Molecular mapping of the broad bean wilt virus 2 resistance locus bwvr in Capsicum annuum using BSR-seq.

KEY MESSAGE: Bulked segregant RNA seq of pools of pepper accessions that are susceptible or resistant to Broad bean wilt virus 2 identifies a gene that might confer resistance to this devastating pathogen. The single-stranded positive-sense RNA virus Broad bean wilt virus 2 (BBWV2) causes substantial damage to pepper (Capsicum annuum) cultivation. Here, we describe mapping the BBWV2 resistance locus bwvr using a F7:8 recombinant inbred line (RIL) population constructed by crossing the BBWV2-resistant pepper accession 'SNU-C' with the susceptible pepper accession 'ECW30R.' All F1 plants infected with the BBWV2 strain PAP1 were susceptible to the virus, and the RIL population showed a 1:1 ratio of resistance to susceptibility, indicating that this trait is controlled by a single recessive gene. To map bwvr, we performed bulked segregant RNA-seq (BSR-seq). We sequenced pools of resistant and susceptible lines from the RILs and aligned the reads to the high-quality 'Dempsey' reference genome to identify variants between the pools. This analysis identified 519,887 variants and selected the region from 245.9-250.8 Mb of the Dempsey reference genome as the quantitative trait locus region for bwvr. To finely map bwvr, we used newly designed high-resolution melting (HRM) and Kompetitive allele specific PCR (KASP) markers based on variants obtained from the BSR-seq reads and the PepperSNP16K array. Comparative analysis identified 11 SNU-C-specific SNPs within the bwvr locus. Using markers derived from these variants, we mapped the candidate bwvr locus to the region from 246.833-246.949 kb. SNU-C-specific variants clustered near DEM.v1.00035533 within the bwvr locus. DEM.v1.00035533 encodes the nitrate transporter NPF1.2 and contains a SNP within its 5' untranslated region. The bwvr locus, which contains four genes including DEM.v1.00035533, could represent a valuable resource for global pepper breeding programs.

Origin and seed coat color differently affect the concentrations of metabolites and antioxidant activities in soybean (Glycine max (L.) Merrill) seeds.

In this study, 54 soybean germplasms of different seed coat colors originated from America, China, Japan, and Korea were cultivated in Korea and analyzed for the contents of total oil, total protein, total phenolic, five fatty acids, and five isoflavones, and antioxidant activities using three assays. The soybeans showed significant variations (p < 0.05) of metabolite contents and antioxidant activities. Origin and seed coat color exhibited a slight or insignificant effect on total protein and total oil contents. In contrast, origin and seed coat color significantly affected the concentration of individual and total isoflavones, and total phenolics, with few exceptions. Whereas fatty acids were significantly affected by origin, seed coat color provided better information regarding the variations in antioxidant capacities. Together, multivariate and correlation analyses revealed important associations between biosynthetically-related metabolites. In general, origin and seed coat color differently influenced the concentration of different classes of metabolites and antioxidant activities.

Nutritional Metabolites of Red Pigmented Lettuce (Lactuca sativa) Germplasm and Correlations with Selected Phenotypic Characters.

Lettuce is an important dietary source of bioactive phytochemicals. Screening and identification of the health beneficial metabolites and evaluating the relationships with phenotypic characters can help consumers adjust their preferences for lettuce plant types. Thus, we explored the major health-beneficial individual metabolites and antioxidant potential of 113 red pigmented lettuce leaf samples. A UV-Vis spectrophotometer and UPLC-DAD-QTOF/MS (TQ/MS) instruments were used for the identification and quantification of metabolites and antioxidant activity accordingly. The metabolites were quantified against their corresponding external standards. The contents of metabolites varied significantly among lettuce samples. Cyanidin 3-O-(6″-O-malonyl)glucoside (4.7~5013.6 µg/g DW), 2,3-di-O-caffeoyltartaric acid (337.1~19,957.2 µg/g DW), and quercetin 3-O-(6″-O-malonyl)glucoside (45.4~31,121.0 µg/g DW) were the most dominant in red pigmented lettuce samples among anthocyanins, hydroxycinnamoyl derivatives, and flavonols, respectively. Lettuces with dark and very dark red pigmented leaves, circular leaf shape, a strong degree of leaf undulation, and highly dense leaf incisions were found to have high levels of flavonoids and hydroxycinnamoyl derivatives. Principal component analysis was used to investigate similarities and/or differences between samples, and the partial least square discriminant analysis classified them into known groups. The key variables that contributed highly were determined. Our report provides critical data on the bioactive constituents of red pigmented lettuce to breeders developing varieties with enhanced bioactive compounds and to nutraceutical companies developing nutrient dense foods and pharmaceutical formulations.

Genome-Wide SNP Markers for Genotypic and Phenotypic Differentiation of Melon (Cucumis melo L.) Varieties Using Genotyping-by-Sequencing.

Melon (Cucumis melo L.) is an economically important horticultural crop with abundant morphological and genetic variability. Complex genetic variations exist even among melon varieties and remain unclear to date. Therefore, unraveling the genetic variability among the three different melon varieties, muskmelon (C. melo subsp. melo), makuwa (C. melo L. var. makuwa), and cantaloupes (C. melo subsp. melo var. cantalupensis), could provide a basis for evolutionary research. In this study, we attempted a systematic approach with genotyping-by-sequencing (GBS)-derived single nucleotide polymorphisms (SNPs) to reveal the genetic structure and diversity, haplotype differences, and marker-based varieties differentiation. A total of 6406 GBS-derived SNPs were selected for the diversity analysis, in which the muskmelon varieties showed higher heterozygote SNPs. Linkage disequilibrium (LD) decay varied significantly among the three melon varieties, in which more rapid LD decay was observed in muskmelon (r2 = 0.25) varieties. The Bayesian phylogenetic tree provided the intraspecific relationships among the three melon varieties that formed, as expected, individual clusters exhibiting the greatest genetic distance based on the posterior probability. The haplotype analysis also supported the phylogeny result by generating three major networks for 48 haplotypes. Further investigation for varieties discrimination allowed us to detect a total of 52 SNP markers that discriminated muskmelon from makuwa varieties, of which two SNPs were converted into cleaved amplified polymorphic sequence markers for practical use. In addition to these markers, the genome-wide association study identified two SNPs located in the genes on chromosome 6, which were significantly associated with the phenotypic traits of melon seed. This study demonstrated that a systematic approach using GBS-derived SNPs could serve to efficiently classify and manage the melon varieties in the genebank.

Isoflavones, anthocyanins, phenolic content, and antioxidant activities of black soybeans (Glycine max (L.) Merrill) as affected by seed weight.

Seed weight is regulated by several genes which in turn could affect the metabolite contents, yield, and quality of soybean seeds. Due to these, seed weight is receiving much attention in soybean breeding. In this study, seeds of 24 black soybean varieties and a reference genotype were grown in Korea, and grouped as small (< 13 g), medium (13-24 g), and large (> 24 g) seeds based on their seed weight. The contents of six anthocyanins, twelve isoflavones, and total phenolic, and the antioxidant activities were determined, and the association of each with seed weight was analyzed. The total anthocyanin (TAC) and total isoflavone (TIC) contents were in the ranges of 189.461-2633.454 mg/100 g and 2.110-5.777 mg/g, respectively and were significantly different among the black soybean varieties. By comparison, the average TAC and TIC were the highest in large seeds than in small and medium seeds while the total phenolic content (TPC) was in the order of small seeds > large seeds > medium seeds. Besides, large seeds showed the maximum 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity, whereas small seeds showed the maximum ferric reducing antioxidant power (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS) scavenging activities. FRAP activity was positively associated with TIC and TAC, the former association being significant. On the other hand, ABTS and DPPH activities were positively correlated to TPC, the later association being significant. Overall, our findings demonstrated the influence of seed weight on anthocyanin, isoflavone, and phenolic contents and antioxidant activities in black soybeans. Besides, the dominant anthocyanins and isoflavones were the principal contributors to the variations observed in the black soybean varieties, and hence, these components could be selectively targeted to discriminate a large population of black soybean genetic resources.

Glucosinolate Content in Brassica Genetic Resources and Their Distribution Pattern within and between Inner, Middle, and Outer Leaves.

Glucosinolates (GSLs) are sulfur-containing secondary metabolites naturally occurring in Brassica species. The purpose of this study was to identify the GSLs, determine their content, and study their accumulation patterns within and between leaves of kimchi cabbage (Brassica rapa L.) cultivars. GSLs were analyzed using UPLC-MS/MS in negative electron-spray ionization (ESI-) and multiple reaction monitoring (MRM) mode. The total GSL content determined in this study ranged from 621.15 to 42434.21 µmolkg-1 DW. Aliphatic GSLs predominated, representing from 4.44% to 96.20% of the total GSL content among the entire samples. Glucobrassicanapin (GBN) contributed the greatest proportion while other GSLs such as glucoerucin (ERU) and glucotropaeolin (TRO) were found in relatively low concentrations. Principal component analysis (PCA) yielded three principal components (PCs) with eigenvalues ≥ 1, altogether representing 74.83% of the total variation across the entire dataset. Three kimchi cabbage (S/No. 20, 4, and 2), one leaf mustard (S/No. 26), and one turnip (S/No. 8) genetic resources were well distinguished from other samples. The GSL content varied significantly among the different positions (outer, middle, and inner) of the leaves and sections (top, middle, bottom, green/red, and white) within the leaves. In most of the samples, higher GSL content was observed in the proximal half and white sections and the middle layers of the leaves. GSLs are regarded as allelochemicals; hence, the data related to the patterns of GSLs within the leaf and between leaves at a different position could be useful to understand the defense mechanism of Brassica plants. The observed variability could be useful for breeders to develop Brassica cultivars with high GSL content or specific profiles of GSLs.

Uncovering Candidate Genes Controlling Major Fruit-Related Traits in Pepper via Genotype-by-Sequencing Based QTL Mapping and Genome-Wide Association Study.

All modern pepper accessions are products of the domestication of wild Capsicum species. However, due to the limited availability of genome-wide association study (GWAS) data and selection signatures for various traits, domestication-related genes have not been identified in pepper. Here, to address this problem, we obtained data for major fruit-related domestication traits (fruit length, width, weight, pericarp thickness, and fruit position) using a highly diverse panel of 351 pepper accessions representing the worldwide Capsicum germplasm. Using a genotype-by-sequencing (GBS) method, we developed 187,966 genome-wide high-quality SNP markers across 230 C. annuum accessions. Linkage disequilibrium (LD) analysis revealed that the average length of the LD blocks was 149 kb. Using GWAS, we identified 111 genes that were linked to 64 significant LD blocks. We cross-validated the GWAS results using 17 fruit-related QTLs and identified 16 causal genes thought to be associated with fruit morphology-related domestication traits, with molecular functions such as cell division and expansion. The significant LD blocks and candidate genes identified in this study provide unique molecular footprints for deciphering the domestication history of Capsicum. Further functional validation of these candidate genes should accelerate the cloning of genes for major fruit-related traits in pepper.

Fruit Morphology, Citrulline, and Arginine Levels in Diverse Watermelon (Citrullus lanatus) Germplasm Collections.

Watermelon (Citrullus lanatus) is a non-seasonal, economically important, cucurbit cultivated throughout the world, with Asia as a continent contributing the most. As part of the effort to diversify watermelon genetic resources in the already cultivated group, this study was devoted to providing baseline data on morphological quality traits and health-beneficial phytonutrients of watermelon germplasm collections, thereby promoting watermelon research and cultivation programs. To this end, we reported morphological traits, citrulline, and arginine levels of watermelon genetic resources obtained from the gene bank of Agrobiodiversity Center, Republic of Korea, and discussed the relationships between each. Diverse characteristics were observed among many of the traits, but most of the genetic resources (>90%) were either red or pink-fleshed. Korean originated fruits contained intermediate levels of soluble solid content (SSC) while the USA, Russian, Tajikistan, Turkmenistan, Taiwan, and Uruguay originated fruits had generally the highest levels of soluble solids. The citrulline and arginine contents determined using the High Performance Liquid Chromatography (HPLC) method ranged from 6.9 to 52.1 mg/g (average, 27.3 mg/g) and 1.8 to 21.3 mg/g (average, 9.8 mg/g), respectively. The citrulline content determined using the Citrulline Assay Kit ranged from 6.5 to 42.8 mg/g (average, 27.0 mg/g). Resources with high citrulline and arginine levels contained low SSC, whereas red- and pink-colored flesh samples had less citrulline compared to yellow and orange.

Mitotypes Based on Structural Variation of Mitochondrial Genomes Imply Relationships With Morphological Phenotypes and Cytoplasmic Male Sterility in Peppers.

Plant mitochondrial genomes characteristically contain extensive structural variation that can be used to define and classify cytoplasm types. We developed markers based on structural variation in the mitochondrial genomes of fertile and cytoplasmic male sterility (CMS) pepper lines and applied them to a panel of Capsicum accessions. We designed a total of 20 sequence characterized amplified region (SCAR) markers based on DNA rearrangement junctions or cytoplasm-specific segments that did not show high similarity to any nuclear mitochondrial DNA segments. We used those markers to classify the mitotypes of 96 C. annuum accessions into 15 groups. Precise genotyping of other Capsicum species (C. frutescens, C. chinense, and C. baccatum) was hampered because of various stoichiometric levels of marker amplicons. We developed a multiplex PCR system based on four of the markers that efficiently classified the C. annuum accessions into five mitotype groups. Close relationships between specific mitotypes and morphological phenotypes implied that diversification or domestication of C. annuum germplasm might have been accompanied by structural rearrangements of mitochondrial DNA or the selection of germplasms with specific mitotypes. Meanwhile, CMS lines shared the same amplification profile of markers with another mitotype. Further analysis using mitochondrial DNA (mtDNA) markers based on single-nucleotide polymorphisms (SNPs) or insertions and deletions (InDels) and CMS-specific open reading frames (orfs) provided new information about the origin of the CMS-specific mitotype and evaluation of candidates for CMS-associated genes, respectively.

Identification and quantification of selected metabolites in differently pigmented leaves of lettuce (Lactuca sativa L.) cultivars harvested at mature and bolting stages.

BACKGROUND: Identification and screening of cultivars rich in bioactive phytoconstituents can be potentially useful to make nutrient-dense dishes and in medicinal formulations. In this study, we have identified, characterized and quantified caffeoylquinic acids, dicaffeoylquinic acid, dicaffeoyltartaric acid, kaempferol conjugates, quercetin malonylglucoside, sesquiterpene lactones, and cyanidin in 22 lettuce cultivars at mature and bolting stages using UPLC-PDA-Q-TOF-HDMS, UPLC, and HPLC. RESULTS: The composition and contents of the studied metabolites and antioxidant activity varied significantly and depend on leaf color, cultivar type and stage of maturity. The main phenolic acid components of lettuce were quinic and tartaric acid derivatives, whereas kaempferol derivatives were the dominant flavonoids. The sum of the content of phenolic acids ranged from 18.3 to 54.6 mg/100 g DW and 15.5 to 54.6 mg/100 g DW, whereas the sum of the contents of flavonoids ranged from 9.2 to 25.9 mg/100 g DW and 14.9 to 83.0 mg/100 g DW in mature and bolting stage cultivars, respectively. The content of cyanidin, lactucin, lactucopicrin, and ABTS radical antioxidant activity were in the range of 0.3 to 9.7 (mature stage) and 0.5 to 10.2 mg/g DW (bolting stage), 1.8 to 41.9 (mature stage) and 9.7 to 213.0 (bolting stage) µg/g DW, 9.9 to 344.8 (mature stage) and 169.2 to 3888.2 (bolting stage) µg/g DW, and 12.1 to 29.0 (mature stage) and 15.7 to 30.3 (bolting stage) mg TE/g DW, respectively. The principal component analysis (PCA) showed that the green and red pigmented lettuce cultivars were grouped to the negative and positive sides of PC1, respectively, while the green/red pigmented cultivars were distributed throughout the four quadrants of the PCA plots with no prominent grouping. The loading plot showed that phenolic acids, flavonoids, and cyanidin are the most potent contributors to the radical scavenging activity of lettuce extracts. CONCLUSIONS: Lettuce at the bolting stage accumulate relatively high amount of sesquiterpene lactones (SLs), quercetin malonylglucoside (QMG), methylkaempferol glucuronide (MKGR), kaempferol malonylglucoside (KMG), and 3-O-caffeoylquinic acid (3-CQA) compared to the mature stage. Higher amount of phytoconstituents were found to be accumulated in the red pigmented lettuce leaves compared to the green lettuce leaves. In addition, the contents of most of the metabolites in lettuce seem to increase with age of the leaves. The presence of the two bitter SLs, lactucin and lactucopicrin, in significantly high amount in lettuce leaves at bolting stage could diminish consumer acceptance. However, alternatively, these leaves could be utilized by nutraceutical companies working to recover these compounds.

Identifying candidate genes for Phytophthora capsici resistance in pepper (Capsicum annuum) via genotyping-by-sequencing-based QTL mapping and genome-wide association study.

Phytophthora capsici (Leon.) is a globally prevalent, devastating oomycete pathogen that causes root rot in pepper (Capsicum annuum). Several studies have identified quantitative trait loci (QTL) underlying resistance to P. capsici root rot (PcRR). However, breeding for pepper cultivars resistant to PcRR remains challenging due to the complexity of PcRR resistance. Here, we combined traditional QTL mapping with GWAS to broaden our understanding of PcRR resistance in pepper. Three major-effect loci (5.1, 5.2, and 5.3) conferring broad-spectrum resistance to three isolates of P. capsici were mapped to pepper chromosome P5. In addition, QTLs with epistatic interactions and minor effects specific to isolate and environment were detected on other chromosomes. GWAS detected 117 significant SNPs across the genome associated with PcRR resistance, including SNPs on chromosomes P5, P7, and P11 that colocalized with the QTLs identified here and in previous studies. Clusters of candidate nucleotide-binding site-leucine-rich repeat (NBS-LRR) and receptor-like kinase (RLK) genes were predicted within the QTL and GWAS regions; such genes often function in disease resistance. These candidate genes lay the foundation for the molecular dissection of PcRR resistance. SNP markers associated with QTLs for PcRR resistance will be useful for marker-assisted breeding and genomic selection in pepper breeding.

QTL mapping and GWAS reveal candidate genes controlling capsaicinoid content in Capsicum.

Capsaicinoids are unique compounds produced only in peppers (Capsicum spp.). Several studies using classical quantitative trait loci (QTLs) mapping and genomewide association studies (GWAS) have identified QTLs controlling capsaicinoid content in peppers; however, neither the QTLs common to each population nor the candidate genes underlying them have been identified due to the limitations of each approach used. Here, we performed QTL mapping and GWAS for capsaicinoid content in peppers using two recombinant inbred line (RIL) populations and one GWAS population. Whole-genome resequencing and genotyping by sequencing (GBS) were used to construct high-density single nucleotide polymorphism (SNP) maps. Five QTL regions on chromosomes 1, 2, 3, 4 and 10 were commonly identified in both RIL populations over multiple locations and years. Furthermore, a total of 109 610 SNPs derived from two GBS libraries were used to analyse the GWAS population consisting of 208 C. annuum-clade accessions. A total of 69 QTL regions were identified from the GWAS, 10 of which were co-located with the QTLs identified from the two biparental populations. Within these regions, we were able to identify five candidate genes known to be involved in capsaicinoid biosynthesis. Our results demonstrate that QTL mapping and GBS-GWAS represent a powerful combined approach for the identification of loci controlling complex traits.

Genetic diversity and population structure analysis to construct a core collection from a large Capsicum germplasm.

BACKGROUND: Conservation of genetic diversity is an essential prerequisite for developing new cultivars with desirable agronomic traits. Although a large number of germplasm collections have been established worldwide, many of them face major difficulties due to large size and a lack of adequate information about population structure and genetic diversity. Core collection with a minimum number of accessions and maximum genetic diversity of pepper species and its wild relatives will facilitate easy access to genetic material as well as the use of hidden genetic diversity in Capsicum. RESULTS: To explore genetic diversity and population structure, we investigated patterns of molecular diversity using a transcriptome-based 48 single nucleotide polymorphisms (SNPs) in a large germplasm collection comprising 3,821 accessions. Among the 11 species examined, Capsicum annuum showed the highest genetic diversity (HE = 0.44, I = 0.69), whereas the wild species C. galapagoense showed the lowest genetic diversity (HE = 0.06, I = 0.07). The Capsicum germplasm collection was divided into 10 clusters (cluster 1 to 10) based on population structure analysis, and five groups (group A to E) based on phylogenetic analysis. Capsicum accessions from the five distinct groups in an unrooted phylogenetic tree showed taxonomic distinctness and reflected their geographic origins. Most of the accessions from European countries are distributed in the A and B groups, whereas the accessions from Asian countries are mainly distributed in C and D groups. Five different sampling strategies with diverse genetic clustering methods were used to select the optimal method for constructing the core collection. Using a number of allelic variations based on 48 SNP markers and 32 different phenotypic/morphological traits, a core collection 'CC240' with a total of 240 accessions (5.2 %) was selected from within the entire Capsicum germplasm. Compared to the other core collections, CC240 displayed higher genetic diversity (I = 0.95) and genetic evenness (J' = 0.80), and represented a wider range of phenotypic variation (MD = 9.45 %, CR = 98.40 %). CONCLUSIONS: A total of 240 accessions were selected from 3,821 Capsicum accessions based on transcriptome-based 48 SNP markers with genome-wide distribution and 32 traits using a systematic approach. This core collection will be a primary resource for pepper breeders and researchers for further genetic association and functional analyses.

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.

Evaluation of Resistance to Ralstonia solanacearum in Tomato Genetic Resources at Seedling Stage.

Bacterial wilt of tomatoes caused by Ralstonia solanacearum is a devastating disease that limits the production of tomato in Korea. The best way to control this disease is using genetically resistant tomato plant. The resistance degree to R. solanacearum was evaluated for 285 tomato accessions conserved in the National Agrobiodiversity Center of Rural Development Administration. These accessions of tomato were originated from 23 countries. Disease severity of tomato accessions was investigated from 7 days to 14 days at an interval of 7 days after inoculation of R. solanacearum under greenhouse conditions. A total of 279 accessions of tomato germplasm were susceptible to R. solanacearum, resulting in wilt and death in 70 to 90% of these plants. Two tomato accessions were moderately resistant to R. solanacearum. Only four accessions showed high resistance against R. solanacearum. No distinct symptom of bacterial wilt appeared on the resistant tomato germplasms for up to 14 days after inoculation of R. solanacearum. Microscopy of resistant tomato stems infected with R. solanacearum revealed limited bacterial spread with thickening of pit membrane and gum production. Therefore, these four resistant tomato germplasms could be used in tomato breeding program against bacterial wilt.