Jin-Kyung Kwon

Molecular mapping and characterization of a single dominant gene controlling CMV resistance in peppers (Capsicum annuum L.)

Cucumber mosaic virus (CMV) is one of the most destructive viruses in the Solanaceae family. Simple inheritance of CMV resistance in peppers has not previously been documented; all previous studies have reported that resistance to this virus is mediated by several partially dominant and recessive genes. In this study, we showed that the Capsicum annuum cultivar ‘Bukang’ contains a single dominant resistance gene against CMVKorean and CMVFNY strains. We named this resistance gene Cmr1 (Cucumber mosaic resistance 1). Analysis of the cellular localization of CMV using a CMV green fluorescent protein construct showed that in ‘Bukang,’ systemic movement of the virus from the epidermal cell layer to mesophyll cells is inhibited. Genetic mapping and FISH analysis revealed that the Cmr1 gene is located at the centromeric region of LG2, a position syntenic to the ToMV resistance locus (Tm-1) in tomatoes. Three SNP markers were developed by comparative genetic mapping: one intron-based marker using a pepper homolog of Tm-1, and two SNP markers using tomato and pepper BAC sequences mapped near Cmr1. We expect that the SNP markers developed in this study will be useful for developing CMV-resistant cultivars and for fine mapping the Cmr1 gene.

Comparative analysis of pepper and tomato reveals euchromatin expansion of pepper genome caused by differential accumulation of Ty3/Gypsy-like elements

BackgroundAmong the Solanaceae plants, the pepper genome is three times larger than that of tomato. Although the gene repertoire and gene order of both species are well conserved, the cause of the genome-size difference is not known. To determine the causes for the expansion of pepper euchromatic regions, we compared the pepper genome to that of tomato.ResultsFor sequence-level analysis, we generated 35.6 Mb of pepper genomic sequences from euchromatin enriched 1,245 pepper BAC clones. The comparative analysis of orthologous gene-rich regions between both species revealed insertion of transposons exclusively in the pepper sequences, maintaining the gene order and content. The most common type of the transposon found was the LTR retrotransposon. Phylogenetic comparison of the LTR retrotransposons revealed that two groups of Ty3/Gypsy-like elements (Tat and Athila) were overly accumulated in the pepper genome. The FISH analysis of the pepper Tat elements showed a random distribution in heterochromatic and euchromatic regions, whereas the tomato Tat elements showed heterochromatin-preferential accumulation.ConclusionsCompared to tomato pepper euchromatin doubled its size by differential accumulation of a specific group of Ty3/Gypsy-like elements. Our results could provide an insight on the mechanism of genome evolution in the Solanaceae family.

Localization of 5S and 25S rRNA genes on Somatic and meiotic chromosomes in Capsicum species of chili pepper

The loci of the 5S and 45S rRNA genes were localized on chromosomes in five species of Capsicum, namely, an-nuum, chacoense, frutescens, baccatum, and chinense by FISH. The 5S rDNA was localized to the distal region of one chromosome in all species observed. The number of 45S rDNA loci varied among species; one in annuum, two in chacoense, frutescens, and chinense, and four in baccatum, with the exceptions that ‘CM334’ of annuum had three loci and ‘tabasco’ of frutescens had one locus. ‘CM334’-derived BAC clones, 384B09 and 365P05, were screened with 5S rDNA as a probe, and BACs 278M03 and 262A23 were screened with 25S rDNA as a probe. Both ends of these BAC clones were sequenced. FISH with these BAC probes on pachytenes from ‘CM334’ plant showed one 5S rDNA locus and three 45S rDNA loci, consistent with the patterns on the somatic chromosomes. The 5S rDNA probe was also applied on extended DNA fibers to reveal that its coverage measured as long as 0.439 Mb in the pepper genome. FISH techniques applied on somatic and meiotic chromosomes and fibers have been established for chili to provide valuable information about the copy number variation of 45S rDNA and the actual physical size of the 5S rDNA in chili.

Evolution of the large genome in Capsicum annuum occurred through accumulation of single‐type long terminal repeat retrotransposons and their derivatives

Although plant genome sizes are extremely diverse, the mechanism underlying the expansion of huge genomes that did not experience whole-genome duplication has not been elucidated. The pepper, Capsicum annuum, is an excellent model for studies of genome expansion due to its large genome size (2700 Mb) and the absence of whole genome duplication. As most of the pepper genome structure has been identified as constitutive heterochromatin, we investigated the evolution of this region in detail. Our findings show that the constitutive heterochromatin in pepper was actively expanded 20.0-7.5 million years ago through a massive accumulation of single-type Ty3/Gypsy-like elements that belong to the Del subgroup. Interestingly, derivatives of the Del elements, such as non-autonomous long terminal repeat retrotransposons and long-unit tandem repeats, played important roles in the expansion of constitutive heterochromatic regions. This expansion occurred not only in the existing heterochromatic regions but also into the euchromatic regions. Furthermore, our results revealed a repeat of unit length 18-24 kb. This repeat was found not only in the pepper genome but also in the other solanaceous species, such as potato and tomato. These results represent a characteristic mechanism for large genome evolution in plants.

A survey of natural and ethyl methane sulfonate-induced variations of eIF4E using high-resolution melting analysis in Capsicum

Allele mining is a method used to find undiscovered natural variations or induced mutations in a plant, and has become increasingly important as more genomic information is available in plants. A high-throughput method is required to facilitate the identification of novel alleles in a large number of samples. In this paper we describe the application of a high-resolution melting (HRM) method to detect natural variations and ethyl methane sulfonate (EMS)-induced mutations in Capsicum. We have scanned single polymorphic mutations in the first exon of the eIF4E gene, wherein the mutations confer resistance to potyviruses. Sixteen allelic variations out of 248 germplasm collections were identified using HRM analysis, and one accession carrying an allelic variation (pvrHRM113) was confirmed to be resistant to the TEV-HAT strain. In addition, five single polymorphic mutations in the eIF4E gene were identified in an EMS-induced mutant population. These results demonstrate that HRM allows for the rapid identification of new allelic variants in both natural and artificial mutant populations.

Biosynthesis of Capsinoid is Controlled by the Pun1 Locus in Pepper

Pungency in pepper (Capsicum annuum L.) has unique characteristics due to the alkaloid compound group, capsaicinoids, which includes capsaicin. Although capsaicinoids have been proved to have pharmacological and physiological effects on human health, the application of capsaicinoids has been limited because of their pungency. Capsinoids found in non-pungent peppers share closely related structures with capsaicinoids and show similar biological effects. Previous studies demonstrated that mutations in the p-AMT gene were related to the production of capsinoids; however, the pathway of capsinoid synthesis has not yet been fully elucidated. In this study, we performed genetic analysis to determine the mechanism of capsinoid synthesis using a F6 recombinant inbred line population. In this population, the presence/absence of capsinoids co-segregated with the genotype of the Pun1 locus, without exception. In addition, we screened the patterns of capsinoid synthesis and the correlation between the Pun1 locus and capsinoid synthesis in p-AMT mutant accessions. In Capsicum germplasms, we selected amino-acid-substituted mutants in the PLP binding domain of the p-AMT gene. Capsinoids were not synthesized with the recessive pun1 gene, regardless of the p-AMT genotype, and no relationship was found between p-AMT mutant type and capsinoid content. We concluded that the Pun1 gene, which is responsible for capsaicinoid synthesis, also controls capsinoid synthesis.

Tomato Male sterile 1035 is essential for pollen development and meiosis in anthers

Summary This study demonstrated that tomato Male sterile 10 35 encodes a basic helix–loop–helix transcription factor involved in meiosis and tapetum development at the early stage of anther development.

New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication

BackgroundTransposable elements are major evolutionary forces which can cause new genome structure and species diversification. The role of transposable elements in the expansion of nucleotide-binding and leucine-rich-repeat proteins (NLRs), the major disease-resistance gene families, has been unexplored in plants.ResultsWe report two high-quality de novo genomes (Capsicum baccatum and C. chinense) and an improved reference genome (C. annuum) for peppers. Dynamic genome rearrangements involving translocations among chromosomes 3, 5, and 9 were detected in comparison between C. baccatum and the two other peppers. The amplification of athila LTR-retrotransposons, members of the gypsy superfamily, led to genome expansion in C. baccatum. In-depth genome-wide comparison of genes and repeats unveiled that the copy numbers of NLRs were greatly increased by LTR-retrotransposon-mediated retroduplication. Moreover, retroduplicated NLRs are abundant across the angiosperms and, in most cases, are lineage-specific.ConclusionsOur study reveals that retroduplication has played key roles for the massive emergence of NLR genes including functional disease-resistance genes in pepper plants.

An ultra-high-density bin map facilitates high-throughput QTL mapping of horticultural traits in pepper (Capsicum annuum)

Most agricultural traits are controlled by quantitative trait loci (QTLs); however, there are few studies on QTL mapping of horticultural traits in pepper (Capsicum spp.) due to the lack of high-density molecular maps and the sequence information. In this study, an ultra-high-density map and 120 recombinant inbred lines (RILs) derived from a cross between C. annuum ‘Perennial’ and C. annuum ‘Dempsey’ were used for QTL mapping of horticultural traits. Parental lines and RILs were resequenced at 18× and 1× coverage, respectively. Using a sliding window approach, an ultra-high-density bin map containing 2,578 bins was constructed. The total map length of the map was 1,372 cM, and the average interval between bins was 0.53 cM. A total of 86 significant QTLs controlling 17 horticultural traits were detected. Among these, 32 QTLs controlling 13 traits were major QTLs. Our research shows that the construction of bin maps using low-coverage sequence is a powerful method for QTL mapping, and that the short intervals between bins are helpful for fine-mapping of QTLs. Furthermore, bin maps can be used to improve the quality of reference genomes by elucidating the genetic order of unordered regions and anchoring unassigned scaffolds to linkage groups.

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum ‘Bukang’ cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.