The genetic architecture of the pepper metabolome and the biosynthesis of its signature capsianoside metabolites.

Capsicum (pepper) is among the most economically important species worldwide, and its fruits accumulate specialized metabolites with essential roles in plant environmental interaction and human health benefits as well as in conferring their unique taste. However, the genetics underlying differences in metabolite presence/absence and/or accumulation remain largely unknown. In this study, we carried out a genome-wide association study as well as generating and characterizing a novel backcross inbred line mapping population to determine the genetic architecture of the pepper metabolome. This genetic analysis provided over 1,000 metabolic quantitative trait loci (mQTL) for over 250 annotated metabolites. We identified 92 candidate genes involved in various mQTLs. Among the identified loci, we described and validated a gene cluster of eleven UDP-glycosyltransferases (UGTs) involved in monomeric capsianoside biosynthesis. We additionally constructed the gene-by-gene-based biosynthetic pathway of pepper capsianoside biosynthesis, including both core and decorative reactions. Given that one of these decorative pathways, namely the glycosylation of acyclic diterpenoid glycosides, contributes to plant resistance, these data provide new insights and breeding resources for pepper. They additionally provide a blueprint for the better understanding of the biosynthesis of species-specific natural compounds in general.

Genomic prediction of morphometric and colorimetric traits in Solanaceous fruits.

Selection of high-performance lines with respect to traits of interest is a key step in plant breeding. Genomic prediction allows to determine the genomic estimated breeding values of unseen lines for trait of interest using genetic markers, e.g. single-nucleotide polymorphisms (SNPs), and machine learning approaches, which can therefore shorten breeding cycles, referring to genomic selection (GS). Here, we applied GS approaches in two populations of Solanaceous crops, i.e. tomato and pepper, to predict morphometric and colorimetric traits. The traits were measured by using scoring-based conventional descriptors (CDs) as well as by Tomato Analyzer (TA) tool using the longitudinally and latitudinally cut fruit images. The GS performance was assessed in cross-validations of classification-based and regression-based machine learning models for CD and TA traits, respectively. The results showed the usage of TA traits and tag SNPs provide a powerful combination to predict morphology and color-related traits of Solanaceous fruits. The highest predictability of 0.89 was achieved for fruit width in pepper, with an average predictability of 0.69 over all traits. The multi-trait GS models are of slightly better predictability than single-trait models for some colorimetric traits in pepper. While model validation performs poorly on wild tomato accessions, the usage as many as one accession per wild species in the training set can increase the transferability of models to unseen populations for some traits (e.g. fruit shape for which predictability in unseen scenario increased from zero to 0.6). Overall, GS approaches can assist the selection of high-performance Solanaceous fruits in crop breeding.

First report of Cucurbit aphid-borne yellows virus causing yellowing disease on pumpkin (Cucurbita pepo) and cucumber (Cucumis sativus) in Bulgaria.

Cucurbit aphid-borne yellows virus (CABYV) was first reported in France in 1992 but has since been observed worldwide (Lecoq et al. 1992; Choi and Choi 2016; Buzkan et al. 2017; Zindovic et al. 2017; Vidal et al. 2018; Khanal and Ali, 2018). This virus has caused severe losses to different crops especially to the members of Cucurbitaceae and yield losses can reach up to 40-50% if infection occurs at early stages (Lecoq et al. 1992). In July 2017, leaf samples showing virus-like symptoms were collected from five pumpkin (Cucurbita pepo L. var. Clypeata Alefield) and two cucumber (Cucumis sativus L. cv. Azuma matsunari) plants, growing in а field near Sadovo, Bulgaria. Nearly all plants in the field were affected and displayed green or yellow mosaic, interveinal yellowing, blisters, and leaf deformation (Fig. 1). The collected samples were all symptomatic and were subjected to double antibody sandwich (DAS) or triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA) to determine the viral agent(s). Specific monoclonal antibodies (Leibniz institute DSMZ, Germany) raised against Cucumber leaf spot virus, Cucurbit chlorotic yellows virus, Cucurbit yellow stunting disorder virus, Cucumber mosaic virus (CMV), Melon necrotic spot virus, Beet western yellows virus (BWYV), CABYV, Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Cucumber green mottle mosaic virus, were used. The total number of tested samples was seven (n=5 from pumpkin and n=2 from cucumber). All of them displayed positive signals for CABYV and BWYV, both belonging to genus Polerovirus, family Luteoviridae. In addition, ZYMV and/or WMV were detected in pumpkins while CMV and/or WMV were detected in cucumber samples, respectively. To confirm the presence of CABYV and/or BWYV, total RNA was isolated from all seven samples by TRI Reagent® (Sigma, St. Louis, USA) and converted to cDNA with First Strand cDNA Synthesis Kit, Thermo Scientific™. Reverse transcription (RT)-polyemerase chain reaction (PCR) was performed using two pairs of primers (CABYV1FW: 5'-TTATCAGGGGACTATGTTTA-3' and CABYV14REV: 5'-GAGGGGATTTTAACTGACTG-3', and BWYV1FW: 5'-AGTAAGTCCTCCCCAACTGA-3' and BWYV2REV: 5'-CTACCCACGACCGTATTCAT-3'), specifically designed to detect CABYV and BWYV, respectively. Amplicons with expected sizes of 1,930 bp were obtained only with CABYV primers for all samples while no fragments were amplified with BWYV primers. The obtained products from two samples (pumpkin and cucumber) were purified and sent to Macrogen Inc., South Korea, for direct sequencing in both directions. High quality nucleotide sequences were submitted to GenBank We have evaluated the quality of the sequencing and trimmed those parts that did not comply the needed quality. The obtained smaller fragments Nucleotide sequences were submitted to GenBank with accession numbers MK671010 (656 bp) and MK671014 (712bp). These sequences contained ORFs encoding CABYV P1-P2 fusion proteins as determined by Blastp analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). A phylogenetic tree constructed by the Neighbor-joining method using 18 CABYV accessions and Potato leafroll virus as an outlier (Fig. 2) showed that the closest accessions to MK671010 and MK671014 were NC003688 (France) and EU636992 (China) with respective nucleotide identity of 98% and 99%. In 2019, another outbreak was observed in the same field near Sadovo and in a field near Plovdiv planted with pumpkins. Nearly 30% of the plants showed leaf yellowing typical for Polerovirus infection. Screening of collected samples (n=17) by RT-PCR confirmed CABYV presence in 15 samples. Based on available reports and according to our knowledge this is the first report of CABYV in Bulgaria.

Double Haploid Development and Assessment of Androgenic Competence of Balkan Pepper Core Collection in Bulgaria.

This study was designed to assess the androgenic potential of 180 pepper accessions and 11 progenies (four F1 and seven BC) possessing PMMoV resistance in order to complement an ongoing pepper breeding program. The experiment was carried out in 10 replications with 20 anthers for each accession in two different induction mediums from 2017 to 2019. The highest androgenic response was observed in culture medium 17-2 but differences between two mediums were nonsignificant. From a total of 191 genotypes, 102 genotypes expressed a potential for direct embryogenesis. Embryo induction was seen to be genotype-dependent and decreased in the following order: Pumpkin > Conical > Bell or blocky > Round > Elongate as the most responsive genotypes with over 10% reacted anthers being observed in CAPS-23, CAPS-29, CAPS-127, CAPS-157, CAPS-169, F1 and BC 887 derived from CAPS-23. The number of regenerated plants was higher in the conical group and least in the round varietal group. Regenerated plants were examined visually and by flow cytometry for identification of spontaneous doubled haploids (DH) and haploids. Those originating from F1 and BC progenies were additionally evaluated by a CAPS marker targeting L4 allele for resistance against PMMoV. Obtained results revealed two groups consisting of homozygous susceptible and resistant plants. Therefore, use of anther culture in ongoing breeding will greatly facilitate the pepper genetic improvement.

Global range expansion history of pepper (Capsicum spp.) revealed by over 10,000 genebank accessions.

Genebanks collect and preserve vast collections of plants and detailed passport information, with the aim of preserving genetic diversity for conservation and breeding. Genetic characterization of such collections has the potential to elucidate the genetic histories of important crops, use marker-trait associations to identify loci controlling traits of interest, search for loci undergoing selection, and contribute to genebank management by identifying taxonomic misassignments and duplicates. We conducted a genomic scan with genotyping by sequencing (GBS) derived single nucleotide polymorphisms (SNPs) of 10,038 pepper (Capsicum spp.) accessions from worldwide genebanks and investigated the recent history of this iconic staple. Genomic data detected up to 1,618 duplicate accessions within and between genebanks and showed that taxonomic ambiguity and misclassification often involve interspecific hybrids that are difficult to classify morphologically. We deeply interrogated the genetic diversity of the commonly consumed Capsicum annuum to investigate its history, finding that the kinds of peppers collected in broad regions across the globe overlap considerably. The method ReMIXTURE-using genetic data to quantify the similarity between the complement of peppers from a focal region and those from other regions-was developed to supplement traditional population genetic analyses. The results reflect a vision of pepper as a highly desirable and tradable cultural commodity, spreading rapidly throughout the globe along major maritime and terrestrial trade routes. Marker associations and possible selective sweeps affecting traits such as pungency were observed, and these traits were shown to be distributed nonuniformly across the globe, suggesting that human preferences exerted a primary influence over domesticated pepper genetic structure.

A step towards Balkan Capsicum annuum L. core collection: Phenotypic and biochemical characterization of 180 accessions for agronomic, fruit quality, and virus resistance traits.

Region-specific local landraces represent a germplasm diversity adapted and acclimatized to local conditions, and are ideal to breed for targeted market niches while maintaining the variability of heirloom traits. A collection of 180 pepper accessions, collected from 62 diverse locations across six Balkan countries, were characterized and evaluated for phenotypic and biochemical variation during a multi-year environment. An assortment of 32 agro-morphological, fruit quality, and virus resistance traits were evaluated, and the top 10% accessions were identified. A wide range of trait variation concerning plant architecture, inflorescence and fruit traits, yield and fruit quality was observed, and appreciable variation was noticed. According to hierarchical clustering, six distinct clusters were established based on pre-defined varietal groups. Divergence among accessions for phenotypic and fruit compositional variability was analyzed, and eight principal components were identified that contributed ~71% of the variation, with fruit shape, width, wall thickness, weight, and fruit quality traits being the most discriminant. Evaluation of the response to tobacco mosaic virus (TMV) and pepper mild mottle mosaic virus (PMMoV) showed that 24 and 1 accession were resistant, respectively while no tomato spotted wilt virus (TSWV) resistance was found. Considerable diversity for agro-bio-morphological traits indicates the Balkan pepper collection as good gene sources for pre-breeding and cultivar development that are locally adapted.