Analysis Of Tandem Repeats In Crab Portunus Trituberculatus Genome And Microsatellite Marker Screening

In this paper, random genome library of crab Portunus trituberculatus was constructed and the lengths of 4164 sequenced clones were between 500 to1500bp. The distribution and frequencies of microsatellite and minisatellite wereanalyzed from the 709 sequences spliced; nine polymorphic microsatellite primer pairs were screened from these clones included short tandem repeat.By sequencing randomly, 4164 clones of sequences in the genomic library of crab Portunus trituberculatus were obtained. This study use software DNASTAR (Version 5. 0) to assemble all of the clones .The length of DNA sequences is about 622,409 bp in total.With the help of the bio-soft Tandem Repeats Finder (Version 2. 02), 697 microsatellite repeat sequences are found in the sequences. In the 697 repeat sequences , the number of the dinucleotide repeat is 445 , and it's the most one(63.84 %) among all of the repeat sequences . The second one is the trinucleotides repeat , 152 (21.81 %) ; the third one is the mononucleotide repeat , 45 (6.46 %) ; the forth one is the tetranucleotides repeat , 31 (4.45 %) ;the fifth one is the petranucleotides repeat , 14 (2.01 %) ; the sixth one is the hexanucleotides repeat , 10 (1.43 %) .Number of repeat sequences that composed of the motif of A is 46, and don't find the motif of C among the mononucleotide repeat. In dinucleotides repeat , the number of AG repeat is 214 , which is the most one, accounting for 48.09 %; and the second and third one are AC and AT repeats, 187 (42.02 %) and 43(9.66 %) respectively. Eight classes of repeat sequences that respectively composed of the motif ACT , AGG, AAT , ACC , AAG, ATC, AAC and AGC , are found in the trinucleotides repeat, in which the number of ACT repeats is 42, the most ; the second one is AGG (35) ; the others are AAT(28),ACC(21),AAG(9),ATC(7),AAC(7) and AGC(3) in turn. AGAC , AACCT and AGGGGA repeats are the most ones in tetranucleotide , pentranucleotide and hexanucleotide respectively. One GC dinucleotide repeat is found in our study and the sequence is referred to the GenBank, and the number of accession is EU113241. The reason of fewer GC repeat is also discussed in the article. Two possible answers are that: one is methylation of C in CpG islands resulting in the mutation of C-T; and another is that it is difficult to sequence the GC repeat sequences.Distributions of copy numbers in different types of repeat sequences are as follows: copy numbers of mononucleotide repeats are mainly between 28～40 and 68～76 , accounting for 80.00%; copy numbers of dinucleotides are mainly between 12and 36 , accounting for 64.04 %; copy numbers of trinucleotides repeats are mainly between 8 and 24 , accounting for 57.90%; copy numbers of tetre- , pentra- and hexanucleotides repeats together are mainly between 4 and 12. In general , the length of microsatellite repeat sequences is mainly between 24 to 72 bp. Based on the above point , it is believed that the nucleotide mutation of microsatellite locations are accumulated largely in a long term of evolution ; and there would be abundant polymorphism in these locations. Therefore , it would be very practical to use microsatellite to study the genome of Portunus trituberculatus and would beapplied to a variety of fields including population differentiation, kinship analysis, linkage analysis, and evolutional and ecological studies. This study provides a base for Portunus trituberculatus microsatellite research.With the help of the bio-soft Tandem Repeats Finder (Version 2. 02), 130 minisatellites were screened in the crab's genome DNA sequences. Their cumulative length occupied 2.55% of the total length of DNA sequences. In the minisatellite sequences, twelve-nucleotide repeats were the most frequent type, accounting for 10.77% of the total number of minisatellites. It showed that the number of sequences decreased with the length of its repeat unit(R=-0.663,p<0.01).Eight-nucleotide repeat had the largest range of copy number of repeat unit (3.9～66.5),the following were thirteen-nucleotide repeat type(2.0～40.6) and twenty-six-nucleotide(2.3～21.0) ,respectively. Descending three repeat types in mean copy number of repeat unit were eight-nucleotide repeat (19.96), twenty-five-nucleotide (16.00) and twenty-two-nucleotide (15.85), respectively. The range of copy number of repeat unit varied from 2 to 66.5, and the copy number mostly ranged from 2 to 25. Moreover, it was showed that the number of corresponding minisatellites decreased as copy number of repeat unit increased. In our research , the 130 minisatellite sequences are composed of 123 kinds of repeat units , so it is very difficult to classify the minisatellite sequences. We think the minisatellite sequences can be classified into three types: dinucleotide minisatellite sequences composed of two kinds of nucleotide, trinucleotide minisatellite sequences composed of three kinds of nucleotide and tetranucleotide minisatellite sequences composed of four kinds of nucleotide. Further, all of above sequences can be divided into many sub-types according to the composition of nucleotide and their number from large to small. Totally, the minisatellite sequences in Portunus trituberculatus are A/T rich. We also discussed the genesis and evolution of minisatellite repeat sequences, and think the minisatellite repeats may come from the microsatellite repeats. It would be very practical to use minisatellite to study the genome of Portunus trituberculatus and would be applied to a variety of fields.In this article , we review isolation methods , design of primer,developmental status and genetic characteristics of microsatellites , and their applications in studies on population study , pedigree analysis , assessment of genetic diversity , and analyzed the causes resulting to null allele , stutter bands , upper allelic dropout and their effects on genotyping of microsatellite.Microsatellite primers were designed from the short tandem sequences which were screened from 709 clones of genomic library. microsatellites which have the 50 bp upstream and downstream flanking sequences were used. Nine primer pairs with high amplified polymorphisms were screened from thirty primers paired designed.