IDENTIFICATION OF TRI- AND TETRA-NUCLEOTIDE REPEAT MICROSATELLITES IN SHRIMP (Litopenaeus vannamei)

Department of Enviromental and Population Health, Tufts University School of Veterinary Medicine, 200 Westboro Road, North Grafton, MA 01536

In an effort to develope a linkage map for penaeidae shrimp, tri- and tetra-nucleotide microsatellites were isolated from a genomic library of L. vannanei by probe hybridization. Clones were obtained by ligating target DNA to vector DNA at different ratios. A total of 1479 positive clones were obtained and used for hybridization with each of the following probes (TAT)₁₀, (CTC)₁₀, (CTTT)₈, and (TGTA)₈. One hundred and seventy eight out of the 1479 clones tested positive to either one or more of the tri-nucleotide and tetra-nucleotide probes and were sequenced. One hundred and twelve clones contained microsatellites. Forty-three out of 112 clones have tri-nucleotide and/or tetra-nucleotide arrays with three or more repeats. Within these 47 clones, a total of 81 trinucleotide and 23 tetranucleotide arrays have been identified. Fifteen out of the 116 clones contained penta-nucleotide repeats either alone or together with tri-nucleotide or tetra-nucleotide repeats. In addition, there are also a total of 7 hexanucleotide arrays with three or more repeats and one nano-nucleotide (CATTATTAT)₃ array. The most abundant tri-nucleotide repeat was (ATT) (n=37) followed by (TAT) (n=11), (CAT) (n=6), (CCT) (n=10), among others. The most abundant in the tetra-nucleotide repeat was (CTTT) (n=11), (ACGC) (n=3) and (GTCT) (n=2). The other 8 repeats were single sequences. The 81 tri-nucleotide microsatellites were classified as perfect (64%), compound imperfect (21%), imperfect (9%), and compound perfect (6%). The 23 tetra-nucleotide microsatellites were classified as perfect (43%), compound perfect (26%), compound imperfect (22%), and imperfect (9%). All clones also contained di-nucleotides in addition to the larger repeats. These microsatellites are being characterized with DNA from a reference family to develope a linkage map for L. vannamei as well as for pedigree tracking in breeding programs and genetic diversity analysis.