Gene Identification using Candidate Gene approach in Pepper
Authors: Arpita Srivastava and Manisha Mangal

Candidate Gene (CG) analysis is based on the hypothesis that known-function genes (the candidate genes) could correspond to loci controlling traits of interest. CGs refer either to cloned genes presumed to affect a given trait (‘functional CGs’) or to genes suggested by their close proximity on linkage maps to loci controlling the trait (‘positional CGs’). In plant genetics, the most common way to identify a CG is to look for map co-segregation between CGs and loci affecting the trait. Statistical association analyses between molecular polymorphisms of the CG and variation in the trait of interest have also been carried out in a few studies. The ?nal validation of a CG will be provided through physiological analyses, genetic transformation and/or sexual complementation. The CG approach has been applied with much success in pepper. Largely this success has been because of the ability to work within the Solanaceae as a highly developed comparative genetic system. Tomato and pepper share DNA sequence similarity, conserved synteny and the information generated from the investments in tomato as model organism. The CG approach has served two purposes. First, it has served to test hypotheses framed around the conservation of gene function in the Solanaceae. Second, it has been used to efficiently characterize traits of agricultural importance in pepper despite the daunting size of the pepper genome. The goal of this article is to present an overview of CG analyses done in pepper for gene identification.

Case Studies:

Tobamovirus Resistance:

The L locus for tobamovirus resistance consists of 4 alleles-L1, L2, L3 and L4. The L locus has been mapped to pepper chromosome 11 and linked markers have been generated but maximum utilization of the L locus requires a marker that identifies the resistance gene itself. The study of the L Locus is aided by the high level of synteny within the Solanaceae. The L locus maps to region that is syntenic to a region of tomato chromosome 11 and contains the I2 gene for Fusarium resistance and also R3 locus for Phytophthora resistance in potato. L gene was hypothesized to be homologous to I2 from tomato and R3a from potato. Thus I2 and R3a served as excellent candidate genes for L locus. Using I2 as candidate gene, L gene was more precisely mapped in an F2 population from a cross between a susceptible C. annuum and a C. annuum with L3 allele introgressed from C. chinense. To find I2 homologs in the pepper genome, a pepper BAC library was screened with an I2 derived probe. Eighty nine positive BAC clones were detected and subsequently screened with primers from R3 in genetic map using simple sequence repeat (SSR) markers derived from the BAC sequences. BACs that mapped to the L locus were used to create SNP markers linked to L.

eIF4E and Potyvirus resistance:

Resistance to potyviruses in pepper is recessive in nature and governed by 5 loci: pvr1, pvr2, pvr3, pvr5 and pvr6. Map based cloning of loss of susceptibility mutants identified mutations in eukaryotic translation (iso) initiation factor 4E (eIF(iso)4E) as responsible for the resistance to potyviruses, including Tobacco etch virus, an important pathogen in pepper. Using this information eIF4E was predicted as candidate for pvr2 in pepper. The eIF4E was found to cosegregate with PVY resistance in a BC1F1 population of 440 individuals. eIF4E was also used as a candidate to prove the allelic nature of pvr1 and pvr2 which mapped to the same genomic region.

Pungency:

Pungency is due to accumulation of capsaicinoids in fruits and was described as a single recessive locus "pun1". Capsaicinoids are synthesized in the placental dissepiments during a finite period early in fruit development. Therefore, transcripts that were differentially expressed in that time and place could be candidates for capsaicinoid biosynthesis genes and thus candidates for pun1. An SSH library developed to obtain transcripts unique to placental tissue identified a gene fragment, SB2-66, that colocalised with Pun 1 in an interspecific mapping population derived from a cross between a pungent pepper and one known to carry the pun1 allele. The full sequence of SB2-66 predicted a gene that was the third acyltransferase found to be expressed in pepper fruit and named as AT3. Expression of AT3 was found to be consistent with the differential expression model for the pungency related genes.

Purple and chocolate colouration (A and cl):

Comparative maps of the Solanaceae showed that genes controlling the presence of anthocyanins in eggplant, potato and pepper colocalised to the same genomic region. The petunia transcription factor mapped to the corresponding position in potato and it was hypothesised that a pepper ortholog of An2 was responsible for anthocyanin accumulation. RFLP blots of F2 populations that segregated for the anthocyanin accumulation were probed with the petunia An2 gene and revealed a single homolog in pepper that further cosegregated with anthocyanin accumulation in all 295 individuals. A fragment of this locus was amplified with PCR primers designed to a conserved region of An2 and the complete sequence was obtained by RACE.

Chocolate peppers are the result of a failure of chlorophyll breakdown during ripening because of a single recessive gene, cl. The mixing of chlorophyll with newly formed red carotenoids gives peppers a brown (chocolate) appearance. Stay green mutants, defective in pheophorbide a oxidase activity, are known in other species and similarities between the chlorophyll metabolites of brown-fruited peppers and stay green mutants lead to the hypothesis that the cl locus in pepper is encoded by a homolog of this characterized gene, pheophorbide a oxidase I (PaO). Using a BC1F1 population of 198 individuals from an interspecific cross between a red fruited C. chinense and a green-fruited C. annuum as the recurrent parent, a pepper stay green homolog (CaSGR) was shown to cosegregate with the chlorophyll retaining phenotype.

About Author / Additional Info:
I am working as a scientist at Indian Agricultural Research Institute, New Delhi with specialization in Genetics and Plant Breeding. Basically involved in hot pepper improvement programs.