Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus

Abstract

Background: Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome.

Results: Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences.

Conclusions: Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our result suggested that it is difficult to analyze the shrimp genome following normal analytical methodology. Hence, it is necessary to avoid repetitive sequence (such as segmental duplications) when studying the other unique structures in the shrimp genome.

Figure 1

Schematic organization of putative genes on the kuruma shrimp BAC clone Mj024A04. Twenty-seven putative genes (boxes) and inter-genic regions (lines) are indicated with transcriptional orientation (arrows). Putative gene 09 is flanked with large GGTTA repeats (double lines).

Figure 2

Amplification of known putative genes using BAC clone samples. F, M and R probes were designed at the 5'-end, middle and 3'-end portion of the Mj024A04-sequence as described in the result. The putative genes (indicated in left column) were detected with BAC clones that showed different hybridization patterns with F, M and R probes (indicated in top line). Three BAC clones for each hybridization group were tested. Reactions with three BAC clones that showed no signal (negative control: neg), Mj024A04 (positive control: pos) and without templates (-) are also included.

Figure 3

Southern blot hybridization of kuruma shrimp genomic DNA. Putative genes (gene 01; Birc-2 Prov protein, 09; Reverse transcriptase, 16; WSSV-like and 27; Semaphorin -1A) used for probe synthesis is indicated at the bottom. Genomic DNA was digested with different combinations of restriction enzymes as indicated at the top.

Figure 4

FISH analysis of Mj024A04-sequence in adult kuruma shrimp testis cells. Multiple fluorescent signals of Alexa Fluor 594-labeled Mj024A04 are indicated as red spots in the nucleus couterstained with Hoechst 33258 (blue).

Figure 5

Copy numbers of 4 putative genes in kuruma shrimp genome. Putative gene 01 (Birc-2 Prov protein), 09 (Reverse Transcriptase), 16 (WSSV-like) and 27 (Semaphorin -1A) were used to calculate copy numbers in different kuruma shrimp tissues as measured by quantitative PCR. Data represent copy numbers of each gene relative to TGase with mean values ± standard deviation (bars) of three experiments.

Figure 6

Amplification of known putative genes using random selected BAC clone samples from different genotypes. All of BAC clones used in the BAC genotyping were selected based on the hybridization pattern against F, M and R probes that correspond to 5'-end (F), middle (M) and 3'-end (R) of the Mj024A04-sequence as described in the result and method. All genotypes were classified based on 3 distinct microsatellite repeats as described in the result. The putative genes (indicated in top line) were detected with BAC clones that showed different genotypes (indicated in left column). Reactions without templates (Nt) and primers (Np) are included as negative control. Reactions with Mj024A04 as template are also included as positive control.