Introduction
Genetic resources are important materials for crop improvement and the bioindustry as sources of valuable alleles, but biodiversity has been eroded with the worldwide reduction in habitats and environmental change. Global efforts have been concentrated on the conservation of threatened plant genetic resources, including landraces and wild species (Ortiz and Engels, Reference Ortiz, Engels and de Vicente2004).
The RDA Genebank at the National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration (RDA), Republic of Korea, is working at preserving agricultural genetic resources based on the Global Action Plan for the Conservation and Sustainable Use of genetic diversity. The total number of orthodox seeds in the RDA Genebank reached 182,013 accessions in 1777 species in 2012 (Table 1), and a total of 28,027 accessions (996 species) of vegetatively propagated resources are maintained in research stations. Of the conserved orthodox seeds, 75.3% are those of cereal crops and 11.7% are those of horticultural crops. Underutilized crops, such as millets, garlic and safflower, comprise a minor group in terms of the numbers conserved and economic importance, but have special nutritional benefits and various bioactive materials, such as essential amino acids, minerals and vitamins (Nelson et al., Reference Nelson, Naylor and Jahn2004).
Table 1 Status of conserved seed genetic resources in the RDA Genebank
Although the genetic diversity of conserved resources is crucial for germplasm management and utilization, the molecular evaluation tools for underutilized crops are insufficient. Specifically, several research groups are developing genomic simple sequence repeat (SSR) markers for species such as buckwheat (Fagopyrum esculentum; Konishi et al., Reference Konishi, Iwata, Yashiro, Tsumura, Ohsawa, Yasui and Ohnishi2006) and safflower (Carthamus tinctorius L.; Hamdan et al., Reference Hamdan, García-Moreno, Redondo-Nevado, Velasco and Pérez-Vich2011), the genus Citrus (Kijas et al., Reference Kijas, Thomas, Fowler and Roose1997; Ahmad et al., Reference Ahmad, Struss and Southwick2003), and garlic (Allium sativum L.; Ma et al., Reference Ma, Kwag, Zhao, Dixit, Lee, Kim, Chung, Kim, Lee and Ji2009b). However, more SSR markers might be required to estimate genetic diversity and population structure compared with those for major crops such as rice and soybean.
Microsatellites are SSRs that are distributed throughout the plant genome and reveal significant allelic diversity. Compared with other genetic markers such amplified fragment length polymorphisms and randomly amplified polymorphic DNA, microsatellite markers are widely used for genetic diversity analysis and genetic map construction in that these markers are easy to detect by polymerase chain reaction (PCR) and have multiallelic nature with high reproducibility (Ni et al., Reference Ni, Colowit and Mackill2002). Furthermore, these markers are adequate for the genetic characterization of several crop species because they are transferable to related species (Peakall et al., Reference Peakall, Gilmore, Keys, Morgante and Rafalski1998; Varshney et al., Reference Varshney, Graner and Sorrells2005; Lee et al., Reference Lee, Kwon, Park, Lee, Kim, Lee, Lee, Gwag, Kim and Ma2011).
In this regard, the Korean National Agrobiodiversity Center has been striving to develop microsatellite markers for the genetic assessment of several underutilized crops. This paper summarizes these activities.
Materials and methods
A total of 956 accessions appertaining to 21 crop species (14 dicotyledonous crops, six monocotyledonous crops and one mushroom) were obtained from the National Agrobiodiversity Center (NAAS, RDA, Republic of Korea) and used for microsatellite marker development and genetic assessment (Table 2). Genomic DNA was extracted basically with a modified cetyltrimethylammonium bromide (CTAB) method (Dellaporta et al., Reference Dellaporta, Wood and Hicks1983) using young leaves and quantified using a UV–Vis spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington, DE, USA).
Table 2 Summary of the microsatellite markers developed for underutilized crops
SSR, simple sequence repeat; N, used accession number; N A, average number of alleles in each analysed set; H E, average expected heterozygosity in each analysed set.
a Motifs among the polymorphic SSR loci.
b SSR clones acquired from the pyrosequencer.
A modified biotin–streptavidin capture method (Dixit et al., Reference Dixit, Jin, Chung, Yu, Chung, Ma, Park and Cho2005) was used for acquiring SSR clone sequences except for two species (C. tinctorius L. and Gerbera hybrida H.) with the pyrosequencing method (Lee et al., Reference Lee, Sung, Lee, Chung, Yi, Kim and Lee2013). The search for SSR motifs and designing of primer pairs were carried out using the ‘SSR Manager’ software (Kim, Reference Kim2004). After checking the proper amplification products, we analysed whether PCR products were polymorphic in a diversity set including eight accessions of each species. The selected polymorphic microsatellite markers were applied to each collection for diversity analysis (Table 2).
Results and discussion
Of the 6310 SSR clones in 21 crop species (14 dicots, six monocots and one mushroom), some motifs were skewed within the acquired sequences and were not appropriate for designing primers. Among the genomic SSR motifs, dinucleotide repeats appeared to be more frequently present in plant genomes, while expressed sequence tag–SSRs involved more trinucleotide repeats (Wang et al., Reference Wang, Weber, Zhong and Tanksley1994; Cardle et al., Reference Cardle, Ramsay, Milbourne, Macaulay, Marshall and Waugh2000; Gao et al., Reference Gao, Tang, Li and Jia2003). In our study, most of the dicots had a higher frequency of dinucleotide motifs, while trinucleotide motifs were more frequently found in most of the monocots (Table 2). Regarding the ratio of GA to GT motifs, GA motifs are more prevalent than GT motifs in plants (Lagercrantz et al., Reference Lagercrantz, Ellegren and Andersson1993; Cardle et al., Reference Cardle, Ramsay, Milbourne, Macaulay, Marshall and Waugh2000; Katti et al., Reference Katti, Ranjekar and Gupta2001), and GA motifs were found to be more prevalent in our study, with some exceptions, such as garlic and unishiu orange (Citrus unshiu).
We designed 3640 primer pairs flanking microsatellite motifs. These ranged from 50 primer pairs for sesame (Sesamum indicum) to 509 for safflower (Table 2). From the designed primer pairs, polymorphic loci across the eight accessions of each species were selected; the number of loci ranged from seven (Vigna radiata L.) to 36 (Pleurotus ostreatus) by species. The average proportion of polymorphic loci to the total number of loci tested was 12% (5% for Zingiber officinale Rosc. to 24% for Actinidia sp.). Further polymorphic loci might be revealed and applied for the genetic assessment of underutilized crops as we searched for polymorphisms in a limited set including only eight accessions per species.
Genetic variability was analysed as an index of allele number and expected heterozygosity in polymorphic loci, while the number of accessions analysed differed among the crop collections. The average allele number was 5.1 (2.8 for C. tinctorius L. to 10.3 for Actinidia sp.) and the expected heterozygosity was 0.51 (0.31 for Ricinus communis L. to 0.74 for Amaranthus hypochondriacus). Peakall et al. (Reference Peakall, Gilmore, Keys, Morgante and Rafalski1998) and Varshney et al. (Reference Varshney, Graner and Sorrells2005) demonstrated that SSRs could be transferred to closely related species and genera. The transferability of genomic SSRs was 36.8–97.1% in the genus Fagopyrum (Ma et al., Reference Ma, Kim, Lee, Lee, Lee, Yi, Park, Kim, Gwag and Kwon2009a) and 47.6–73.0% in the genus Allium (Lee et al., Reference Lee, Kwon, Park, Lee, Kim, Lee, Lee, Gwag, Kim and Ma2011), and SSRs were applicable to related species in the genera Citrus (Dixit et al., Reference Dixit, Chung, Zhao, Lee, Lee, Ma, Lee, Gwag, Kim and Park2010) and Prunus (Kwon et al., Reference Kwon, Chung, Park, Lee, Ma, Lee and Park2012).
As we focused on the development of molecular markers for underutilized crops, we investigated the genetic diversity in a limited number of accessions from the total collection. Now, we are trying to resolve the genetic diversity in the total usable collection, including the geographical and phenotypic variability of each crop based on the developed microsatellite markers, including assessments of genetic diversity and relationships in the mungbean (V. radiata L.; Gwag et al., Reference Gwag, Dixit, Park, Ma, Kwon, Cho, Lee, Lee, Kang and Lee2010), Job's tear (Coix lacryma-jobi L.; Ma et al., Reference Ma, Kim, Dixit, Chung, Gwag, Kim and Park2010), Chinese matrimony vine (Lycium chinense Mill.; Zhao et al., Reference Zhao, Chung, Cho, Rha, Lee, Ma, Han, Bang, Park and Kim2010) and garlic (A. sativum L.; Lee et al., Reference Lee, Kwon, Park, Lee, Kim, Lee, Lee, Gwag, Kim and Ma2011).
These activities will help us to determine the genetic diversity in conserved crop collections. The diversity detected in underutilized crops could be used for the preservation and sustainable utilization of these crops.
Acknowledgements
This work was supported by the RDA and by a grant (code no. PJ008625) from the National Academy of Agricultural Science, RDA, Republic of Korea.
