Nonhost resistance is a plant immune response to resist most pathogens. The molecular basis of nonhost resistance remains poorly understood but recognition of pathogen effectors by immune receptors, a response known as effector-triggered immunity, has been proposed as a component of nonhost resistance. We performed transient expression of 54 P. infestans RXLR effectors in pepper accessions using optimized heterologous expression methods and analyzed the inheritance of effector-induced cell death in an F2 population derived from a cross between two pepper accessions. Pepper showed a localized cell death response upon inoculation with P. infestans, suggesting that recognition of effectors may contribute to nonhost resistance in this system. Nonhost pepper accessions recognized from 2 to 36 effectors. Among the effectors, PexRD8 and Avrblb2 induced cell death on a broad range of pepper accessions. Segregation of effector-induced cell death in an F2 population derived from a cross between two pepper accessions fit a 15:1, 9:7 and 3:1 depending on the effector. Our genetic data suggests that single or two independent/complementary dominant genes are involved in the recognitions RXLR effectors. Our findings indicate that multiple loci recognizing a series of effectors underpin nonhost resistance of pepper to P. infestans and may confer resistance durability.

Sessile organism, plants constitutively challenged with pathogens have been developed various strategies for protection, such as preformed and inducible defense mechanisms. Receptor-like Proteins(RLPs) play critical roles in defense response as well as in plant development and growth. The domain structure of RLPs consists of extracellular leucine–rich repeats, a transmembrane domain, and a short cytoplasmic tail. Here, we identified putative 170 RLP genes from pepper genome using in-house bioinformatics pipeline. The distribution of RLPs on pepper pseudomolecule showed uneven spread and a number of RLPs were physically clustered by tandem array in the specific chromosome. Motifs analysis of pepper RLPs showed conserved LRR sequences (LxxLxxLDLxxNxxxGxIP). To understand further functional and evolutionary characteristics, evolutional relationship and gene profiling analysis are on progress.

Plants have evolved elaborate innate immune systems against invading pathogens, such as bacteria, fungi, oomycetes, viruses and insects. Among them, intracellular immune receptors known as nucleotide-binding site and leucine-rich repeat (NB-LRR) play critical roles in effector-triggered immunity (ETI) regarding to plant defense. Here, we identified potential NB-LRR coding sequences from pepper genome using bioinformatics analysis and performed comparative analysis with Solanaceae plants. As a result, we identified 267, 443, and 755 NBS-encoding genes in the genome of tomato, potato, and pepper, respectively. These may indicate that the Solanaceae NB-LRRs were evolved through species-specific unequal-duplication event. Further phylogenetic and clustering analyses revealed that Solanaceae NB-LRRs were classified into the 14 subgroups with 1 TNL and 13 CNL types. We found that the genes in CNL-G1 and CNL-G2 subgroup were highly expanded compared to other subgroup showing a large portion of NB-LRR in pepper genome. Among 755 NB-LRRs in pepper genome, 623 were physically mapped on all 12 pepper chromosome pseudomolecules. Furthermore, a number of NB-LRRs in the same group were physically clustered by tandem array in the specific chromosome. Genome-wide identification of pepper NB-LRR family and their evolutionary analysis could provide an important resource for identification and characterization of genes for breeding of disease resistance crops.

sy-2 (Seychelles-2) is a temperature sensitive natural mutant of Capsicum chinense and native to Seychelles Island in Africa. Previously we showed that sy-2 leaves were irregularly shaped and defective in chlorophyll development at temperatures below 24℃. A segregation test revealed that the sy-2 gene is controlled by a single recessive gene. To identify the sy-2 gene, we performed a map-based cloning approach using a total 600 individual F2 plants derived from crossing sy-2 and the wild type C. chinense ‘No.3341’. Fine-mapping of the locus allowed us to position sy-2 to an approximately 170-kb region flanked by markers IN2-1-1 and SNP-3-7 on chromosome 1. Among the approximately 36 hypothetical genes in this region several candidate genes including: HSP90-like ATPase family proteins, lipid-transfer proteins, calmodulin-domain protein kinases, and zinc finger proteins (ZFPs) were identified. RT-PCR and sequencing of the hypothetical genes are under way to identify sy-2.

Platycodon grandiflorum, which is the only species in the genus Platycodon of the family Campanulaceae, is an herbaceous flowering perennial. P. grandiflorum is generally known as bellflower or balloon flower indicating its ornamental uses. It has also been traditionally used as a medicinal crop in East Asia, which is widely employed as an antiphlogistic, antitussive, and expectorant. However, marker-assisted selection and molecular breeding in P. grandiflorum has lagged behind other plants such as pepper and tomato because of the lack of genetic information and effective molecular markers. Transcriptome sequencing provides an effective way to obtain large amount of sequence data when there is no available genome sequence. In this study, we performed a transcriptome analysis in platycodons, which has not been attempted previously. We analyzed simple sequence repeats (SSRs) using RNA-seq data. Di-nucleotide motifs were the most abundant repeats (39% ~ 40%) followed by mono- (26% ~ 32%), tri- (25% ~ 31%), tetra- (1.5% ~ 1.9 %), penta- (0.3% ~ 1%) in three platycodon accessions. Based on the SNP information obtained from RNA-seq analysis, we developed 12 PCR-based markers in Platycodon. The number of alleles ranged from two to seven with the average PIC value of 0.373. These 12 markers were applied to 21 platycodon accessions and a phylogenetic tree was constructed. The markers developed in this study could be introduced in molecular breeding program of platycodons. The SSR information obtained from RNA-seq analysis could be further utilized for developing genic-SSR markers in platycodons. Since platycodon is considered as an orphan crop, which has not been actively deployed for genetic study, the sequence information obtained from this study will contribute to further genetic improvements, genomic information and gene discovery in platycodon

sy-2 (Seychelles-2) is a temperature sensitive mutant of Capsicum chinense and native to Seychelles Island in Africa. Previously we showed that sy-2 leaves were irregularly shaped and defective in chlorophyll development at temperatures lower than 24℃. A segregation test revealed that the sy-2 gene is controlled by a single recessive gene. To identify the sy-2 gene, we performed a map-based cloning approach using a total of 1,010 F2 plants derived from crossing sy-2 and the wild type C. chinense ‘No.3341’. sy-2 gene is located on chromosome 1, 0.3 cM and 0.1cM away from cosII markers C2_At4g29120 and C2_At1g09070, respectively. The tomato genome sequence between those two markers was compared with pepper genome sequence. We found three of pepper scaffold sequences in this region. We developed seven ingle nucleotide polymorphism (SNP) markers on the pepper scaffold sequences, among which five SNP markers were co-segregated with sy-2. To fill the gap between the scaffolds which contains co-segregating markers, we screened a bacterial artificial chromosome (BAC) library, and end-sequences of total of 22 AC clones were i. We found that five clones were overlapped to cover the gap. We fully sequenced four AC clones and found that the physical distance between C2_At4g29120 and C2_At1g09070 is 343kb. This region contains 70 putative genes such as HSP90-like ATPase family proteins, lipid-transfer proteins, calmodulin-domain protein kinases, and zinc finger proteins (ZFPs). To identify the sy-2 gene, we performed RT-PCR and found that a ZFP-like gene is differentially expressed between WT and sy-2 leaves. This result suggests that the ZFP-like gene is a strong candidate for the sy-2 gene. We are currently characterizing this candidate gene.

The perturbation of the steady state of reactive oxygen species due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 promoter with/without a selection marker, the bar gene. A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol. An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice

Pepper mottle virus (PepMoV) is frequently occurring virus in pepper field. PepMoV infected plants show symptoms including mosaic leaf, distortion of foliage and fruit deformation. The dominant gene Pvr7 from Capsicum annuum ‘9093’ confers resistance to PepMoV. Previous research reveals that Pvr7 is located in 10 chromosome and linked to the dominant potyvirus resistance gene Pvr4 and Tomato spotted wilt virus (TSWV) resistance gene Tsw. To identify the Pvr7 gene, we constructed an intraspecific F2 mapping population from a cross between C. annuum ‘9093’ (PepMov resistant) and C. annuum ‘Jejujaerae’ (PepMoV susceptible). Resistance of F2 plants were screened with green flouorescent protein (GFP) tagged PepMoV. Genomic DNA was extracted from F2 individuals and markers were developed using C.annuum ‘CM334’ whole genome sequence (WGS). Several single nucleotide polymorphism (SNP) markers that were co-segregated with Pvr7 were developed. We are expecting that this Pvr7 SNP marker can be used to breeding PepMoV resistant cultivars and fine mapping of Pvr7.

Chili pepper (Capsicum annuum) is one of the most important vegetable crop for human being as a rich source of nutritions and spicy condiments. To make reference genome sequence of pepper, we sequenced the whole genome of Capsicum annuum, CM334 using Illumina/Solexa Genome Analyzer GA2. The genome size of CM334 is estimated as 3.5 Gb. A total of 716 Gb (205.96x coverages of the whole genome) of raw sequences were generated. After filtering out the low quality sequences, a total of 233 Gb (66.7x) of the raw sequences were used for assembly. Total assembled contig length and number were 2.93 Gb and 295,502, respectively. N50 and average length were 25.72 kb and 6,5 kb, respectively. By sequencial scaffolding with mate-pair sequences of 2 kb - 20 kb sizes, a total of 3.04 Gb of scaffold which is approximately 90% of the whole genome was assembled. The total number of scaffolds was 33,876 with N50 length of 1,605 kb. For annotation of the pepper genome, a total of 46 Gb of transcriptome sequences were generated from 12 different tissues using Illumina GA2 and Hi-seq 2000. We are under way of analyzing the characteristic traits of pepper using transcriptome data. The progress of pepper genome sequencing project including gene annotation, gene family analysis, comparative genomics studies on evolution of hot taste, genome expansion and fruit development will be presented in the meeting.