Pepper Mapping & Major Genes - Department of Plant Sciences

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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 P1 159234
• 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 tomato Zygier 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 P1 152225 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
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