Characterization and Development of EST-SSR Markers Derived from Transcriptome of Yellow Catfish

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater fish due to its delicious flesh and high nutritional value. However, lack of sufficient simple sequence repeat (SSR) markers has hampered the progress of genetic selection breeding and molecular research for yellow catfish. To this end, we aimed to develop and characterize polymorphic expressed sequence tag (EST)–SSRs from the 454 pyrosequencing transcriptome of yellow catfish. Totally, 82,794 potential EST-SSR markers were identified and distributed in the coding and non-coding regions. Di-nucleotide (53,933) is the most abundant motif type, and AC/GT, AAT/ATT, AAAT/ATTT are respective the most frequent di-, tri-, tetra-nucleotide repeats. We designed primer pairs for all of the identified EST-SSRs and randomly selected 300 of these pairs for further validation. Finally, 263 primer pairs were successfully amplified and 57 primer pairs were found to be consistently polymorphic when four populations of 48 individuals were tested. The number of alleles for the 57 loci ranged from 2 to 17, with an average of 8.23. The observed heterozygosity (HO), expected heterozygosity (HE), polymorphism information content (PIC) and fixation index (FIS) values ranged from 0.04 to 1.00, 0.12 to 0.92, 0.12 to 0.91 and −0.83 to 0.93, respectively. These EST-SSR markers generated in this study could greatly facilitate future studies of genetic diversity and molecular breeding in yellow catfish.


Introduction
Molecular marker systems, such as simple sequence repeats (SSRs) or microsatellites [1], single nucleotide polymorphism (SNPs) [2], amplified fragment length polymorphisms (AFLPs) [3] and random amplification of polymorphic DNAs (RAPDs) [4] have been developed and are applied to fisheries and aquaculture. Yellow catfish is an important freshwater fish for its delicious flesh and high market value, whereas overfishing is decreasing its number and genetic diversity [5]. Applying genomic tools in the selection of elite broodstock has the potential to improve the productivity and commercial value of this species. In populations of yellow catfish, males grow faster than females by two to three folds. For this reason, an all-male monosex population has been massively produced for commercial purpose [3,6,7]. However, genetic resources and suitable molecular markers are still scarce in yellow catfish.
SSRs are tandem repeating sequences of 1-6 nucleotides and distributed throughout vertebrate genomes [8]. Based on their locations, SSRs can be classified into genomic SSRs (gSSRs) and Expressed Sequence Tag-SSRs (EST-SSRs) [9]. Because of high level of polymorphism, SSRs have wide applications in population genetics, such as parentage analysis [10], Quantitative Trait Locus (QTL) mapping [11], marker assisted selection (MAS) [12], and phylogenetic studies [13]. Traditional methods of developing gSSR markers require fragmented genomic DNA and are usually time-consuming and labor-intensive. With the advent of high-throughput sequencing technology, the development of EST-SSRs has become a fast, efficient, and low-cost option for economical fish species [14,15].
The transcriptome of yellow catfish was acquired using a 454 GS-FLX Titanium platform and 540 Mbp of raw data were generated. In this study, we analyze the frequency and distribution of 82,794 potential EST-SSRs in the yellow catfish transcriptome. Sixty of 300 validated primer pairs were selected and further characterized for polymorphism analysis. Recently, we have performed genetic selection breeding on four wild populations of yellow catfish collected from Chang Lake (Jingzhou), Hong Lake (Honghu), South Lake (Zhongxiang) and Dongting Lake (Hunan) as previously reported [16]. These EST-SSR markers should provide a promising genetic resource for molecular breeding of yellow catfish.

SSR Marker Development and Genetic Diversity Analysis
A total of 300 SSR primers located on 280 assembled congtigs and singletons were randomly selected and amplified using DNA templates extracted from four wild populations of yellow catfish from Chang Lake, Hong Lake, South Lake and Dongting Lake. Of these SSR primers, 263 (87.7%) pairs of primers exhibited stable and repeatable amplification, and 57 (19%) of them were identified as polymorphic loci in all 48 individuals. Although we tried multiple PCR reactions under different amplification conditions, the 37 pair of primers still did not produce any PCR fragment, which probably due to assembly errors in sequences or primer pairs designed across a splice site with a large intron [20]. Among the 263 worked and 37 not-worked SSRs, there are 122 (46.4%) and 11 (29.7%) SSRs in the 3'-UTR, 71 (27.0%) and 12 (32.4%) SSRs in the 5'-UTR, 66 (25.1%) and 13 (35.1%) SSRs in the coding region, respectively. Further, there are 106 polymorphic and 157 unpolymorphic SSR markers, in which 41 (38.7%) and 81 (51.6%), 33 (31.1%) and 38 (24.2%), 30 (28.3%) and 36 (22.9%) SSRs were respectively located in the 3'-UTR, 5'-UTR and coding region. Moreover, tetra-nucleotide repeat is the most frequent form in both polymorphic SSRs (67.0%, 24 in the 3'-UTR, 21 in the 5'-UTR and 26 in the coding region) and unpolymorphic SSRs (51.6%, 36 in the 3'-UTR, 22 in the 5'-UTR and 23 in the coding region). A representative set of yellow catfish accessions amplified by primer pair H86 was shown in Figure 3. The selected 57 polymorphic primer pair sequences were characterized and deposited in GenBank to provide a foundation for breeding and genetic research of yellow catfish ( Table 2).
Across the four populations of 48 individuals surveyed, the number of alleles (NA) per locus varied widely among the markers ( Table 2) and ranged from 2 to 17, with an average of 8.23 alleles. We made an analysis of the observed (Ho) and expected heterozygosity (HE). The former value was ranged from 0.04 to 1.00 with an average of 0.52, while the latter varied from 0.12 to 0.92 with an average of 0.70. The high value of mean Ho and HE suggests that there is a relatively high heterozygosity. The overall polymorphic index content (PIC) values were ranged from 0.12 to 0.91 with an average of 0.66. According to the criterion previously described, three categories were defined as high (PIC > 0.5), moderate (0.25 < PIC < 0.5) and low (PIC < 0.25) [21,22]. So these 57 primers exhibited high levels of PIC. Lastly, the fixation index (FIS) values were ranged from −0.83 to 0.93 with an average of 0.25.     Figure 3. PCR amplification profiles of 48 yellow catfish accessions using primer pair H86. The PCR amplified products were separated on 7% polyacrylamide gel. M indicated the molecular markers.

Fish Samples
Four wild populations of yellow catfish (2-3 years old) were collected from Chang Lake (Jingzhou), Hong Lake (Honghu), South Lake (Zhongxiang) and Dongting Lake (Hunan), as described previously [16]. 12 individuals were randomly selected from each population. Experimental protocols used here were approved by the institution animal care and use committee of Huazhong Agricultural University.

SSR Identification and Development of Primer Pairs
We have carried out 454 pyrosequencing technology to perform high-throughput deep sequencing of the yellow catfish transcriptome, with a cDNA library constructed by one RNA pool which has an equal quantity of total RNA extracted from ovary, testis, liver, kidney, muscle, brain, spleen and heart of yellow catfish (accession number of NCBI archive database: SRP032172). All types of SSRs from dinucleotides to hexanucleotides were identified from the assembled contigs and singletons using MISA software under default parameter settings: a minimum of ten repeats for dinucleotide SSRs, six repeats for dinucleotide SSRs, five repeats for trinucleotide, tetranucleotide pentanucleotide and hexanucleotide SSRs. Then we designed primers for the microsatellite sequences using the software Primer Premier 5.0.

Genomic DNA Extraction, PCR Amplification and Electrophoresis
Genomic DNA was extracted from the tail fin following the traditional proteinase K and phenol-chloroform extraction method, as described by Wang et al. [1]. The concentration of DNA was adjusted to 100 ng/μL, and DNA was stored at −20 °C until used.
To initially evaluate the polymorphism of the identified microsatellite markers, polymerase chain reaction (PCR) was performed using a 10 μL total volume that contained 0.5 mM each primer, 0.25μL each dNTP, 0.25 μL PCR buffer, 1 μL MgCl2, 0.5 units of Taq polymerase, and approximate 50 ng DNA. The following conditions were used for the PCR: 1 cycle of denaturation at 95 °C for 5 min and 35 cycles of 30 s at 94 °C, 30 s at a primer-specific annealing temperature, and 45 s at 72 °C. In the final step, the products were extended for 7 min at 72 °C. The PCR products were separated on 7% native polyacrylamide gel and visualized via silver staining. The allele size was estimated according to the pUC18 marker (TianGen Biotech, Beijing, China).

Evaluation of SSR Polymorphism and Genetic Diversity Analysis
To determine the polymorphism of these SSR loci, optimized primers were used to perform PCR reaction with genomic DNA extracted from 48 individuals of these four populations. PCR amplification was performed to accurately screen population-level variation, and PCR products were subjected to electrophoresis 7.0% non-denaturing polyacrylamide gels. To test the level of polymorphism at each EST-SSR locus in four populations , the number of observed alleles (NA), observed heterozygosities (HO) and expected heterozygosities (HE), fixation index (FIS) and polymorphism information content (PIC) values were calculated using POPGENE (Version 1.31) and CERVUS (Version 3.0.3).

Conclusions
By exploiting 454 transcriptome sequencing database, we obtained much information of EST-SSR makers. We not only developed 57 available EST-SSR makers, but also evaluated the population genetics of wild yellow catfish. This is the first report of a comprehensive study on the development and analysis of SSR markers by high-throughput sequencing in yellow catfish. Our results will provide a set of available EST-SSR markers that will be essential for future molecular breeding and genetic studies of yellow catfish.