Pepper Mapping & Major Genes Mapping of chlorophyll retainer (cl) mutation in pepper The Pun1 gene for pungency QTL mapping for fruit size and shape

Molecular mapping of the chlorophyll retainer (cl) mutation in pepper (Capsicum spp.) and screening for candidate genes using tomato ESTs homologous to structural genes for the chlorophyll catabolisim pathway Efrati, Eyal, and Paran 2005 Goals: Locate the cl mutation in the molecular genetic map of pepper Determine whether known structural genes in chlorophyll catabolisim pathway correspond to the CL locus –CHLOROPHYLLASE gene from citrus –PHEOPHORBIDE  OXYGENASE from barley Shown to correspond to the accelerated cell death1 gene in Arabidopsis

Pepper Fruit Color Genotypes Red, Yellow, Orange fruit result from accumulation of carotenoid pigments Chocolate brown color results from lack of chlorophyll degradation & accumulation of red pigments during ripening Controlled by single recessive gene, chlorophyll retainer (cl) Double recessive mutants cl x y yielded olive green recessive fruit

Genetic Mapping of cl in Pepper Constructed BC1 population - C. annuum inbred 4590 x C. chinense P cl mutation segregated as single recessive gene (expected) Out of 198 progeny - –105 mutant phenotype (olive green or brown) –93 wild type (red or yellow) Bulk segregant analysis approach - screened 500 primers on bulks of mutant and wt BC1 individuals - no polymorphisim

Genetic Mapping of cl in Pepper Similar mutant gf mapped to chromosome 8 in tomato –Scored RFLP markers from syntenic chromosome 1 in BC1 population Reciprocal translocation invovling chromosome 1 & 8 differentiates C. annum & C. chinense –Pepper chromosome 8 shows pseudo linkage with chromosome 1 Homologous tomato chromosome 8 in the pepper interspecific cross is chromosome 1

Mapping candidate genes in chlorophyll catabolism pathway Mapped 4 loci in tomato linkage map –3 tomato EST clones homologous to CHLOROPHYLLASE CLET-8-F17 - CHLASE1 - chromosome 6 CLET-26-G11 - CHLASE2 - chromosome 9 CLED-28-N13 - CHLASE3 - chromosome 12 –Mapped PHEOPHORBIDE  OXYGENASE to chromosome 11-D

Conclusions Mapping of CL, CHLOROPHYLLASE, and PHEOPHORBIDE  OXYGENASE to different chromosomes indicates that the cl mutation is not caused by these genes Although CL was mapped in pepper and the genes encoding for the catabolism enzymes were mapped in tomato, the comparative pepper-tomato map indicates that these chromosomal regions are syntenic between the 2 species The location of the genes in the pepper map can be extrapolated based on the their position in the tomato map CL possibly corresponds to a different structural gene or to a gene that regulates the pathway

The Pun1 gene for pungency in pepper encodes a putative acyltransferase Stewart et al., 2005 Pungency in Capsicum pepper fruits due to accumulation of alkaloid capsaicin (capsaicinoids) –Produced by the condensation of vanillylamine, derived from phenylalanine with a branched chain fatty acid Absence of pungency controlled by single recessive gene pun1

Identification of SB2-66 as a candidate gene for Pun1 SB2-66 co-localized with Pun1 in F2 mapping population C. frutescens BG 2816 (Pun1/Pun1) x C. annum ‘Maor’ (pun1/pun1) Mapped to Capsicum chromosome 2 Surveyed pungent and non-pungent genotypes to detect polymorphisims

Genomic DNA sequence and structure at Pun1 locus Sequenced genomic DNA at Pun1 locus in pungent and non-pungent phenotypes - Comparison indicated 2.5kb deletion in pun1 genotypes Conservation of deletion between Bell and Jalapeno peppers indicates the deletion is widespread throughout C. annum

AT3 as a candidate for Pun1 AT3 acyltransferase identified as candidate for Pun1 –Mapped to chromosome region containing Pun1 –Hybridization pattern and amino acid sequences consistent with pungency Silencing of AT3 decreased capsaicinoid accumulation and AT3 protein accumulation - consistent with pun1 phenotype AT3 expressed only in placenta of Pun1 fruit - also exclusively where capsaicinoids expressed –Expression highest during early fruit development - decreases as fruit matures Pun1 encodes acyltransferase critical to capsaicinoid biosynthesis –Recessive allele pun1 due to deletion in promoter and first exon

QTLs mapping for fruit size and shape in chromosomes 2 and 4 in pepper and a comparison of the pepper QTL map with that of tomatoZygier et al., 2005 Use pepper ILs to map QTLs for fruit size and shape in chromosomes 2 and 4 To determine the degree of QTL conservation in the two Capsicum species C. chinense and C. frutescens Are QTLs in these regions orthologous in pepper and tomato

Construction of C. chinense chromosome 2 and 4 ILs Small-fruited P x C. annum blocky type inbred 100/63 Back-crossing & marker assisted selections BC2S3 Homo lines IL-37 & IL 315 Crossed IL with 100/63 constructed F2 populations 68 IL-37 & 125 IL-315 individuals Additional selfing and marker assisted selction resulted in fixed F4 lines

Construction of C. frutescens populations Advanced backcross progenies of Maor x wild-type C. frutescens BG 2816 Back-crossing to BC2 population BC2 plants - BC2-117 & BC2-29 Selfed 99 BC2S1-117 & 123 BC2S1-29

Fruits of the Parents

Conservation of QTLs in chromosome 2 in pepper Fruit weight QTL (fw2.1) with a large effect detected in same genomic region in both mapping populations Localization to same genomic region supports conclusion that same QTL is segregating in both populations Detected single fruit-shape in BC2S1-117 that coincided with fw2.1

Conservation of QTLs in chromosome 2 in pepper and tomato Fruit shape QTL ovate in tomato is most closely linked marker to fruit weight QTL fw2.1 Pepper fw2.1 could also correspond to tomato fw2.1 Linkage of the chromosome region containing fw2.3 to fw2.1 –Possible that 2 tightly linked QTL account for large phenotypic effect on fruit weight in pepper

Conservation of QTLs in chromosome 4 in pepper and tomato 2 fruit weight QTLs detected –Similar positions in both mapping populations –Greater effect in BC2S1-29 than F2 of IL-315 F2 IL-315 fs4.1 co-localized with fw4.1 & BC2S1-29 fs4.2 co-localized with fw4.2

Conclusions Cultivated alleles at both fruit shape QTLs increased the degree of fruit elongation Possible consevation of fw2.1 and fw4.2 conserved in 3 Capsicum species Conservation of fruit weight QTLs in chromosomes 2 and 4 in pepper and tomato Low level of QTL conservation controlling fruit shape in pepper and tomato