Koeun Han

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.

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.

Development of a Genetic Map for Onion (Allium cepa L.) Using Reference-Free Genotyping-by-Sequencing and SNP Assays

Single nucleotide polymorphisms (SNPs) play important roles as molecular markers in plant genomics and breeding studies. Although onion (Allium cepa L.) is an important crop globally, relatively few molecular marker resources have been reported due to its large genome and high heterozygosity. Genotyping-by-sequencing (GBS) offers a greater degree of complexity reduction followed by concurrent SNP discovery and genotyping for species with complex genomes. In this study, GBS was employed for SNP mining in onion, which currently lacks a reference genome. A segregating F2 population, derived from a cross between ‘NW-001’ and ‘NW-002,’ as well as multiple parental lines were used for GBS analysis. A total of 56.15 Gbp of raw sequence data were generated and 1,851,428 SNPs were identified from the de novo assembled contigs. Stringent filtering resulted in 10,091 high-fidelity SNP markers. Robust SNPs that satisfied the segregation ratio criteria and with even distribution in the mapping population were used to construct an onion genetic map. The final map contained eight linkage groups and spanned a genetic length of 1,383 centiMorgans (cM), with an average marker interval of 8.08 cM. These robust SNPs were further analyzed using the high-throughput Fluidigm platform for marker validation. This is the first study in onion to develop genome-wide SNPs using GBS. The resulting SNP markers and developed linkage map will be valuable tools for genetic mapping of important agronomic traits and marker-assisted selection in onion breeding programs.

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

Summary 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.

Identification and molecular genetic mapping of Chili veinal mottle virus (ChiVMV) resistance genes in pepper ( Capsicum annuum )

Chili veinal mottle virus (ChiVMV) threatens the agricultural production of peppers (Capsicum annuum) in Asia and Africa. In this study, we evaluated ChiVMV resistance in the four pepper varieties CV3, CV4, CV8, and CV9. Segregation analyses revealed that CV3 and CV8 contain the single dominant resistance gene Cvr1, and CV9 contains the single recessive resistance gene cvr4. SNP markers were developed and used to map the Cvr1 gene in CV3 to the short arm of chromosome 6 where NLR genes are clustered. In CV4 oligogenic resistance loci were detected. A genotyping-by-sequencing (GBS) combined with modified sliding window approach mapped two resistance loci, to chromosomes 6 and 10. The development of SNP markers and the resulting knowledge of genomic positioning will assist in breeding ChiVMV-resistant pepper varieties and in the fine mapping of ChiVMV resistance genes.

Development of SNP markers using genotyping-by-sequencing for cultivar identification in rose (Rosa hybrida)

In this study, we developed single nucleotide polymorphism (SNP) markers to identify cultivars of the polyploid Rosa hybrida using genotyping-by-sequencing (GBS). Sequences obtained from GBS libraries of the genomes of 79 rose cultivars were aligned to contigs created by de novo assembly, and these contigs ranged from 200 to 2,591 bp, with an average of 305 bp per contig. We selected 1,778 SNPs from 13,488 putative SNPs. SNP markers that were present in more than 70% of the 79 cultivars were selected and evaluated, both on the basis of polymorphism information content values and level of heterozygosity, and 20 SNP markers were ultimately selected for high- throughput analysis. From these 20 SNP markers, 4 were successfully converted to markers for DNA chip assays. High resolution melting analysis was carried out to further distinguish rose genotypes. Using a set of seven SNP markers, we identified 70.9% of the 79 rose cultivars, and 87.5% of the 16 new cultivars developed in Goyang City. This paper is the first to report the development of a SNP marker set to identify Rosa hybrida cultivars by GBS.