The Transcriptome Of Grass Carp And Evolution Of Type-Ⅰ Microsatellite

Grass carp, Ctenopharyngodon idellus (Valenciennes, 1844), is an economically important species cultured in Asia, but a very aggressive invasive species in North America and Europe. As such, the situation of grass carp in Asia and in the west hemisphere is highly differential. As one of the most important aquaculture species, in Asia and elsewhere it is cultured, grass carp has gone through much inbreeding and is facing a serious genetic decline and disease problems; and its wild populations have been threatened by habitat degradation in much of Asia and the Middle East. On the other hand, as an exotic species with powerful ability for weed digestion, grass carp has been widespread with extreme reproductive success in watersheds of North America and Europe, posing serious ecological challenge, threatening most of the native and local species. Therefore, the understanding of its genetic background is very important for both aquaculture industry and ecosystems. However, its genomic resources are lacking. To date, there are only few expressed sequence tags (ESTs) developed from this species. The objectives of this study were to construct normalized cDNA libraries for efficient EST analysis, to generate ESTs from these libraries, and to identify EST-related molecular markers such as microsatellites and single nucleotide polymorphisms (SNPs) for genetic analysis of this species. A total of 6,269 ESTs were generated representing 4,815 unique sequences, from which 105 microsatellites and 5,228 SNPs were identified. These genome resources provide the material basis for future microarray development, identification of gene function, and genetic linkage and QTL analysis.Type-I microsatellite means those microsatellite which are linked to genes of known function. However, there are already have some reports of microsatellite polymorphism about fishes, however, the main function of those microsatellite is unknown. Therefore, it is important for us to research the evolution of microsatellite as the second step of study the genetic information of grass carp. After investigate eight sequence which containing microsatellite, we found gene MKLN1 and RRP7A have mRNA alternative splicing.Muskelin coded by MKLN1 participates in signal transduction, cell-matrix adhesion and cellular component movement as an intracellular protein. In previous study, MKLN1 containing 15 repeats of dinucleotide (CT) was identified from a cDNA library of grass carp, Ctenopharyngodon idellus. To further investigate the polymorphism of the dinucleotide, we examed ten individuals from three different grass carp population in this research and identified other different repeats of dinucleotide as (CT)9, (CT)10, (CT)11 and (CT)13. Each individual owning at most two locus indicates only one copy MKLN1 in the genome of grass carp. Compared with its orthologure in Zebrafish (Danio rerio) or Fugu (Takifugu rubripes) genome, the dinucleotide microsatellite was found not in an exon, insteading of in one intron corresponding 10th intron of MKLN1 in Danio rerio or Takifugu rubripes. Further investigating mRNA fragment spanning from 10th to 11th exons showed that there existed mRNA alternative splicing in MKLN1 and the microsatellite indeed located in unspliced 10th intron in grass carp. Terminal code“TGA”locating in unspliced intron leads the transcript to translate into truncated muskelin. The truncated muskelin might lost partial function because of kelch domain broken. The abundance of unspliced transcript was higher than that of spliced transcript in gill, intestine, head kidney, skin, hepatopancreas, intestine, muscle and kidney. Taken together, the microsatellite we found in one EST of MKLN1 is from an unspliced intron, and the unspliced intron lead the transcript encode truncate muskelin.RRP7A belongs to the RRP7 family,it’s protein participate in the pro-rRNA processing. In previous study, RRP7A containing 14 repeats of dinucleotide (AC) was identified from a cDNA library of grass carp, Ctenopharyngodon idellus. To further investigate the polymorphism of the dinucleotide, we examed ten individuals from three different grass carp population in this research and identified other different repeats of dinucleotide as (AC)15 and (AC)16. Compared with it’s cDNA transcripts, the genome of RRP7A include six introns and seven exons and the dinucleotide microsatellite was found in intron 3. Further investigation on mRNA expression showed that there existed six mRNA alternative splicing transcripts in RRP7A and the microsatellite (AC)19 indeed located in unspliced 3th intron in transcript S2 of grass carp. Terminal code“TAA”locating in unspliced intron leads the transcript to translate into truncated RRP7A protein and lost partial function because of RRM domain broken. Other transcript of S3~S5 may lost structure because of the alternative splicing, so transcripts of S2~S5 may lost their function.In the research of microsatellite polymorphism, we found the microsatellite in MKLN1 and RRP7A are all in introns, the main reason of the protein polymorphism is due to mRNA alternative splicing other than polymorphic microsatellite and the alternative splicing also influence the function of some transcripts. On the other hand, it is indicate that RNA alternative splicing is another way of microsatellite being contained in the ESTs.