Abstract
Fifty-seven polymorphic loci were isolated from a microsatellite-enriched goose (Anser cygnoides) genomic library. The number of alleles per locus ranged from 2 to 10. The expected and observed heterozygosities ranged from 0.05 to 0.95 and from 0.04 to 1.00, respectively. Forty-seven out of 57 loci conformed to the Hardy–Weinberg equilibrium, and no significant linkage disequilibrium was found in the pairwise comparisons among the 57 loci. The microsatellite markers isolated and characterised in this study will contribute to the construction of a genetic map and mapping of quantitative trait loci in geese, as well as to phylogenetic studies of this species.
Introduction
Goose (Anser cygnoides) is an agriculturally important bird that is widespread and abundant in China (Li et al. Citation2007). Construction of the goose genetic map will provide insight into the genome organisation and chromosomal localisation of cloned genes, and provide a framework for the identification and location of major genes associated with economically important traits (Mohsen and Ghodrat Citation2007). Many polymorphic genetic markers must be isolated before constructing this map, including genomic microsatellite markers in particular. Microsatellites, also known as single sequence repeats (SSRs), are small arrays of two to six bases arranged in tandem repeats spread throughout the genomes (Gao et al. Citation2003). Microsatellites are advantageous over many other genetic markers because they are highly polymorphic, highly abundant, codominantly inherited, analytically simple and readily scored (Olowofeso et al. Citation2005). SSRs have now been recommended as standard markers to be used in the construction of highly saturated maps, and in some cases, in marker-assisted selection (Mohsen and Ghodrat Citation2007; Zhang et al. Citation2010). Although microsatellite markers have been isolated in goose (Cathey et al. Citation1998; Du et al. Citation2005; Tu et al. Citation2005), the number and availability are still limited. In this study, we have isolated and characterised 57 novel microsatellite markers in goose.
Materials and methods
DNA extraction and purification
Blood samples were taken from the wing vein of 30 unrelated geese (15 males and 15 females) of a breeding population (Changxing City, China) and stored at 20°C. Genomic DNA was extracted from whole blood using the Tadano et al. (Citation2007) method.
Enrichment of SSR by magnetic beads
We constructed a goose genomic library enriched with (CA)n repeats, following a combination of modified procedures according to Carleton et al. (Citation2002) and Huang et al. (Citation2005). Briefly, genomic DNA derived from one female Landaise goose was digested with Hae III and Rsa I (Promega, Madison, WI, USA). Fragments 400–1400 bp in length were recovered from 1% low melting point agarose gels, and ligated to an adaptor (consisting of a 21-mer: 5′-CTCTTGCTTGAATTCGGACTA-3′; and a phosphorylated 25-mer: 5′-pTAGTCCGAATTCAAGCAAGAGCACA-3′) with T4 DNA ligase (Takara, Dalian, China). A pre-hybridisation polymerase chain reaction (PCR) amplification was then performed in a thermal cycler (Bio-Rad) using the above 21-mer oligonucleotide as unique primer (10 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min). For enrichment, the DNA was denatured and hybridised to a biotinylated probe [B-ATAGAATAT(CA)12] in 3×SSC/0.1% SDS at 68°C for 20 min. The DNA hybridised to the probe was then captured with streptavidin magnetic beads and washed (twice in 6×SSC/0.1% SDS at room temperature, twice in 3×SSC/0.1% SDS at 68°C, and twice in 6×SSC at room temperature). Following purification, the DNA was denatured from the beads in 0.1×TE at 95°C and PCR-amplified using the 21-mer oligonucleotide primer with the following thermal profile: 20 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min.
Positive clone sequencing and primer design
The PCR products were purified, ligated to pMD 19-T vector (Takara) and transformed into JM109 cells. Plasmids were extracted from positive clones of the microsatellite-enriched library and sequenced on an ABI PRISM 377 sequencer according to the manufacturer's instructions. When the sequence data were available, unique SSR primers were designed from the regions flanking the repeat motifs using Primer 3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and were synthesised by Takara Biotechnology Co, Ltd. (Dalian, China).
PCR amplification and genotyping
All the primer pairs were tested in 30 unrelated individuals (15 males and 15 females) from a breeding population (Changxing City, China). The PCRs were performed in a 20-µL volume containing 2.5U Taq DNA polymerase (Takara), 1×PCR buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM of each primer and 50 ng goose DNA. Thermal profile for the PCR was: 5 min at 95°C, 30 cycles of 40 s at 94°C, 30 s at primer-specific annealing temperature (), 30 s at 72°C, and a final 10 min elongation step at 72°C. Electrophoresis was performed using polyacrylamide gel electrophoresis (6% gel) with a 100-bp ladder molecular size standard (Takara) to estimate allele sizes via silver staining. The allele number, size range and number of bands per individual were quantified using QUANTITY ONE v. 4.4 (Bio-Rad).
Table 1. Characterisation of 57 polymorphic microsatellites loci isolated from Anser cygnoides.
Statistical analysis
The expected (H E) and observed heterozygosity (H O) were calculated using POPGENE32 v. 1.32 (Yeh et al. Citation2000). Tests for Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were conducted using GENEPOP v. 3.4 (Raymond and Rousset Citation1995). Sequential Bonferroni correction was used to adjust the P-values for multiple statistical comparisons (Rice Citation1989). Significance of scoring error, due to stuttering, large allele dropout, and null alleles, was analysed at 95% confidence interval by MICRO-CHECKER 2.2.3 software (Van Oosterhout et al. Citation2004).
Results and discussion
SSR enrichment, cloning and sequencing
To efficiently isolate microsatellites, a library of (CA)n enriched microsatellites was constructed with the procedure described in the Materials and Methods section. PCR screening was amplified to detect positive clones from the enriched library using (CA)12 and the 21-mer oligonucleotide as primers. Out of 331 recombinant clones, the PCR products of 318 (96.1%) were found to contain two bands, which indicated that these clones were likely to be positive (Lench et al. Citation1996). Positive clones were sequenced in both directions, and a consensus sequence was created. Of the 318 sequenced clones, 240 (75.5%) contained (CA)n or (TG)n microsatellites with five or more repeats (Carleton et al. Citation2002; Huang et al. Citation2005). Compared with previous reports, the efficiency of microsatellite isolation by the method of magnetic bead enrichment is much higher than other traditional protocols (Cathey et al. Citation1998; Du et al. Citation2005). We also found other kinds of microsatellites in this microsatellite-enriched library [e.g. (ACT)n, (TTC)n]. Similar results were also observed in previous studies (Huang et al. Citation2005; Wang et al. Citation2008). Of the isolated microsatellite sequences, 197 unique sequences had repeats of sufficient length and adequate flanking sequence to design primers (GenBank accession numbers: GU130819–GU131015).
Characterisation of microsatellites
The PCR conditions of each primer pair were optimised using a goose genomic DNA with Gene Amp Gradient PCR System (Bio-Rad), and polymorphisms were detected with genomic DNA of 30 unrelated individuals. A total of 57 primer pairs exhibited length polymorphisms in 30 detected individuals (). Allele number ranged from 2 to 10, with an average of 4.2 per locus, which was consistent with the result of Du et al. (Citation2005). The H E and H O ranged from 0.05 to 0.95 and from 0.04 to 1.00, respectively. Ten loci significantly deviated from HWE (), which may have been due to the small sample size or the existence of null alleles. The MICRO-CHECKER analysis suggested that there were no indications for scoring error due to stuttering and large allele dropout. However, it did show evidence for null alleles presenting in the 10 loci. No loci showed significant LD. The microsatellite markers characterised here will contribute to the construction of genetic linkage maps and the identification of quantitative trait loci for maker-assisted selection in this economically important species.
Acknowledgements
The Science and Technology Department of Zhejiang province (2007C12058; 2009C12079) and the earmarked fund supported this work for Modern Agro-industry Technology Research System, China (NYCYTX-45-02).
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