preface
Higher crustaceans are the most diverse and morphologically diverse non-insect arthropods, and many of their members are commercially important. Although crustacean DNA sequence information is growing exponentially, little is known about the genomic organization of Malacostraca. Here, we construct a bacterial artificial chromosome (BAC) library.
BAC terminal sequencing was performed to provide genomic information for the Kuruma shorma shrimp, one of the most widely farmed species in crustaceans, and the presence of redundant sequences was found in the BAC library. We examined BAC clones containing redundant sequences to further analyze their length, copy number, and location in the kuruma shrimp genome.
The Mj024A04 BAC clone includes a redundant sequence containing 27 putative genes that appear to show normal genomic DNA structure. Notably, among the putative genes, 3 genes encode homologous proteins as apoptosin inhibitors and 7 genes encode homologous proteins as leukoplakia virus, a deadly pathogen known to affect crustaceans.
Colony hybridization and PCR analysis of 381 BAC clones revealed that almost half of the BAC clones retained DNA fragments with sequences homologous to representative BAC clones Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome, and the calculated copy number was at least 100. Microsatellite-based BAC genotyping clearly shows that the Mj024A04 homologous sequence was cloned from at least 48 different chromosomal sites. The lack of micro-commonality with available genome sequences of Daphnia and Drosophila suggests that these fragments are unique in kuruma shrimp in current arthropod genome sequences.
summary
The genome size of crustaceans varies greatly, with the smallest crustacean having a C value of 0.14 pg and the largest genome weighing 64.62 pg, a difference of 460 times. Despite the economic importance of crustaceans and huge biomass production, little is known about the genomic organization of crustaceans, especially Marakostellaka (including shrimp and crab), except for the presence of many repeating sequences.
Recently, although the genomic DNA sequence of the crustacean Daphnia dulva has been determined, it seems inappropriate to draw any conclusions about the crustacean genome, as recent phylogenetic analyses based on DNA sequence data and morphological comparisons between hexapods (including insects) and crustaceans have provided an unexpected finding that cladades (including Daphnia daffia) are phylogenically closer to hexapods than to Malacostraca. Therefore, there is a need to elucidate the genetic differences between the crustacean genome, particularly cladic and hexapod groups and other sister groups.
Penaaid shrimp classified as Decapoda in Marakosstraka has been the subject of in-depth research. Due to its commercial value, several papers have been published in the past few years on expression sequence tag (EST) analysis and genetic linkage mapping. However, the depth information of their large genomes is largely unknown, estimated to be about 70% the size of the human genome, and rich in AT and AAT sequences.
As a first step in understanding shrimp genome organization, we constructed a BAC library (named MjBL2) from kuruma shrimp (Marsupenaeus japonicus) and sequenced the BAC ends. The results clearly show that some sequences are extremely redundant in many BAC clones of the MjBL2 library. We chose a BAC clone (Mj024A04) for detailed analysis of its entire sequence and redundancy in the shrimp genome, and found many copies of DNA fragments containing Mj024A04 sequences. This suggests that the superamplification of this particular DNA fragment occurred through segmental replication events during the evolution of the Kuruma shrimp genome.
BAC library construction and BAC terminal sequencing
To outline the composition and organization of the Kuruma shrimp nuclear genome, we constructed the BAC library (MjBL13) using the Kuruma shrimp genome DNA prepared from the blood cells of 2 shrimp, and analyzed the BAC terminal sequence (BES). MjBL2 consists of 49,152 BAC clones, arranged in 128 microtiter plates and stored at -80 °C. By Not I digestion of 135 randomly selected BAC clones, the average insert size is estimated at 205 kb.
Further BES analysis was performed using 2 randomly selected BAC clones from MjBL192 and the retrieved readings were assembled for continuity [DDBJ: AG993477-AG993734]. The resulting BES is divided into 29 singletons and 51 consecutive sequences, including 2 to 24 reading segments. Notably, BLASTN and BLASTX analyses showed that many of these BES (20 readings in BLASTN and 55 readings in BLASTX) contained protein sequences encoding "apoptosin inhibitors (IAPs)" reported in black tiger shrimp (Penaeus monodon).
Representative DNA sequences of BAC clones
BAC clones (Mj04A2) with the gene similar to "Black Tiger Shrimp IAP" were randomly selected from MjBL024, and the entire DNA sequence was analyzed in detail by shotgun sequencing. The 120 kb genomic DNA sequence (Mj024A04 sequence) obtained by computer annotation analysis revealed 27 putative genes that apparently appear to be normal genomic regions with exon intron structure [DDBJ:AP010878].
Large GGTTA repeats were found in the middle of sequence flanking gene 09, which encodes a protein similar to the reverse transcriptase of Konitaki pufferfish. Notably, of the other 26 genes, three genes (genes 01, 06 and 24) encode proteins homologous to IAP in three species, the Xenopus frog, Drosophila melanogastered (Drosophila) and the Norwegian rat (Norwegian rat), and seven genes (Genes 11, 13, 14, 15, 16, 17 and 18) are homologous to ORF in "Leukoplakia Syndrome Virus (WSSV)", the main shrimp pathogenic dsDNA virus, Shrimp, as well as other crustaceans such as crabs and crayfish, are highly toxic.
Detection of Mj024A04 sequence and its copy number in the genome of intermediary shrimp
Next, we used Southern blot hybridization to detect multiple copies of the Mj024A04 sequence in the genome of Kuruma shrimp using several different restriction enzymes. The results showed that each of the 4 putative genes (genes 01, 09, 16, and 27) had multiple DNA bands, confirming the existence of multiple copies of these genes. In addition, we performed fluorescence in situ hybridization (FISH) using labeled Mj024A04 BAC clones.
The FISH image clearly shows many fluorescent spots in the nucleus of the testicular of adult shrimp. With the repetition of large repeats, we further examined the copy number of the putative gene using genomic DNA prepared from 01 different organs (brain, blood cells, heart, testes, muscles, swimming legs, and intestines) and 09 larvae by quantitative PCR of genes 16, 27, 7, and 3.
Our results show that these putative genes have 100 times the copy number of the putative single-copy gene glutaminyl aminotransferase (TGase), except for gene 09 (reverse transcription transposon). This indicates the presence of multiple copies of the Mj024A04 sequence. Taken together, our results show that the large DNA fragment Mj024A04 appears multiple times in the genome.
BAC genotyping and PCR detection of putative genes in Mj024A04 sequences
To rule out possible cloning bias, we performed BAC genotyping using three microsatellite polymorphisms. 292 distinct genotypes were detected in 201 F, M, R-positive BAC clones screened from 2 to 008 plates of MjBL008, representing 0.2 coverage of the shrimp genome. PCR-based presumptive gene testing of eight independent BAC clones selected from different genotypes revealed the presence of almost all 17 putative genes. Assuming that all shrimp have heterogeneous chromosomes, both genotypes from one allele should be detected.
Since we constructed the MjBL13 library from 2 individuals, a chromosomal locus can be expected to have up to 26 different haplotypes. The 299 different genotypes detected indicate that at least 11 different chromosomal sites contain duplicates of the entire Mj024A04 sequence.
BAC library construction and BAC terminal sequencing for the first characterization of the kuruma shrimp genome
The content of nuclear DNA in Kuruma shrimp is reported to be 2.83 pg, indicating that the genome size of Kuruma shrimp is almost the same as that of other prawns, such as P. vannamei and Penaeus monodon, with a genome size of about 2,000Mbp reported. In this study, we first constructed BAC libraries from kuruma shrimp. The average insert size of the MjBL2 BAC clone is estimated at 135 kb, and the total MjBL2 insert size can be calculated to be about 6,600 Mbp, indicating that MjBL2 represents 3.3 times the genome coverage of kuruma shrimp.
Although MjBL2 is not suitable for physical localization and genome sequencing because it was constructed from 13 shrimp, MjBL2 is useful as a first step in characterizing the kuruma shrimp genome. We performed BES analysis to understand for the first time the sequence composition of the unsequenced kuruma shrimp genome. The results of the BES analysis were very surprising because even after analyzing only 192 clones, we detected 51 contigs, each containing multiple readings, ranging from 2 to 24.
This shows that following a typical BAC construction method, we obtained multiple copies of the same DNA fragment in the kuruma shrimp genome. However, presumptive genes such as the black tiger shrimp IAP gene homologue annotated with BLAST do not appear to have potentially repeatable activity like transposable elements. To further identify abnormalities in the Kuruma shrimp genome, we further analyzed these DNA fragments in the Kuruma shrimp genome.
Representative genetic content of BAC clone Mj024A04
Randomly selected Mj024A04 BAC clones with black tiger shrimp IAP sequences were fully sequenced and 27 genes were predicted in vitro. Of the 27 genes predicted, we found three genes that were homologous to IAP. It is well known that apoptosis is a genetic pathway of controlled cell suicide and plays a key role in multiple processes such as development, tissue homeostasis, DNA damage response, and pathological processes.
IAP has been shown to block apoptosis by directly binding to the baculovirus IAP repeat (BIR) domain present in IAP to inhibit the proteolytic activity of caspase, a core component of the apoptosis mechanism. Cellular homologues called BIR domain-containing proteins (BIRPs) are characterized by the presence of a variable number of BIR domains. These congeners have been found in yeast, nematodes, flies and higher vertebrates.
In fruit flies, 1 IAP congener (Thread or IAP2, IAP12265, Bruce and Deterin or CG24) has been identified. We analyzed the phylogenetic relationship of putative genes 06, 24, and 9 with other BIR domains in several organisms. The BIR domain in putative gene 24 is clustered with the BIR domain in the black tiger shrimp IAP gene, indicating that the putative gene 25 may have the same function as the black tiger shrimp IAP.
Of particular interest is the fact that the putative gene 22 contains five BIR domains, the first report on BIRP containing more than three BIR domains. BIR domains are known to play an important role in protein-protein interactions, and it has been shown that the presence of multiple BIR domains in a single protein molecule increases the affinity of BIRP for the protein of interest.
In addition, the range of target molecules that BIRP can interact with also increases with the number of BIR domains. Therefore, a putative gene <> with five BIR domains may be a novel BIRP with different functions.
conclusion
Our results show that superamplification of large DNA fragments occurs in the kuruma shrimp genome. Although we only analyzed partially repeated DNA fragments, our results show that it is difficult to analyze the shrimp genome with normal analytical methods. Therefore, it is necessary to avoid duplicate sequences when studying other unique structures in the shrimp genome.
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