Development of a CAPS marker for the Pvr4 locus: a tool for pyramiding potyvirus resistance genes in pepper.

The Pvr4 resistance gene in pepper confers a complete resistance to the three pathotypes of potato virus Y (PVY) and to pepper mottle virus (PepMoV). In order to use this gene in a marker-assisted selection (MAS) program and to permit the pyramiding of several potyvirus resistance genes in the same cultivar, tightly linked amplified fragment length polymorphism (AFLP) markers were obtained by the bulked segregant analysis method. Eight linked AFLP markers were mapped in an interval from 2.1 +/- 0.8 to 13.8 +/- 2.9 cM around this locus. The closest codominant AFLP marker was converted into a codominant CAPS (cleaved amplified polymorphic sequence) marker using data from the alignment of the two allele sequences. We have further characterized the relevance of the CAPS marker for MAS programs in different pepper breeding lines.     

Non-pungent Capsicum contains a deletion in the capsaicinoid synthetase gene, which allows early detection of pungency with SCAR markers

The capsaicinoid synthetase (CS) gene cosegregated perfectly with the C locus, which controls the presence of pungency, in 121 F2 individuals from a cross between 'ECW123R' and 'CM334', both of Capsicum annuum. We concluded that CS and C are tightly linked. Sequence analysis of the genes of four pungent and four non-pungent pepper lines showed that the non-pungent peppers had a 2,529 bp-deletion in the 5' upstream region of CS. We have developed molecular markers of the C locus to detect pungency at the seedling stage. Based on the deleted sequence, we developed five SCAR markers, two of them being codominant. These SCAR markers will be useful for easy, accurate, and early detection of non-pungent individuals in breeding programs.     

Marker development for erect versus pendant-orientated fruit in Capsicum annuum L

The erect habit of fruit setting is a unique characteristic of ornamental peppers and wild pepper species. The erect habit is known to be controlled by the up locus on pepper (Capsicum annuum L.) chromosome 12. The result of a genetic analysis using Saengryeog 211 (pendant), Saengryeog 213 (erect), and their F1 and BC1 progeny demonstrated that up is a recessive gene. To develop an up-linked marker, bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) were employed using 108 F2:3 individuals. The closest AFLP marker, A2C79, was located at a genetic distance of 1.7 cM from the up locus and was converted into a cleaved amplified polymorphic sequence (CAPS) marker. This marker was mapped at a genetic distance of 4.3 cM from the up locus. When the CAPS was applied to seven ornamental lines and 27 breeding lines with erect fruit, these genotypes of 28 lines were correctly predicted. Thus, the CAPS marker will be useful for marker-assisted selection (MAS) of pepper breeding lines with the up allele at the early seedling stage.     

Development of RAPD and SCAR markers linked to the Pvr4 locus for resistance to PVY in pepper ( Capsicum annuum L.)

Potato Virus Y (PVY) is the only potyvirus infecting pepper ( Capsicum annuum L.) in Europe. Currently, the development of pepper varieties resistant to PVY seems to be the most-efficient method to control PVY damage. Among the sources of resistance, a monogenic dominant gene Pvr4 confers resistance against all known PVY pathotypes. In this work, bulked segregant analysis (BSA) was used to search for randomly amplified polymorphic DNA (RAPD) markers linked to the Pvr4 gene, using segregating progenies obtained by crossing a homozygous resistant ('Serrano Criollo de Morelos-334') with a homozygous susceptible ('Yolo Wonder') cultivar. Eight hundred decamer primers were screened to identify one RAPD marker (UBC19(1432)) linked in repulsion phase to Pvr4. This marker was converted into a dominant sequence characterised amplified region (SCAR) marker (SCUBC19(1423)). This marker was mapped into a dense Capsicum genetic map in a region where several genes for resistance to different diseases are located. This marker can be useful to identify PVY-resistant genotypes in segregating progenies of pepper in marker-assisted selection (MAS) breeding programs.     

A versatile PCR marker for pungency in Capsicum spp.

Pungency in Capsicum spp. is an important quality trait for pepper breeding. The perception of pungency in pepper is due to the presence of a group of compounds named capsaicinoids, only found within the Capsicum genus. How pungency is controlled at genetic and molecular levels has not been completely elucidated. The use of molecular markers to assess pungency trait is required for molecular breeding, despite the difficulty of development of universal markers for this trait. In this work, a DNA sequence possibly related to pungency with a high similarity to Pun1 locus was studied, and sequence analysis of this homolog revealed a 15?bp deletion in non-pungent pepper accessions. An allele-specific pair of primers was designed and specific fragments of 479?bp from non-pungent and 494?bp from pungent accessions were obtained. Polymorphism of this marker, named MAP1, was tested in a wide range of accessions, belonging to several Capsicum species, including pungent and non-pungent accessions of C. annuum L., and pungent accessions of C. chinense, C. baccatum, C. frutescens, C. pubescens, C. galapagoense, C. eximium, C. tovarii, C. cardenasii, and C. chacoense. All these Capsicum accessions were correctly discriminated. The marker suitability to assess pungency in domesticated and wild Capsicum species was demonstrated, and therefore it will be very useful in marker assisted selection (MAS). Moreover, MAP1 was located in a saturated pepper linkage map and its possible relationship with the Pun1 locus has been discussed. Among the available markers for this complex quality trait, the marker developed in this study is the most universal so far.     

BAC-derived markers converted from RFLP linked to Phytophthora capsici resistance in pepper (Capsicum annuum L.)

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.     

A CAPS marker to assist selection of tomato spotted wilt virus (TSWV) resistance in pepper.

The hypersensitive resistance to tomato spotted wilt virus (TSWV) in pepper is determined by a single dominant gene (resistant allele: Tsw) in several Capsicum chinense genotypes. In order to facilitate the selection for this resistance, four RAPD (among 250 10-mer primers tested) were found linked to the Tsw locus using the bulked segregant analysis and 153 F2 individuals. A close RAPD marker was converted into a codominant cleaved amplified polymorphic sequence (CAPS) using specific PCR primers and restriction enzymes. This CAPS marker is tightly linked to Tsw (0.9 +/- 0.6 cM) and is helpful for marker-assisted selection in a wide range of genetic intercrosses.     

Cleaved amplified polymorphic sequence and amplified fragment length polymorphism markers linked to the fertility restorer gene in chili pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS) in plants, which is due to failure to produce functional pollen, is a maternally inherited trait. Specific nuclear genes that sup-press CMS, termed fertility restorer (Rf) genes, have been identified in several plants. In this study, Rf-linked molecular markers in pepper (Capsicum annuum L.) were detected by bulked segregant analysis of eight amplified fragment length polymorphisms (AFLPs). Only AFRF8 was successfully converted to a cleaved amplified polymorphic sequence (CAPS) marker. This was named AFRF8CAPS and genotype determination using it agreed with that obtained with the original AFRF8. A linkage map with a total size of 54.1 cM was constructed with AFRF8CAPS and the seven AFLP markers using the Kosambi function. The AFRF8CAPS marker was shown to be closest to Rf with a genetic distance of 1.8 cM. These markers will be useful for fast and reliable detection of restorer lines during F(1) hybrid seed production and breeding programs in pepper.     

Identification of Gene-Specific Polymorphisms and Association with Capsaicin Pathway Metabolites in Capsicum annuum L. Collections

Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Production of capsaicin is an important quantitative trait with high environmental variance, so the development of markers regulating capsaicinoid accumulation is important for pepper breeding programs. In this study, we performed association mapping at the gene level to identify single nucleotide polymorphisms (SNPs) associated with capsaicin pathway metabolites in a diverse Capsicum annuum collection during two seasons. The genes Pun1, CCR, KAS and HCT were sequenced and matched with the whole-genome sequence draft of pepper to identify SNP locations and for further characterization. The identified SNPs for each gene underwent candidate gene association mapping. Association mapping results revealed Pun1 as a key regulator of major metabolites in the capsaicin pathway mainly affecting capsaicinoids and precursors for acyl moieties of capsaicinoids. Six different SNPs in the promoter sequence of Pun1 were found associated with capsaicin in plants from both seasons. Our results support that CCR is an important control point for the flux of p-coumaric acid to specific biosynthesis pathways. KAS was found to regulate the major precursors for acyl moieties of capsaicinoids and may play a key role in capsaicinoid production. Candidate gene association mapping of Pun1 suggested that the accumulation of capsaicinoids depends on the expression of Pun1, as revealed by the most important associated SNPs found in the promoter region of Pun1.     

Metabolite biodiversity in pepper (Capsicum) fruits of thirty-two diverse accessions: variation in health-related compounds and implications for breeding

A comprehensive study on morphology and biochemical compounds of 32 Capsicum spp. accessions has been performed. Accessions represented four pepper species, Capsicum annuum, Capsicum frutescens, Capsicum chinense and Capsicum baccatum which were selected by their variation in morphological characters such as fruit color, pungency and origin. Major metabolites in fruits of pepper, carotenoids, capsaicinoids (pungency), flavonoid glycosides, and vitamins C and E were analyzed and quantified by high performance liquid chromatography. The results showed that composition and level of metabolites in fruits varied greatly between accessions and was independent of species and geographical location. Fruit color was determined by the accumulation of specific carotenoids leading to salmon, yellow, orange, red and brown colored fruits. Levels of both O- and C-glycosides of quercetin, luteolin and apigenin varied strongly between accessions. All non-pungent accessions were devoid of capsaicins, whereas capsaicinoid levels ranged from 0.07 up to 80 mg/100g fr. wt. in fruit pericarp. In general, pungent accessions accumulated the highest capsaicinoid levels in placenta plus seed tissue compared to pericarp. The non-pungent capsaicinoid analogs, capsiates, could be detected at low levels in some pungent accessions. All accessions accumulated high levels of vitamin C, up to 200 mg/100g fr. wt. The highest vitamin E concentration found was 16 mg/100g fr. wt. Based on these metabolic data, five accessions were selected for further metabolic and molecular analysis, in order to isolate key genes involved in the production of these compounds and to assist future breeding programs aimed at optimizing the levels of health-related compounds in pepper fruit.     

Construction of an integrated pepper map using RFLP,SSR, CAPS,AFLP, WRKY,rRAMP, and BAC end sequences

Map-based cloning to find genes of interest, markerassisted selection (MAS), and marker-assisted breeding (MAB) all require good genetic maps with high reproducible markers. For map construction as well as chromosome assignment, development of single copy PCR-based markers and map integration process are necessary. In this study, the 132 markers (57 STS from BAC-end sequences, 13 STS from RFLP, and 62 SSR) were newly developed as single copy type PCR-based markers. They were used together with 1830 markers previously developed in our lab to construct an integrated map with the Joinmap 3.0 program. This integrated map contained 169 SSR, 354 RFLP, 23 STS from BAC-end sequences, 6 STS from RFLP, 152 AFLP, 51 WRKY, and 99 rRAMP markers on 12 chromosomes. The integrated map contained four genetic maps of two interspecific (Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’) and two intraspecific (C. annuum ‘CM334’ and C. annuum ‘Chilsungcho’) populations of peppers. This constructed integrated map consisted of 805 markers (map distance of 1858 cM) in interspecific populations and 745 markers (map distance of 1892 cM) in intraspecific populations. The used pepper STS were first developed from end sequences of BAC clones from Capsicum annuum ‘CM334’. This integrated map will provide useful information for construction of future pepper genetic maps and for assignment of linkage groups to pepper chromosomes.     

Molecular mapping and genetic analysis of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene

The brown planthopper (BPH), Nilaparvata lugens St?l, is a serious insect pest of rice (Oryza saliva L.). We have determined the chromosomal location of a BPH resistance gene in rice using SSR and RFLP techniques. A rice line 'B14', derived from the wild rice Oryza latifolia, showed high resistance to BPH. For tagging the resistance gene in 'B14X', an F2 population and a recombinant inbred (RI) population from a cross between Taichung Native 1 and 'B14' were developed and evaluated for BPH resistance. The results showed that a single dominant gene controlled the resistance of 'B14' to BPH. Bulked segregant SSR analysis was employed for identification of DNA markers linked to the resistance gene. From the survey of 302 SSR primer pairs, three SSR (RM335, RM261, RM185) markers linked to the resistance gene were identified. The closest SSR marker RM261 was linked to the resistance gene at a distance of 1.8 cM. Regions surrounding the resistance gene and the SSR markers were examined with additional RFLP markers on chromosome 4 to define the location of the resistance gene. Linkage of RFLP markers C820, R288, C946 with the resistance gene further confirmed its location on the short arm of chromosome 4. Closely linked DNA markers will facilitate selection for resistant lines in breeding programs and provide the basis for map-based cloning of this resistance gene.     

Origin of plantlets and callus obtained from chile pepper anther cultures

Summary Androgenesis occurred from chile pepper (Capsicum annuum L.) anthers incubated in a continuous warm environment (29° C) with continuous light. Forty plantes and embryoids were retrieved from anther cultures and anllyzed for isozyme markers. Of these, 35 exhibited a single allele for markers suggesting microspore origin, while 5 were heterozygous indicating somatic tissue origin. Chromosome numbers were confirmed for 21 plantlets, of which 16 were haploid and 5 were diploid. However, two plants exhibited a single allele for an isozyme marker but possessed the diploid chromosome number, suggesting spontaneous doubling. Anther cultures also produced callus. Nearly 92% of the slow-growing calli sampled were heterozygous for the isozyme marker, suggesting somatic tissue origin. More than 46% of the fast-growing calli exhibited only one allele for the marker, indicating microspore origin. Callus did not regenerate plantlets. The occurrence of both heterozygous and homozygous diploid plantlets from pepper anther cultures has important implications for applied breeding programs.     

Molecular mapping of the male-sterile, female-sterile mutant gene (st8) in soybean

Soybean male-sterile, female-sterile mutant genes have been identified by genetic and cytological studies. The St8 gene has been identified as an asynaptic mutation resulting in male and female sterility. This mutant gene was derived from a gene-tagging study using the soybean w4-mutable line. In this report we identified the genetic map position of st8 via restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers. The St8 gene mutation was located between RFLP marker E107 and SSR markers Satt132, Sct_065, and Satt414 on molecular linkage group J and linked to each by 7.8 cM and 3.4 cM, respectively.     

Construction of an intraspecific integrated linkage map of pepper using molecular markers and doubled-haploid progenies.

An integrated molecular linkage map of pepper (Capsicum annuum L.), including mainly RFLP and RAPD markers, has been constructed by alignment of three intraspecific linkage maps generated by segregating doubled-haploid progenies. A total of 85 markers covered approximately 820 cM in 14 linkage groups. Four linkage groups were assigned to 4 chromosomes. Two new genes of agronomic interest were located: L controlling hypersensitive resistance to TMV and up controlling the erect habit of the fruits. The C gene controlling the fruit pungency was more precisely located. This map is estimated to represent from 36 to 59% of the total pepper genome. An examination of segregation data has revealed several genomic regions with aberrant segregation ratios often favouring the agronomic big-fruited parents, particularly in crosses involving the exotic parent CM334, suggesting that these genome regions are subjected to selection during the process of doubled-haploid production. The suitability of doubled-haploid progenies for mapping projects and the differences observed between this intraspecific integrated map with earlier published interspecific pepper maps are discussed.     

An integrated genetic linkage map of pepper (Capsicum spp.)

An integrated genetic map of pepper including 6 distinct progenies and consisting of 2262 markers covering 1832 cM was constructed using pooled data from six individual maps by the Keygene proprietary software package INTMAP. The map included: 1528 AFLP, 440 RFLP, 288 RAPD and several known gene sequences, isozymes and morphological markers. In total, 320 anchor markers (common markers in at least two individual maps) were used for map integration. Most anchor markers (265) were common to two maps, while 27, 26 and 5 markers were common to three, four and five maps, respectively. Map integration improved the average marker density in the genome to 1 marker per 0.8 cM compared to 1 marker per 2.1 cM in the most dense individual map. In addition, the number of gaps of at least 10 cM between adjacent markers was reduced in the integrated map. Although marker density and genome coverage were improved in the integrated map, several small linkage groups remained, indicating that further marker saturation will be needed in order to obtain a full coverage of the pepper genome. The integrated map can be used as a reference for future mapping studies in Capsicum and to improve the utilization of molecular markers for pepper breeding.These authors contributed equally to the work described in this paper(e-mail: