Identification of pigmentation genes in skin, muscle and tail of a Thai-flag variety of Siamese fighting fish Betta splendens.

Pigmentation genes expressed in skin, body muscle and tail of Thai-flag compared with Blue, White and Red varieties of Siamese fighting fish Betta splendens were identified. In total, 22,919 new unigenes were found. Pearson correlation and PCA analysis revealed that expression profiles of genes in muscle, skin and tail across solid color variety were similar. In contrast, those in skin and red tail part of Thai-flag were closely related but they showed different expression profiles with the white tail part. Moreover, 21,347-64,965 SNPs were identified in exonic regions of identified genes. In total, 28,899 genes were differentially expressed between paired comparisons of libraries where 13,907 genes (48.12 %) were upregulated and 14,992 genes (51.88 %) were downregulated. DEGs between paired libraries were 106-5775 genes relative to the compared libraries (56-2982 and 50-2782 for upregulated and downregulated DEGs). Interestingly, 432 pigmentation genes of B. splendens were found. Of these, 297 DEGs showed differential expression between varieties. Many DEGs in melanogenesis (Bsmcr1r, Bsmcr5r, and Bsslc2a15b), tyrosine metabolism (Bstyr, Bstyrp1b and Bsdct), stripe repressor (BsAsip1 and BsAsip2b), pteridine (Bsgch2) and carotenoid (BsBco2) biosynthesis were downregulated in the Thai-flag compared with solid color varieties. Expression of Bsbco1l, Bsfrem2b, Bskcnj13, Bszic2a and Bspah in skin, muscle and tail of Thai-flag, Blue, Red and White varieties was analyzed by qRT-PCR and revealed differential expression between fish varieties and showed anatomical tissue-preferred expression patterns in the same fish variety. The information could be applied to assist genetic-based development of new B. splendens varieties in the future.

Assessing Genetic Diversity of Wild Populations and Different Color Varieties for Genetic Improvement of Siamese Fighting Fish Betta splendens in Thailand.

To establish sustainable resources and founder populations for genetic improvement of the Siamese fighting fish Betta splendens, genetic diversity in wild and hatchery stocks was examined using mitochondrial (mt) DNA genes cytochrome b (cytb), 16S ribosomal DNA (16S rDNA), and cytochrome oxidase subunit I (COI), and eight microsatellite loci. Based on mtDNA sequences, restrictive levels of polymorphism (0, 3, and 1 substitutions) were observed in this study. For analysis of microsatellites, fluorescent multiplex PCR was developed, and subsequently identifying moderate levels of observed (Ho = 0.4488) and expected (He = 0.6627) heterozygosities and a high number of alleles per locus (15.125 alleles) for overall samples. Comparison of Siamese fighting fish from different sources revealed large genetic differences between pairs of farmed fish (eight groups) and between wild (three geographic locations) and farmed fish (P < 0.0031 following Bonferroni correction). This suggested limited exchanges of genetic resources between commercial farms. When different color varieties of B. splendens were compared, large genetic distances and significant FST estimates and genetic heterogeneity were found (P < 0.0031). Effective population sizes (Ne) were estimated and two farms (NP2-2BS and BK1-4BS) showed Ne greater than 10. Among color varieties, Multi-colors and Blue revealed reasonable Ne (large and 27.9), but lower Ne values (3.6-8.4) were found for the remaining color varieties. These results indicate an urgent need for the establishment of gene pool resources of B. splendens for effective genetic improvement of Siamese fighting fish in Thailand.

Transcriptome comparison for identification of pigmentation-related genes in different color varieties of Siamese fighting fish Betta splendens.

Transcriptome comparison was performed to identify genes expressed in skin, muscle and tails of mono-color (Red, Blue, Black, White and Yellow), bi-color (Cambodian) and multi-color (Marble) varieties of Siamese fighting fish Betta splendens. In total, 163,140 unigenes covering 26.348 Gb were found. Of these, 93,899 (57.55 %) unigenes significantly matched at least one database. In total, 5039 differentially expressed genes (DEGs) were found where 2415 genes (47.93 %) showed higher expression and 2624 genes (52.07 %) showed lower expression for all pairwise comparisons. DEGs between paired color varieties were 133-443. Of these, 38-220 genes were more highly expressed while 37-280 genes were more lowly expressed relative to the compared varieties. A total of 897 sequences (148 genes) significantly matched pigmentation-related genes of Danio rerio (E-value < 1e-06). Of these, 19 DEGs were identified. Examples are tyrosinase-related protein 1a (BsTyrp1a), epidermal growth factor receptor (BsEgfr) and neurofibronin 1a (BsNf1a). Moreover, 711,123 SNPs were identified and 1365 of these were located in pigmentation-related genes. Interestingly, an A > C474 SNP in the gene BsTrpm7 and an indel (position 3571) in the BsItgb1a gene were found only in Cambodian. A C > T2520 SNP in BsFzd4 and 10 of 11 SNPs in BsTyrp1a were found only in Black. Different expression levels (P < 0.05) were found for tyrosinase (BsTyr), BsTyrp1a, BsNf1a and BsEgf1 among skin, body muscle and tails of the same variety and among the same tissues of different varieties (Red, Green, Blue, Black, Cambodian and Multi-colors, N = 5 each).

Identification of a Growth-Associated Single Nucleotide Polymorphism (SNP) in Cyclin C of the Giant Tiger Shrimp Penaeus monodon.

The full-length cDNA of cyclin C of the giant tiger shrimp Penaeus monodon (PmCyC) was isolated by RACE-PCR. It was 1443 bp in length containing an open reading frame (ORF) of 804 bp and 267 deduced amino acids. Tissue distribution analysis indicated that PmCyC was more abundantly expressed in ovaries and testes than other tissues of female and male juveniles (P < 0.05). A pair of primers was designed, and an amplification product of 403 bp containing an intron of 123 bp was obtained. Polymorphism of amplified PmCyC gene segments of the 5th (3-month-old G5, N = 30) and 7th (5-month-old G7, N = 18) generations of domesticated juveniles was analyzed. Four conserved SNPs (T>C134, T>C188, G>A379, and T>C382) were found within the examined sequences. A TaqMan genotyping assay was developed for detection of a T>C134 SNP. Association analysis indicated that this SNP displayed significant association with body weight (P < 4.2e-10) and total length (P < 2e-09) of the examined G7 P. monodon (N = 419) with an allele substitution effect of 5.02 ± 0.78 g and 1.41 ± 0.19 cm, respectively. Juveniles with C/C134 (22.80 ± 2.51 g and 12.97 ± 0.53 cm, N = 19) and T/C134 (20.41 ± 0.93 g and 12.77 ± 0.21 cm, N = 129) genotypes exhibited a significantly greater average body weight and total length than those with a T/T134 genotype (14.72 ± 0.53 g and 11.37 ± 0.13 cm, N = 271) (P < 0.05).

A SNP of the hemocyanin gene (LvHc) is a marker for high growth and ammonia-tolerance in Pacific white shrimp Litopenaeus vannamei.

Expression levels of hemocyanin (LvHc), activating transcription factor 4 (LvAtf4), glutathione S-transferase (LvGst), caspase 2 (LvCasp2) and anti-lipopolysaccharide factor (LvAlf) were examined in the hepatopancreas of Pacific white shrimp Litopenaeus vannamei juveniles exposed to a lethal concentration of ammonia-N (32.15 mg/l). The expression levels of all transcripts except LvAlf were significantly greater (P < 0.05) in tolerant shrimp (Lv-AT; N = 30) that survived up to 72 h post treatment (hpt) than in susceptible shrimp (Lv-AS24 and Lv-AS72; N = 45 and 15), that died within 24 h or between 24 and 72 hpt, respectively. Subsequently, effects of non-lethal concentrations of ammonia-N (control, 10 and 20 mg/l) on the expression of LvHc in juvenile shrimp were examined. Compared to the control, expression levels of LvHc transcripts in hemocytes and the hepatopancreas of tested shrimp changed after exposure to ammonia-N. One SNP (C > T545) was found in the LvHc322 gene segment. Real-time PCR amplification of specific alleles (real-time PASA) was developed for detection of C > T545 genotypes. Juveniles in the lethal exposure test that carried a C/T545 genotype showed a greater average body weight and total length (8.46 ± 0.36 g and 10.05 ± 0.16 cm) than those with a C/C545 genotype (7.48 ± 0.31 g and 9.60 ± 0.13 cm) (P < 0.05). Similar results were found in the second generation (G2) of a growth-improved stock (3 and 4 families of BIOTEC-G2-L1 and BIOTEC-G2-L2) and in commercially farmed shrimp (2 groups). Accordingly, expression levels and SNP of LvHc can serve as markers for selection high growth performance in ammonia-tolerant L. vannamei.

Isolation and expression analysis of Bystin 1 transcript and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

The full-length cDNA of bystin isoform 1 (PmBys1) of the giant tiger shrimp Penaeus monodon was characterized. It was 1553 bp in length containing an ORF of 1365 bp corresponding to a polypeptide of 454 amino acids. The level of PmBys1 mRNA in ovaries was greater than that in other tissues of females and in testes of males in both juveniles and wild broodstock (P < .05). In non-ablated wild female broodstock, PmBys1 mRNA significantly and progressively increased in ovaries from stage I of development, peaking at stage IV (P < .05). Its level in stages I-IV of eyestalk-ablated broodstock was greater than that in non-ablated broodstock (P < .05). Injection of exogenous serotonin (50 µg/g body weight) into 18-month-old shrimp resulted in a significantly increase of ovarian PmBys1 mRNA at 6-48 h post injection (hpi) (P < .05). PmBys1 protein (52 kDa) was found in ovarian stages I-V of non-ablated wild broodstock and II-IV of ablated wild broodstock, respectively. Along with the 52 kDa band, immunoreactive bands of 50 and 43 kDa were also observed in ovarian stages II-IV of both non-ablated and ablated broodstock and in ovaries of post-spawning broodstock. The 43 KDa band was not observed in ovarian stage I of wild female broodstock or in premature juveniles. PmBys1 protein was localized in the ooplasm of previtellogenic oocytes, nucleo-cytoplasmic compartments of vitellogenic oocytes and cortical rods of mature oocytes in wild broodstock. The results implied a possible role for PmBys1 during ovarian development in P. monodon.

Characterization of transforming growth factor beta regulator 1-like and association between its expression levels and growth of the giant tiger shrimp Penaeus monodon.

Molecular markers that allow selection of juveniles and broodstock with improved growth performances are useful for the shrimp industry. Here, the full-length cDNA of transforming growth factor beta regulator 1-like (PmTbrg1-l) in the giant tiger shrimp Penaeus monodon was determined. It was 1184 bp in length and contained an open reading frame (ORF) of 975 bp corresponding to a deduced polypeptide of 324 amino acids. Successful RNA interference (RNAi) carried out using juveniles injected with PmTbrg1-l dsRNA revealed reduced levels of PmTbrg1-l and myostatin (PmMstm) in hemocytes when compared to shrimp injected with saline solution and GFP dsRNA (P < .05). Associations between single-strand conformational polymorphism (SSCP) patterns or single nucleotide polymorphism (SNP) patterns and growth-related parameters (average body weight and total length) were examined. Juveniles with pattern III (corresponding to A/A918; N = 37) showed a trend for greater average body weight and total length than those with patterns II (G/G918; N = 42) and IV (A/G918; N = 75). The expression level of PmTbrg1-l in the hepatopancreas of females was significantly higher than that in males (P < .05) in two sample sets of three-month-old domesticated juveniles (N = 59 and 50; P < .05). Moreover, its expression level in large-size juveniles was significantly higher than that in medium-size and small-size juveniles in both groups of samples (P < .05). Results indicated that PmTbrg1-l is functionally related with growth of P. monodon.