Genes & Genomics (2011) 33: 313-316 DOI 10.1007/s13258-011-0021-5
SHORT COMMUNICATION
Development, characterization, and inheritance of 113 novel EST-SSR markers in the Pacific oyster (Crassostrea gigas) Qi Li · Qingzhi Wang · Mingjun Qi · Jianlong Ge · Rihao Cong 1)
Received: 24 January 2011 / Accepted: 06 March 2011 / Published online: 30 June 2011 © The Genetics Society of Korea and Springer 2011
Abstract A total of 113 novel EST-derived simple sequence repeat (EST-SSR) markers were developed in the Pacific oyster (Crassotrea gigas). Polymorphisms of these markers were evaluated in a wild population of 30 individuals. The number of alleles per locus ranged from 2 to 27 with an average of 6.3, and the observed and expected heterozygosities varied from 0 to 0.9667 and from 0.0333 to 0.9701, respectively. Mendelian segregations were tested for 24 of the markers that were polymorphic in one family produced by single-pair mating. Null alleles were discovered at four loci. Nine tests of segregation ratios revealed significant departures from expected Mendelian ratios. As a useful addition to the collection of the microsatellites that are now available for C. gigas, these EST-SSR markers will help the advance in investigation of QTL mapping and genetic diversity in this species.
Keywords Pacific oyster; Crassotrea gigas; EST-SSR markers
Introduction The Pacific oyster (Crassotrea gigas), primarily originated from China, Japan and Korea of East Asia, is a commercially important species. Since the 1940s, the Pacific oyster has been introduced to many oyster-culturing countries all over the world because of its fast growth rate, high production and well acclimatization. It has had the highest worldwide production of any cultured aquatic species since 1993; in 2007, the world production of this species was 4.2 million metric tons (FAO, 2009). China produced over 3.5 million tons of cultured oysters in 2007 (DOF, 2008), and C. gigas is one of the most
Q. Li( ) · Q. Wang · M. Qi · J. Ge · R. Cong Key Laboratory of Mariculture Ministry of Education, Ocean University of China, Qingdao 266003, China e-mail:
[email protected]
important species. C. gigas is cultured in all parts of the coast by different methods depending on the environment, but the major producers are Liaoning and Shandong provinces in the north, and Fujian and Guangdong provinces in the south. Although China has a long history of oyster aquaculture, oysters are in an early stage of domestication. Genetic studies, which offer great potential to detect associations between allelic forms of a gene and phenotypes, will accelerate the development of the oyster industry. Simple sequence repeats (SSRs), or microsatellites, are extremely useful markers for genetic linkage mapping because of their high polymorphism, abundance, codominance and small length, which facilitates genotyping using polymerase chain reaction (PCR). To date, about 378 SSRs were developed in C. gigas (Magoulas et al., 1998; Huvet et al., 2000; McGoldrick et al., 2000; Li et al., 2003; Sekino et al., 2003; Yamtich et al., 2005; Yu and Li, 2007, 2008; Wang et al., 2008; Qiu et al., 2009a, 2009b; Sauvage et al., 2009; Li et al., 2009; Qi et al., 2009; Yu et al., 2010; Bai et al., 2011), including 164 genomic SSRs and 214 expressed sequence tag derived SSRs (EST-SSRs). These SSR markers provide sufficient resource in this species to evaluate wild and cultured genetic resources, but are still deficient for SSR-based mapping studies, which is necessary to the identification and mapping of quantitative trait loci (QTL), and marker-assisted selection (MAS) (Hubert and Hedgecock, 2004). SSRs linked to genes (type I markers) are generally more conserved than anonymous type II markers, associated with unannotated genomic regions, which enable comparative mapping among distant evolutionarily organisms and permit locating the genes in linkage map (Serapion et al., 2004). Expressed sequence tag databases are a rich source for obtaining SSR markers for a variety of organisms including aquaculture species. In this study, we developed and characterized a set of 113 new EST-SSRs for C. gigas. To ensure accuracy of genotyping results, the Mendelian inheritance for the markers is determined from offspring collected from a full-sib family.
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Materials and Methods DNA extraction from C. gigas To evaluate polymorphism, 30 wild Pacific oysters collected from coastal waters in Weihai, Shandong province, China, were used. Genomic DNA was extracted from adductor muscle by standard proteinase K digestion, phenol-chloroform extraction, and DNA precipitation. Primer design A total of 28150 ESTs obtained from the GenBank (May 10, 2010) database were screened using SSRHUNTER program (Li and Wan, 2005) that was designed to find regions containing SSRs. The parameters were set for detection of di-, triand tetranuclotide motifs with a minimum of five repeats, respectively. A total of 1063 SSR loci were identified and 300 were selected for SSR marker optimization. Primers flanking microsatellite were designed using the PRIMER PREMIER 5.0 program (PREMIER Biosoft International, USA). The major parameters for primer design were set as follows: primer length from 18 to 24 nucleotides, PCR product size from 100 to 350 bp, optimum annealing temperature at 55-65℃, and GC contents from 40% to 60%. Microsatellite analysis PCR amplifications were performed in a total of 10-μl volumes containing 0.25 U Taq DNA polymerase (Takara, Japan), 1× PCR buffer, 0.2 mM dNTP mix, 1 μM of each primer set, 1.5 mM MgCl2, and about 100 ng template DNA. PCR was performed on a GeneAmp 9700 PCR System (Applied Biosystems, USA) as follows: 3 min at 94℃, 35 cycles of 45 sec denaturation at 94℃, 45 sec at the annealing temperatures described in Table S1, and 45 sec extension at 72℃, and an additional 5 min extension at 72℃ at the end of the 35 cycles. The amplification products were then resolved by 6% denaturing polyacrylamide gel, and visualized via silver staining. A 10-bp DNA ladder (Invitrogen, USA) was used as a reference marker for allele size determination. Statistical analysis The number of alleles and the levels of expected, observed heterozygosity (HE, HO) of these SSR loci were estimated by using the Microsatellite Analyzer software (Dieringer and Schlötterer, 2003). Tests of deviation from Hardy-Weinberg equilibrium (HWE) in SSR markers were performed using GENEPOP 4.0 (Rousset, 2008). Segregation analysis The inheritance patterns of EST-SSRs alleles were studies in up to 85 F1 offspring in one family produced by single-pair mating in 2008. To ascertain if alleles are inherited in a Mendelian fashion, all observed progeny ratios of each primer set were tested against the expected Mendelian segregation ra-
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tios (1:1, 1:2:1, and 1:1:1:1) using chi-square analysis (with n - 1 degrees of freedom, where n = number of phenotypic classes).
Results and Discussion Of the 300 potential SSR makers, 242 primer pairs amplified the expected products, and 58 were not easily amplified. There were several reasons for failing in amplification. Priming site polymorphism in the template leads to inferior or erratic amplification, introns in the target amplicon are too large and numerous to allow effective amplification under standard screening conditions, and a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template (Kim et al., 2010). Of the 242 primer pairs, 113 showed polymorphism in the population of C. gigas (Table S1), while the others were monomorphic. Five loci (CgEH84, CgEH143, CgEH148, CgEH183, and CgEX67) had a bigger size than expected, which might be caused by the presence of introns. Because the cDNAs from which ESTs are derived lack introns, one possible concern with EST-SSRs is that unrecognized intron splice sites could disrupt priming sites, resulting in failed amplification. Alternatively, large introns could fall between the primers, resulting in a product that is either too large or, in extreme cases, failed amplification. All the polymorphic loci are different from the published EST-SSRs (Yu and Li, 2007, 2008; Wang et al., 2008; Qiu et al., 2009a,b; Sauvage et al., 2009; Li et al., 2009; Yu et al., 2010; Bai et al., 2011). The number of alleles per locus ranged from 2 to 27 with an average of 6.3. The observed and expected heterozygosities varied from 0 to 0.9667 and from 0.0333 to 0.9701, respectively. Although the variability observed in the EST-SSRs is possibly underestimated due to small sample size (30 individuals), it was still much higher than that of allozymes in the Pacific oyster population (Ozaki and Fujio, 1985). The high level of length variation of the EST-SSRs found here is similar to previous reports in the Pacific oyster (Wang et al., 2008; Sauvage et al., 2009), and comparable to those of the eastern oyster, blue mussel, and zhikong scallop (Wang and Guo, 2007; Yu and Li, 2007; Zhao and Li, 2008). GenBank (BLAST) searches indicated that 36 of 113 SSR-ESTs matched to genes of known functions at E values less than 10-4, whereas the other 77 had no significant matchs to known genes (Table S1). Forty-five of the 113 loci deviated significantly from HWE after Bonferroni correction due to homozygote excess. The departure from HWE with an excess of homozygotes may be the result of one or more of the following reasons. (1) Large allele “dropout” artifacts in the PCR amplification process: In heterozygous individuals, preferential amplification of a smaller allele over a larger allele would result in the mis-scoring of heterozygotes for homozygotes even though larger alleles may indeed exist (Banks et al., 1999). (2) Small sample size:
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Table 1. Segregation of 24 C. gigas EST‐SSRs in a full‐sib family. Locus Sire Dam Genotypes of progeny Expected ratio Observed ratio CgEH03 AB AB AA:AB:BB 1:2:1 4:33:23 CgEH07 AB AC AA:AB:AC:BC 1:1:1:1 0:26:12:37 CgEH11 AB BB AB:BB 1:1 50:35 CgEH22 AB BB AB:BB 1:1 42:23 CgEH34 BC AB AB:AC:BB:BC 1:1:1:1 12:23:17:32 CgEH51 AB AB AA:AB:BB 1:2:1 5:43:22 CgEH99 AB AN AA (AN):AB:BN 2:1:1 21:26:24 CgEH103 AB BC AB:AC:BB:BC 1:1:1:1 15:13:13:44 CgEH123 AC BC AB:AC:BC:CC 1:1:1:1 30:28:14:13 CgEH126 AB BB AB:BB 1:1 53:28 CgEH134 BN AB AB:AN:BB (BN) 1:1:2 20:28:36 CgEH141 AB AC AA:AB:AC:BC 1:1:1:1 0:16:22:16 CgEH142 AB BC AB:AC:BB:BC 1:1:1:1 12:14:17:37 CgEH148 AB BC AB:AC:BB:BC 1:1:1:1 31:22:9:17 CgEH169 AB AB AA:AB:BB 1:2:1 26:36:6 CgEH172 AC AB AA:AB:AC:BC 1:1:1:1 3:25:24:29 CgEH173 AB BC AB:AC:BB:BC 1:1:1:1 15:28:6:22 CgEH183 AB AB AA:AB:BB 1:2:1 21:41:20 CgEH187 AB BN AB:AN:BB (BN) 1:1:2 37:21:26 CgEX11 BN AN AB:AN:BN:NN 1:1:1:1 40:18:20:2 CgEX51 AB AB AA:AB:BB 1:2:1 11:44:30 CgEX101 AB AB AA:AB:BB 1:2:1 7:46:32 CgEH184 AA AB AA:AB 1:1 13:17 CgEX82 AA AB AA:AB 1:1 3:27 * Significant deviation (P < 0.05) from expected Mendelian ratios after Bonferroni correction (k = 24). N represents inferred null alleles.
As microsatellite DNA has a rapid mutation rate, resulting large number of alleles, a large sample size is needed for accurate reflection of genotypic frequencies (Ruzzante, 1998). (3) Presence of null alleles: Null alleles of microsatellite regions, which occasionally fail to yield an amplification product, may arise through mutations such as point mutations in the primer annealing site (Pemberton et al., 1995). (4) Inbreeding effects: Extensive heterozygote deficiency has also been reported at allozyme loci in natural populations of the Pacific oyster (Ozaki and Fujio, 1985). All the 113 polymorphic EST-SSRs were analyzed in one full-sib family. Eighty-nine loci were monomorphic (AA × AA genotype), and thus resulted in offspring identical to the parents. The remaining 24 loci were polymorphic and segregated in the family. Genotypic frequencies in parents and offspring at each of 24 loci are shown in Table 1. Fifteen genotypic ratios were in accordance with Mendelian expectations after Bonferroni correction. Two genotypic ratios (CgEH99 and CgEH134) conformed to Mendelian expectations when we assumed that both parents carried a null allele in the heterozygote state, and unexpected offspring genotypes were homozygotes and heterozygotes for null alleles. Null alleles for single-copy, PCR-based DNA markers are often the result of polymorphisms in the microsatellite flanking regions of DNA
P‐value 0.0018* 0.0000* 0.1037 0.0184 0.0143 0.0026 0.0025 0.9776 0.0096 0.0055 0.1981 0.0002* 0.0550 0.0049 0.0025 0.0002* 0.0017* 0.9879 0.0001* 0.0000* 0.0136 0.0005* 0.4652 0.0000*
to which PCR primers are designed to bind (Callen et al., 1993; Jones et al., 1998). Genoptypic ratios of the nine SSRs (CgEH03, CgEH07, CgEH141, CgEH172, CgEH173, CgEH187, CgEX11, CgEX101, and CgEX82) showed significant deviation from expected genotype frequencies (Table 1). Many marine invertebrates, and particularly bivalve molluscs, frequently exhibit non-Mendelian segregation ratios of alleles, which can confound the creation of a linkage map (Reece et al., 2004). Naciri et al. (1995) suggested that high genetic load with resulting strong zygotic selection during the larval stage was the cause of segregation distortion in the flat oyster (Ostrea edulis), a phenomenon recently demonstrated experimentally in C. gigas families by Launey and Hedgecock (2001). These EST-SSR markers should prove to be a useful addition to the collection of the microsatellites that are now available for C. gigas. Given the levels of polymorphism, the markers will help the advance in investigation of QTL mapping and genetic diversity in this species. Acknowledgement This study was supported by the grants from 973 Program (2010CB126406), Marine Public Welfare Research Program (200905020) and National Natural Science Foundation of China (31072207).
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