Genome-wide association study of resistance to anthracnose in pepper (Capsicum chinense) germplasm.

BACKGROUND: Anthracnose is a fungal disease caused by Colletotrichum spp. that has a significant impact on worldwide pepper production. Colletotrichum scovillei is the most common pathogenic anthracnose-causing species in the Republic of Korea. RESULTS: The resistances of 197 pepper (Capsicum chinense) accessions deposited in Korea's National Agrobiodiversity Center were evaluated for their response against the virulent pathogens Colletotrichum acutatum isolate 'KSCa-1' and C. scovillei isolate 'Hana') in the field and in vitro methods for three consecutive years (2018 to 2020). The severity of the disease was recorded and compared between inoculation methods. Six phenotypically resistant pepper accessions were selected based on three years of disease data. All of the selected resistant pepper accessions outperformed the control resistant pepper in terms of resistance (PI 594,137). A genome-wide association study (GWAS) was carried out to identify single nucleotide polymorphisms (SNPs) associated with anthracnose resistance. An association analysis was performed using 53,518 SNPs and the disease score of the 2020 field and in vitro experiment results. Both field and in vitro experiments revealed 25 and 32 significantly associated SNPs, respectively. These SNPs were found on all chromosomes except Ch06 and Ch07 in the field experiment, whereas in the in vitro experiment they were found on all chromosomes except Ch04 and Ch11. CONCLUSION: In this study, six resistant C. chinense accessions were selected. Additionally, in this study, significantly associated SNPs were found in a gene that codes for a protein kinase receptor, such as serine/threonine-protein kinase, and other genes that are known to be involved in disease resistance. This may strengthen the role of these genes in the development of anthracnose resistance in Capsicum spp. As a result, the SNPs discovered to be strongly linked in this study can be used to identify a potential marker for selecting pepper material resistant to anthracnose, which will assist in the development of resistant varieties.

Variability of Anthocyanin Concentrations, Total Metabolite Contents and Antioxidant Activities in Adzuki Bean Cultivars.

In this study, adzuki bean cultivars including Arari, Chilbopat, Geomguseul, and Hongeon were recently cultivated, and the concentrations of seven individual anthocyanins were determined in their seed coats for the first time. Moreover, the variations of total saponin content (TSC), total phenolic content (TPC), 1,1-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, Trolox equivalent antioxidant capacity (TEAC), and ferric reducing antioxidant power (FRAP) between defatted and undefatted extracts of whole seeds, seed coats, and dehulled seeds of each were analyzed. The anthocyanins were detected only in the black seed-coated cultivars and delphinidin-3-O-glucoside was dominant in both Geomguseul (12.46 mg/g) and Chilbopat (10.88 mg/g) followed by delphinidin-3-O-galactoside. TSC and TPC were in the ranges of 16.20−944.78 mg DE/g and 0.80−57.35 mg GAE/g, respectively, and each decreased in the order of seed coats > whole seeds > dehulled seeds regardless of extract type. The antioxidant activities also showed similar patterns of variation. Geomguseul seed coats outweighed the remaining cultivars in terms of TPC and FRAP activity (p < 0.05). Generally, significant variations of metabolite contents and antioxidant activities were observed between cultivars and across their seed parts (p < 0.05). Thence, black seed-coated adzuki beans could be excellent sources of anthocyanins and antioxidants.

Classification of 17 species Aegilops using DNA barcoding and SNPs, reveals gene flow among Aegilops biuncialis, Aegilops juvenalis, and Aegilops columnaris.

Rapid changes in agricultural environments caused by global warming pose a major challenge to food production and safety. Common wheat (Triticum aestivum) is a hexaploid plant (AABBDD) that shares large numbers of quantitative traits and resistance genes with B and D genomes of Aegilops species, which are responsible for several metabolic functions and biosynthetic processes, particularly in plant adaptation to biotic as well as abiotic stresses. Comparatively, the abundance of the Aegilops gene pool is much higher than that of Triticum. Therefore, we used four universal DNA barcodes for plants (ITS2, matK, rbcL, and psbM-petN) to construct a phylogenetic tree to classify the genus Aegilops. Fourteen species were distinguished among a total of 17 representative species. Aegilops biuncialis, Aegilops juvenalis, and Aegilops umbellulata could not be grouped into any of the clusters in the phylogenetic tree, indicating that these three species could not be distinguished by four DNA barcodes. Therefore, from 2408 SNPs obtained using genotyping by sequencing (GBS), we manually screened 30 SNPs that could be potentially used to classify these three species. The results of gene flow and genetic differentiation index (Fst) showed that the genetic differentiation among the three species was small, and there was bidirectional horizontal gene transfer between the three species, which was consistent with our results that the three species were difficult to classify by DNA barcode.

Phylogeography and Antioxidant Activity of Proso Millet (Panicum miliaceum L.).

Proso millet (Panicum miliaceum L.) or broomcorn millet is among the most important food crops to be domesticated by humans; it is widely distributed in America, Europe, and Asia. In this study, we genotyped 578 accessions of P. miliaceum using 37 single-sequence repeat (SSR) markers, to study the genetic diversity and population structure of each accession. We also investigated total phenolic content (TPC) and superoxide dismutase (SOD) activity and performed association analysis using SSR markers. The results showed that genetic diversity and genetic distance were related to geographic location and the fixation index (Fst). Population structure analysis divided the population into three subpopulations. Based on 3 subpopulations, the population is divided into six clusters in consideration of geographical distribution characteristics and agronomic traits. Based on the genetic diversity, population structure, pairwise Fst, and gene flow analyses, we described the topological structure of the six proso millet subpopulations, and the geographic distribution and migration of each cluster. Comparison of the published cluster (cluster 1) with unique germplasms in Japan and South Korea suggested Turkey as a possible secondary center of origin and domestication (cluster 3) for the cluster. We also discovered a cluster domesticated in Nepal (cluster 6) that is adapted to high-latitude and high-altitude cultivation conditions. Differences in phenotypic characteristics, such as TPC, were observed between the clusters. The association analysis showed that TPC was associated with SSR-31, which explained 7.1% of the total variance, respectively. The development of markers associated with TPC and SOD will provide breeders with new tools to improve the quality of proso millet through marker-assisted selection.

Cryopreservation of hairy roots of Rubia akane (Nakai) using a droplet-vitrification procedure.

An efficient protocol for the cryopreservation of madder (Rubia akane Nakai) hairy root cultures was developed using droplet-vitrification and alternative loading and vitrification solutions formulated previously in our laboratory. Among eight preculture treatments tested, the highest post-cryopreservation regeneration was obtained for explants incubated in liquid half-strength MS medium with progressively increased sucrose concentration (0.3 M for 54 h, then 0.5 M for 16 h). Loading of precultured explants improved their post-cryopreservation regeneration by 50-75% compared with non-loaded control. Combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective, while applying six PVS2-based solutions at room temperature resulted in low post-cryo regeneration. Treatment duration was optimized to 30 min for loading and to 10-20 min for vitrification solution. Apices of primary and secondary hairy roots showed similar post-cryo regeneration (88 and 95%, respectively), which was significantly higher than regeneration of root sections without apices (65%). Droplet-vitrification produced higher post-cryo regeneration than 'classical' vitrification in cryovials. Our results suggest that droplet-vitrification using alternative loading and vitrification solutions is an efficient method for cryopreservation of R. akane hairy root cultures.

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.

Development of SSR markers for studies of diversity in the genus Fagopyrum.

The numbers of SSR markers and their utilization have not been determined and investigated as extensively in Fagopyrum species as compared to other crop species. The current report presents 136 new SSR markers in Fagopyrum esculentum ssp. esculentum and their application to related species in the genus Fagopyrum. Of the 136 SSRs, 10 polymorphic SSR markers were utilized in a genetic diversity analysis of a common buckwheat population consisting of 41 accessions of diverse origin. The study showed observed (H(O)) and expected (H(E)) heterozygosities ranging from 0.071 to 0.924 (mean = 0.53) and from 0.073 to 0.902 (mean = 0.412), respectively. Forty-one of the 136 SSRs amplified sequences in other Fagopyrum species, including the cymosum and urophyllum groups. The phylogenetic relationships revealed using the SSRs was consistent with results obtained using other marker systems, with one exception. The sequence and diversity information obtained using these new SSRs and their cross-transferability to related Fagopyrum species will increase our understanding of genetic structures and species relationships within the Fagopyrum genus.

Genetic assessment of safflower (Carthamus tinctorius L.) collection with microsatellite markers acquired via pyrosequencing method.

A genetic evaluation of safflower germplasm collections derived from different geographical regions and countries will provide useful information for sustainable conservation and the utilization of genetic diversity. However, the molecular marker information is limited for evaluation of genetic diversity of safflower germplasm. In this study, we acquired 509 putative genomic SSR markers for sufficient genome coverage using next-generation sequencing methods and characterized thirty polymorphic SSRs in safflower collection composed of 100 diverse accessions. The average allele number and expected heterozygosity were 2.8 and 0.386, respectively. Analysis of population structure and phylogeny based on thirty SSR profiles revealed genetic admixture between geographical regions contrary to genetic clustering. However, the accessions from Korea were genetically conserved in distinctive groups in contrast to other safflower gene pool. In conclusion, these new genomic SSRs will facilitate valuable studies to clarify genetic relationships as well as conduct population structure analyses, genetic map construction and association analysis for safflower.

Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots.

In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22-71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39-45 %) was already observed after the sucrose pretreatment; it increased to 54-68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment.

Plant defense gene promoter enhances the reliability of shiva-1 gene-induced resistance to soft rot disease in potato.

PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.