracterización y herencia de microsatélites y su aplicación como marcadores moleculares en un programa de mejoramiento de camarón blanco

As an integral part of a genetic breeding program of Pacific white shrimp (Litopenaeus vannamei) the genetic variability was evaluated with molecular markers, specifically microsatellites identified de novo, including their characterization, sequencing, primers design, PCR optimization, and agreement to Mendelian inheritance expectations. A Sau3A1 partial genomic library with 2000 clones was produced. Two strategies were followed to identify microsatellites: A) direct sequencing of clones, and B) sequencing of positive clones to biotinylated probes (CT)10 and (GT)10. The most abundant identified microsatellites were dinucleotides, 58.82 % (1/0.042 kb), followed by trinucleotides, 27.73 % (1/0.88 kb), tetranucleotides, 9.25 % (1/2.7 kb), and pentanucleotides, 4.2 % (1/5.84 kb). The most abundant microsatellite was (CT)n followed by (GT)n, in agreement with a previous report in L. vannamei and contrasting with Penaeus monodon and most of the arthropods species where (GT)n is the most abundant dinucleotide. There was a previously reported satellite/microsatellite region (PVS1) in 43.33 % of the clones, resulting in a high abundance in the L. vannamei genome (1/0.8 kb). Because the presence of this microsatellite in the shrimp genome reduces the possibility of finding new microsatellites, we propose the construction of libraries by cutting with restriction enzymes that do not recognize any sequence in the PVS1 region (i.e. HaeIII, AluI, or HincII, vector SmaI). After sequencing 90 clones (29,199 bp), and identifying 119 microsatellite regions in 56 clones, an abundance of one microsatellite per 0.25 kb, the amplification by PCR of 25 designed primers pairs was evaluated. Seven microsatellites were amplified, and the banding pattern was clearly interpreted in five of them. These microsatellites have a high potential in genetic variation studies of populations of L. vannamei. The PCR amplification of the most polymorphic microsatellites (Pvan1758 y Pvan1815) was optimized to identify alleles and genotypes. Both microsatellites agreed with Mendelian expectations when the offsprings of three families were evaluated. The maternal genotype was verified and the paternal was inferred. The locus Pvan1815 showed null alleles, which were resolved decreasing the stringency of the PCR reaction increasing MgCl2 concentration. These analyses and modifications increased the confidence in the use of these microsatellites in genetic variation studies. Genetic diversity in a shrimp-breeding program with an established pedigree was monitored for three generations by Pvan1578 and Pvan1815 microsatellites. Two consecutive generations were produced (G1 and G2) using as a founder stock (G0) a captive population in domestication from Los Melagos, Son.- Venezuela (MV line). The main goal was to monitor these generations to establish levels of genetic variation and proceed with a selection program. The amount of genetic variation, expressed as observed heterozygosity (Ho) and expected heterozygosity (He) did not change significantly through generations (Ho = 0.65 - 0.72; He = 0.77 - 0.71) with an average Ho around that reported for wild stocks (0.666) and higher than that observed in cultured stocks (0.594). The genetic variation expressed in number of alleles (nA) increased through the gain of rare alleles. Nevertheless, the mean number of effective alleles (ne) decreased 16.6% from G0 to G2. The average number of alleles at G0 and G2 (7.5 and 10), and the average number of effective alleles (4.16 and 3.47) were less variable than those from wild populations, but higher than those from inbred stocks. There were significant differences in allele frequencies in one (G0-G1) or both (G1-G2) microsatellites, caused by a significant frequency increase or decrease of certain alleles through the contribution of the newly introduced MV male breeders. The MV male variability was not included in G0 but was already considered at the G2 as a new introduction of variability. In spite of the acceptable genetic variation in heterozygosity, the decreased effective number of alleles (ne) through generations highlights the previous trend of 3-4 alleles at both loci to dominate. These reduced ne might indicate that a certain reduction in variability already existed in the initial MV line used as broodstock, which could be corrected during this early stages of the breeding program by introducing new and different organisms. To increase the genetic variation at a G3, several individuals in the breeding program were mated with breeders from a Colombia line. A preliminary analysis of the Colombian breeders (n = 23) revealed higher values of variability (nA = 10, ne = 6.85, Ho = 0.74, He = 0.87), different allele frequencies, and two exclusive alleles in both microsatellites that allow the distinction between the lines. The monitoring of genetic variability has proved useful and should be maintained in this and other breeding programs, especially before and after selection is applied. The inclusion of other genetic markers will be useful, not only to complement the evaluations of genetic variability, but also for pedigree assessments and possible association with productive traits.