Development of trinucleotide (GGC)n SSR markers in peanut (Arachis hypogaea L.) 

Mei Yuan¹ · Limin Gong² · Ronghua Meng³ · Shuangling Li¹ · Phat Dang⁴ · Baozhu Guo⁵ · Guohao He*² 

¹Shandong Peanut Research Institute, Qingdao, China
²Department of Agricultural Sciences, College of Agricultural, Environmental and Natural Sciences, Tuskegee University, Tuskegee, AL, USA
³Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, PA, USA
⁴USDA-ARS, National Peanut Research Laboratory, Dawson, GA, USA
⁵USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GA, USA

*Corresponding author: hguohao@tuskegee.edu

Financial support: This work was supported by a grant from the National High Technology Research and Development Program of China (No. 2006AA100106) and partly supported by USDA/CSREES/CBG (No. 00-38814-9541).

Keywords: cultivated peanut, microsatellites, polymorphism, simple sequence repeat.

Cultivated peanut (Arachis hypogaea L.) is an oilseed crop of economic importance. It is native to South America, and it is grown extensively in the semi-arid tropics of Asia, Africa, and Latin America. Given an extremely narrow genetic base, efforts are being made to develop simple sequence repeat (SSR) markers to provide useful genetic and genomic tools for the peanut research community. A SSR-enriched library to isolate trinucleotide (GGC)n SSRs in peanut was constructed. A total of 143 unique sequences containing (GGC)n repeats were identified. One hundred thirty eight primer pairs were successfully designed at the flanking regions of SSRs. A suitable polymerase was chosen to amplify these GC-rich sequences. Although a low level of polymorphism was observed in cultivated peanut by these new developed SSRs, a high level of transferability to wild species would be beneficial to increasing the number of SSRs in wild species.

Cultivated peanut (Arachis hypogaea L.) is one of the most important oilseed crops due to its valuable source of vegetable oil and protein. However, improving peanut production by molecular tools has been difficult because of its narrow genetic base which originated from a single and recent polyploidization event (Young et al. 1996). Simple sequence repeat (SSR) markers are more informative because they are multi-allelic, co-dominant, and abundant in plant genome, compared to other molecular marker systems, such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). Thus, SSR markers are favored as genetic and genomic tools for plant genetic linkage mapping, diversity study, and plant breeding programs. Recent studies have shown that SSRs in different positions of a gene can play important roles in determining protein function, genetic development, and regulation of gene expression (Lawson and Zhang, 2006).

A considerable number of SSR sequences have been identified from peanut genome by several research groups (Hopkins et al. 1999; He et al. 2003; Ferguson et al. 2004; Moretzsohn et al. 2005; Proite et al. 2007; Cuc et al. 2008). SSR markers developed from these repeat sequences offer promising genetic and genomic tools in peanut research. Using SSR markers, genetic diversity of peanut germplasm has been studied in Valencia (Krishna et al. 2004), in mini-core collection (Barkley et al. 2007), and in Chinese (Tang et al. 2007) and Japanese peanut germplasm collections (Naito et al. 2008). SSR markers have been identified and characterized for association with resistance traits such as rust and late leaf spot resistance (Mace et al. 2006), and resistance against Ralstonia solanacearum (Jiang et al. 2007) and Sclerotinia minor (Chenault et al. 2008). Genetic linkage maps with SSR markers have been constructed for diploid AA genome (Moretzsohn et al. 2005), BB genome (Moretzsohn et al. 2009), tetraploid AABB genome derived from a cross of cultivated with amphidiploids (Foncéka et al. 2009), and tetraploid AABB genome in the cultivated peanut (Hong et al. 2008, Varshney et al. 2009; Hong et al. 2010). Although an exceedingly large number of SSRs have been identified, the polymorphic SSR markers may not be sufficient for the construction of a saturated linkage map in the cultivated peanut, provide enough meaningful markers for marker-assisted selection in peanut breeding programs, or sufficient coverage of important domains of the peanut genome for functional genomics research.

A survey of peanut SSR sequences in public data either from genomic DNA or that derived from expressed sequence tags (ESTs) has shown that AG/TC repeats are predominant, followed by AC/TG in dinucleotide repeats. The AAT/TTA repeat is abundant among trinucleotide repeats in peanut (Ferguson et al. 2004; Moretzsohn et al. 2005). The trinucleotide (AAT)n SSRs also showed high frequencies in other legume species such as soybean (Gao et al. 2003) and Medicago (Eujayl et al. 2004). However, CCG/GGC repeat is the most common repeats in monocot species (Cordeiro et al. 2001; Morgante et al. 2002; Varshney et al. 2002). Although Zhao and Kochert (1993) have mentioned that (GGC)n microsatellites were present in peanut, no detailed information on this type of SSR was available because only a few (GGC)n SSRs were isolated either from genomic DNA or ESTs to date. Therefore, the objectives of this study were to isolate (GGC)-SSR sequences to assess their abundance in peanut genome, and to test their informative content.

Construction of SSR-enriched library

A method of obtaining SSR-enriched library modified from the procedures of Kijas et al. (1994), Hakki and Akkaya (2000), and Reddy et al. (2001) was used to develop SSR markers (He et al. 2003). Briefly, genomic DNA was isolated from peanut leaves using the MasterPure^TM Plant Leaf DNA purification kit (Epicentre, Madison, WI). The DNA was digested by HindIII and MseI, and the restriction fragments were ligated by corresponding adapters and amplified following the AFLP protocol (Vos et al. 1995). The biotinylated SSR probe (GGC)₁₅ was used to hybridize the denatured pre-amplified fragments. The hybridized mixture was added to streptavidin-coated paramagnetic beads. The DNA-probe hybrids were incubated at room temperature, and a magnetic field was applied to precipitate the beads, which were attached by SSR-containing fragments that hybridized to biotinylated probes. The SSR-enriched fragments were amplified by polymerase chain reaction (PCR), products were cloned into the TA-cloning vector pCR4-TOPO utilizing topoisomerase-mediated ligation (Invitrogen, San Diego, CA), ligated vectors were transformed into chemically competent E. coli TOP10 and plated onto Luria-Bertani plates (LB) with antibiotic selection. Single colonies were selected, grown overnight in LB. Plasmids were purified and sequenced.

Marker development

SSR-containing sequences were used for primer designing using the software Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The designed primers were tested for amplification using one genotype DNA. Because amplicons are GC-rich sequences, PrimeSTAR HS DNA polymerase produced by TaKaRa Bio Inc. (Shiga, Japan) was used for PCR amplification instead of Taq DNA polymerase. PCR reactions were performed in 10 µl volumes, containing 1 x PrimeSTAR GC buffer (100 mM Tris-HCl, pH 8.3, 10 mM KCl, 2 mM MgCl₂, (NH4)₂SO₄), 2.5 mM of each dNTP, 0.25 U of PrimeSTAR HS DNA polymerase, 0.375 µM of each primer and 10 ng of genomic DNA. Amplifications were conducted using a DNA Engine Dyad (BioRad, CA, USA) thermal cycler, with the PCR program: 98ºC for 3 min (1 cycle), 98ºC for 10 sec, 54-60ºC for 5-10 sec, 72ºC for 1 min (30 cycles), and 72ºC for 7 min (1 cycle). The annealing temperature and time were optimized for each primer. The PCR products were separated on 6% non-denaturing polyacrylamide gels stained with ethidium bromide.

Plant material and DNA extraction

A subset of 20 genotypes was selected from the mini core collection in peanut (Holbrook and Dong, 2005). Four wild genotypes were added to this material panel for the polymorphism test. The panel represents diverse genotypes collected from both different original countries and botanical varieties (Table 1). Total genomic DNA was isolated from young leaves of each plant using the same DNA purification kit as described above. The DNA concentration was diluted to 10 ng/µl for PCR amplification.

A total of 768 clones from the (GGC)n SSR-enriched library were sequenced, of which 156 (20.3%) contained SSRs. After removing redundancy, 143 unique SSR sequences were deposited in GenBank (accession number AY526357 - AY526456; AY731521 - AY731558). Using (GGC)₁₅ as a probe, the isolated SSRs were all GGC/CCG repeats except one containing CAT/GTA repeats. Among (GGC)n SSRs, 90.6% were perfect repeats, 3.6% imperfect, and 5.8% were compound repeats. The number of (GGC) repeats was in the range of (GGC)₅ to (GGC)₂₁, with 80% repeat number between (GGC)₉ and (GGC)₁₅. The frequency of (GGC)n repeats showed a bell-shape distribution (Figure 1).

One hundred forty three primer pairs were designed based on the flanking sequences of SSRs. The primer sequences, their melting temperature (Tm), and product size are listed in Table 2. Using regular Taq DNA polymerase, only 47.1% of the primer pairs yielded amplicons. This is due to the fact that GC-rich sequences can form a complex secondary structure preventing PCR amplification. In order to resolve the complex structure formation, some additives and enhancing agents including dimethyl sulfoxide (DMSO), betaine, formamide, and glycerol were used in many reports (Henke et al. 1997; Kang et al. 2005; Musso et al. 2006). In this study, the PrimeSTAR DNA polymerase (TaKaRa Bio Inc. Japan) was used because it is especially useful in the PCR amplification of GC-rich sequences. Therefore, of the 143 primer pairs, 138 could produce amplicons with this polymerase and its specific buffer, and only 5 (PM630, PM694, PM695, PM711 and PM714) did not show any PCR products for both wild and cultivated genotypes.

Further, of the 138 (GGC)n SSRs, the majority of them detected a polymorphism among 4 wild genotypes (Figure 2a), while only 6 revealed variation among 20 cultivated accessions (Figure 2b). The level of polymorphism detected by (GGC)n markers was surprisingly low in this study.

The enrichment approach was used to isolate trinucleotide SSRs using (GGC)₁₅ as a probe, with the efficiency of isolation being as low as 20.3%. Our previous report on isolation of dinucleotide GA/CT repeat SSRs (He et al. 2003) showed a high efficiency (61%) of capturing sequences containing SSRs using the similar approaches. Low efficiency in this study may indicate that there is less abundance of (GGC)n SSRs in peanut genome, thereby lessening the chance for the probe to be hybridized with DNA sequences containing GGC/CCG repeats.

The low frequency of (GGC)n SSR in this study, as well as in other legume species, could be explained by two facts (Mun et al. 2006). The methylation of cytosine could have increased the rates of mutation to thymine, or the (GC)n repeats were selected against due to the increased stability of (GC)n hairpin structures. However, (GGC)n SSRs were predominant in monocot species. It is believed that the majority of variation of repeats in a SSR locus has resulted from slippage during DNA replication (Levinson and Gutman, 1987). But, strand-slippage theories alone are insufficient to explain the differential abundance of specific repeat types in different genomes (Mun et al. 2006). In rice, (GGC)n SSRs were observed preferentially in exons (Cho et al. 2000), while Mun et al. (2006) suggested that a positive selection pressure, such as a preference of codon usage in exons or a regulatory effect of specific repeats in noncoding regions, may underlie the taxa-specific accumulation of certain repeat types. The taxa-specific SSRs originated after divergence of legume from monocot (Mun et al. 2006).

In three previous reports on peanut, the frequencies of repeats showed a bias towards low number in both dinucleotide and trinucleotide SSRs (Moretzsohn et al. 2005; Proite et al. 2007; Guo et al. 2009). These results were calculated from the combination of all types of SSR repeats. The mutation rate of SSRs increases with repeat number, but long SSRs in eukaryotic genomes have a mutation bias to become shorter SSRs (Hong et al. 2007). The SSRs with repeat number less than a certain threshold are stable during mitosis and meiosis, whereas above a certain threshold the SSRs become extremely unstable as shown in humans (Strachan and Read, 1999). The current result was derived from one type of SSR repeat (GGC)n. The bell-shape (no bias to either end) distribution of repeat number in (GGC)n SSRs in this study might suggest a very low and random mutation rate of all repeats in (GGC)n SSR in the peanut genome.

Optimum annealing times could also improve PCR amplification of GC-rich sequences. Mamedov et al. (2008) pointed out that shorter annealing times are not only sufficient but also necessary for efficient PCR amplification when GC-rich templates are being used. They demonstrated that 3-6 sec was sufficient, but depended on annealing temperature. When annealing time was increased, a smear was observed when products were separated using gel-electrophoresis. The manufacturer of PrimeSTAR DNA polymerase also recommended using 5 sec as annealing time. The annealing time was optimized in this study for those GGC-SSR primers. We have tested various annealing time from 5 sec to 10 sec and annealing temperature from 54 to 60ºC. The result showed that annealing time (10 sec) and annealing temperature (58ºC) were suitable for amplification of most primers. When annealing temperature was increased, more bands appeared compared to low temperatures.

A comparison of trinucleotide with dinucleotide repeats for polymorphism in peanut has resulted in different opinions by several peanut research groups. Mace et al. (2006) have reported that markers for dinucleotide repeats tend to detect a greater number of different alleles than trinucleotide repeat markers. Proite et al. (2007) also stated that dinucleotide repeats are more polymorphic than trinucleotide repeats. However, Cuc et al. (2008) reported otherwise, stating that trinucleotide SSRs showed higher allele numbers than dinucleotide SSRs. In this study, only 4.3% GGC SSRs were polymorphic based on 24 diverse genotypes. Compared to our previous results showing 33.9% polymorphic markers in dinucleotide SSRs (He et al. 2003), the current result corroborates that dinucleotide SSRs detect more DNA variation than trinucleotide SSRs.

To date, a large number of SSRs have been detected in peanut, but only about 10% of them are polymorphic (unpublished data). The effort has been made to obtain SSR markers from both genomic and EST sources. The (GGC)n SSRs identified in this study represents a new type of SSR markers in peanut with related insights into their abundance and information utility. Despite the low number of polymorphic markers in cultivated peanut, two SSR markers were mapped to an existing linkage map of cultivated peanut (data not shown). A high level of transferability of (GGC)n SSRs to wild species facilitates the construction of genetic linkage maps in wild species, and accelerates the introgression of wild useful genes into cultivated peanut.

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