Ho-Sun Lee

Development and Molecular Characterization of 55 Novel Polymorphic cDNA-SSR Markers in Faba Bean (Vicia faba L.) Using 454 Pyrosequencing

Faba bean (Vicia faba L.) is a major food source and fodder legume, popularly known for its high content of seed-protein. Its role is critical in crop rotation, and for fixing nitrogen effectively. Polymorphic simple sequence repeat markers from transcript sequences (cDNA; simple sequence repeat [SSR]) were developed for faba bean (Vicia faba). We found that 1,729 SSR loci from 81,333 individual sequence reads and 240 primer pairs were designed and synthesized. In total, 55 primer pairs were found to be polymorphic and scorable consistently when screened in 32 accessions. The number of alleles ranged from 2 to 15, frequency of major alleles per locus varied from 0.17 to 0.91, the genotypes number ranged from 2 to 17, observed and expected heterozycosity values ranged from 0.00 to 0.44 and 0.17 to 0.89 and overall PIC values ranged from 0.16 to 0.88 respectively. These markers will be a useful tool for assessing the genetic diversity, understanding the population structure, and breeding patterns of faba bean.

Incidence, Molecular Characteristics and Pathogenicity of Gibberella fujikuroi Species Complex Associated with Rice Seeds from Asian Countries

Abstract Gibberella fujikuroi species complex (GFSC) was isolated from rice (Oryza sativa L.) seed samples from ten Asian countries and investigated for incidence of GFSC, molecular characteristics, and pathogenicity. Regardless of geographic origin, GFSC was detected with incidences ranging from 3% to 80%. Four species, Fusarium fujikuroi, F. concentricum, F. proliferatum, and F. verticillioides, were found to show an association with rice seeds, with F. fujikuroi being the predominant species. In phylogenetic analyses of DNA sequences, no relationship was found between species, isolates, and geographic sources of samples. Unidentified fragments of the β-tubulin gene were observed in ten isolates of F. fujikuroi and F. verticillioides. With the exception of three isolates of F. fujikuroi, F. fujikuroi, F. proliferatum, and F. verticillioides were found to have FUM1 (the fumonisin biosynthetic gene); however, FUM1 was not found in isolates of F. concentricum. Results of pathogenicity testing showed that all isolates caused reduced germination of rice seed. In addition, F. fujikuroi and F. concentricum caused typical symptoms of bakanae, leaf elongation and chlorosis, whereas F. proliferatum and F. verticillioides only caused stunting of seedlings. These findings provide insight into the characteristics of GFSC associated with rice seeds and might be helpful in development of strategies for management of bakanae.

New cDNA-SSR markers in the narrow-leaved vetch (Vicia sativa subsp. nigra) using 454 pyrosequencing

Transcriptomes from high-throughput sequencing technology provide a good resource for molecular markers. Here, we report the development of polymorphic simple sequence repeat (SSR) markers from transcript sequences via 454 pyrosequencing for Vicia sativa subsp. nigra (narrow-leaved vetch). In contrast to V. sativa subsp. sativa, the most commonly grown vetch species, the narrow-leaved vetch survives in diverse soil and climate conditions, serving as a valuable breeding resource of environmental tolerance. We found 2,429 SSR loci from 17,971 individual sequence reads, and 100 primer pairs were designed and synthesized. In total, 49 primer pairs were polymorphic when screened in 32 accessions. The number of alleles was 2–19, the frequency of major alleles per locus varied from 0.19 to 0.87, the genotype numbers ranged from 2 to 19, and observed and expected heterozygosity values ranged from 0.00 to 0.78 and from 0.23 to 0.92, respectively. Polymorphism index content values ranged from 0.20 to 0.86. Considering the relatively high polymorphism in the selected cDNA-SSR marker sets, these markers can be applied to assess genetic diversity, population structure, and positional cloning for the narrow-leaved vetch to facilitate vetch breeding programs.

New Genetic Loci Associated with Preharvest Sprouting and Its Evaluation Based on the Model Equation in Rice

Preharvest sprouting (PHS) in rice panicles is an important quantitative trait that causes both yield losses and the deterioration of grain quality under unpredictable moisture conditions at the ripening stage. However, the molecular mechanism underlying PHS has not yet been elucidated. Here, we explored the genetic loci associated with PHS in rice and formulated a model regression equation for rapid screening for use in breeding programs. After re-sequencing 21 representative accessions for PHS and performing enrichment analysis, we found that approximately 20,000 SNPs revealed distinct allelic distributions between PHS resistant and susceptible accessions. Of these, 39 candidate SNP loci were selected, including previously reported QTLs. We analyzed the genotypes of 144 rice accessions to determine the association between PHS and the 39 candidate SNP loci, 10 of which were identified as significantly affecting PHS based on allele type. Based on the allele types of the SNP loci, we constructed a regression equation for evaluating PHS, accounting for an R2 value of 0.401 in japonica rice. We validated this equation using additional accessions, which exhibited a significant R2 value of 0.430 between the predicted values and actual measurements. The newly detected SNP loci and the model equation could facilitate marker-assisted selection to predict PHS in rice germplasm and breeding lines.

Variation in Antioxidant Activity and Polyphenol Content in Tomato Stems and Leaves

Tomato was considered as one of the most widely cultivated vegetable crops in the world. Tomato plant has high antioxidant capacity which can be attributed to the high levels of carotenoids, phenols, vitamins C and E. However, most of tomato plants have been discarded as waste after fruit harvesting. In order to identify genetic resources with high antioxidant level for use in food or as feed additives, we investigated the ABTS, DPPH antioxidant activity and polyphenol content in tomato leaves and stems. A total of 112 tomato accessions were classified into three groups by latitude of their collected countries: 30°~60° North (50 accessions), 0°~30° North (40 accessions), and 0°~30° South (22 accessions). Stem and leaf extracts showed wide variation in ABTS antioxidant activity ranging from 1.6 ± 1.0 to 48.4 ± 6.1 μg Trolox mg -1 dw. The antioxidant activity of DPPH was in the range of 6.3 ± 0.2 to 40.0 ± 0.3 μg ASC mg -1 dw. Total polyphenol content ranged from 6.1 ± 0.2 to 38.9 ± 0.7 μg GAE mg -1 dw. ABTS, DPPH antioxidant activities and polyphenol content in accessions from 30°~60°N latitude were significantly higher (P<0.05) than those from 0°~30°N latitude. ABTS values showed a significant positive correlation (r = 0.700**) with DPPH activity. IT100506 (KOR) and 702959 (UKR) were recommended as potential sources of natural antioxidants due to their highest antioxidant activity among accessions. This study will provide valuable information for tomato breeders in developing and producing functional food or feed additives resources.

The Complete Chloroplast Genome of Capsicum frutescens (Solanaceae)

Premise of the study: We report the complete sequence of the chloroplast genome of Capsicum frutescens (Solanaceae), a species of chili pepper. Methods and Results: Using an Illumina platform, we sequenced the chloroplast genome of C. frutescens. The total length of the genome is 156,817 bp, and the overall GC content is 37.7%. A pair of 25,792-bp inverted repeats is separated by small (17,853 bp) and large (87,380 bp) single-copy regions. The C. frutescens chloroplast genome encodes 132 unique genes, including 87 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. Of these, seven genes are duplicated in the inverted repeats and 12 genes contain one or two introns. Comparative analysis with the reference chloroplast genome revealed 125 simple sequence repeat motifs and 34 variants, mostly located in the noncoding regions. Conclusions: The complete chloroplast genome sequence of C. frutescens reported here is a valuable genetic resource for Capsicum species.

Variation in pre-harvest sprouting resistance, seed germination and changes in abscisic acid levels during grain development in diverse rice genetic resources

Among domesticated traits, pre-harvest sprouting (PHS) caused by the early breakage of dormancy leads to severe economic losses. Therefore, regulating PHS is important for cereal crop improvement against changes in climate. In this study, we surveyed naturally occurring variations in seed germination in diverse rice germplasm for the available resources of this trait, and investigated the changes of abscisic acid (ABA) levels during grain development by the distinguished PHS-resistant groups. We discovered wide variations in germination among the 205 rice accessions examined and found that 90 accessions are resistant (germination

Effects of Temperature, Light and Chemical Reagent on Dormancy Breaking and Seed Germination of Three Species in Apiaceae

The seeds of Apiaceae have been known as having morphophysiological dormancy (MPD). It is a class of seed dormancy in which the embryo is both underdeveloped and physiologically dormant. This study was conducted to investigate the effects of the temperature, light and chemical reagent on dormancy breaking and seed germination of three species in Apiaceae (Peucedanum japonicum Thumb, Angelica tenuissima Nakai, Glehnia littoralis F. Schmidt et Miq.). Seed size of three species ranged from 5.57-9.7 mm in length and 3.7 mm in width. The 1000-seed weight ranged from 0.21 g in A. tenuissima to 17.53 g in G. littoralis. Maximum percent-germinations of P. japonicum, A. tenuissima, and G. littoralis were 62.6%, 43.3%, and 36.4%, respectively. In P. japonicum and A. tenuissima, prechilling (4C for 7 days) were only best effective on breaking dormancy and germination. The GA3 or KNO3 treatments were ineffective for breaking dormancy. The proper temperature for germination was constant 20C. In G. littoralis, gibberellin treatments were effective for germination. The seeds showed relatively good germinations at alternative 15/20C (16/8h) and constant 20C (dark).

Development of SSR Markers and Their Use in Studying Genetic Diversity and Population of Finger Millet (Eleusine coracana L. Gaertn.)

Finger millet (Eleusine coracana L. Gaertn.) is an important cereal crop in eastern Africa and southern India with excellent grain storage capacity and the unique ability to thrive in extreme environmental conditions. In this study, we analyzed the genetic diversity and population structure of finger millet using 12 developed microsatellites. By sequencing 815 clones from an SSR-enriched genomic DNA library, we obtained 12 polymorphic SSR markers, which also revealed successful amplicons in finger millet accessions. Using the developed SSR markers, we estimated genetic diversity and population structure among 76 finger millet accessions in Asia, Africa, and unknown origins. The number of alleles ranged from 2 to 9, with an average of 3.3 alleles. The mean values of observed heterozygosity and expected heterozygosity were 0.27 and 0.35, respectively. The average polymorphism information content was 0.301 in all 76 finger millet accessions. AMOVA analysis showed that the percentage of molecular variance among the populations was 1%, that among individuals was 5%, and that within individuals was 94%. In STRUCTURE analysis, the 76 finger millet accessions were divided into two subpopulations which had an admixture of alleles. There was a correspondence among PCoA, AMOVA, and population structure. This study may form the basis for a finger millet breeding and improvement program.

Development of microsatellite markers at the National Agrobiodiversity Center in Korea for the genetic assessment of underutilized crops

The RDA Genebank at the National Agrobiodiversity Center (NAAS, RDA, Republic of Korea) has conserved about 182,000 accessions in 1777 species and is working at preserving agricultural genetic resources for the conservation and sustainable utilization of genetic diversity. The detection of genetic variability in conserved resources is important for germplasm management, but the molecular evaluation tools providing genetic information are insufficient for underutilized crops, unlike those for major crops. In this regard, the Korean National Agrobiodiversity Center has been developing microsatellite markers for several underutilized crops. We designed 3640 primer pairs flanking simple sequence repeat (SSR) motifs for 6310 SSR clones in 21 crop species. Polymorphic loci were revealed in each species (7–36), and the mean ratio of polymorphic loci to all the loci tested was 12%. The average allele number was 5.1 (2.8–10.3) and the expected heterozygosity 0.51 (0.31–0.74). Some SSRs were transferable to closely related species, such as within the genera Fagopyrum and Allium . These SSR markers might be used for studying the genetic diversity of conserved underutilized crops.