Candidate gene analysis on self incompatibility in cocoa (Theobroma cacao L.)

Abstract

Cocoa is a perennial tree with typical plant habit and specific fruit characteristics. It is highly influenced by climate changes and growing environment, which makes long term and dynamic breeding programme necessary (Malhotra and Hubali, 2016). Physiological and genetic investigations have unveiled that the yield potential of cocoa is not yet fully exploited (Bertus, 2004). Demand for chocolate is increasing at a rate of 15-20 per cent every year. To meet this demand, more area has to be brought under cocoa cultivation using improved genetic stock. Development of superior hybrids have significantly contributed to improve the cocoa productivity in many countries (Kennedy et al., 1987; Dias et al., 2003). Cocoa hybrids showed wide adaptability, low environmental interaction and improved yield, when compared to traditional cultivars (Dias et al., 2003). Self-incompatibility is a pollination control mechanism which prevents self-fertilization. Hence, this can be exploited in hybrid production by avoiding emasculation, which is a cumbersome process (Minimol and Amma, 2013). Moreover, emasculation will damage the flowers leading to reduced success rate. Conventionally, the self-incompatibility is measured by selfing 100 flowers per tree. If no fruit set is observed, then the plant is classified as self-incompatible (Mallika et al., 2006). This is a tedious process which will reduce the pace of breeding programme. In various other crops, many candidate genes have been reported for self-incompatibility (McCormick, 1998). However, the actual sequence variations in candidate genes are yet to be studied in cocoa. Identification of appropriate genes involved in self-incompatibility will help to identify its mechanism at an early stage and quicken the breeding programme. In this study, 10 candidate genes viz. Serine Receptor Kinase (SRK), S Locus Glycoprotein (SLG), Barely Any Meristem 1 (BAM1), Barely Any Meristem 2 (BAM2), COMPASS-like H3K4 histone methylase component (WDR5a), Voltage-dependent L-type calcium channel subunit (alpha-1F), Gamete Expressed Protein (GEX1), Zinc finger AN1 domain-containing stress-associated protein 12 (PMZ), ARM repeat-containing protein (ARC1) and Hapless 2 (McCormic, 1998; Lanaud et al., 2017) were characterized. These genes were reported to have involved in self-incompatibility in other crops. Genomic nucleotide sequences from reported host plant species were retrieved from the NCBI GenBank database. Using this information, homologous gene sequences of the candidate genes in cocoa were retrieved. Primer sets targeting major exonic regions for each of the candidate genes were designed. Genomic DNA was isolated from self-compatible genotype (GVI-167 x GIV-18.5) and self-incompatible genotype (IMC20) and the candidate genes were PCR amplified. Amplified products were sequenced and the variations in the sequences between the self-incompatible and self-compatible genotypes were analyzed, in comparison with a self-compatible reference genome (Argout et al., 2010). Between the self-incompatible and self-compatible genotypes, a total of 31 different SNPs were discovered among the genes studied. All of them were found to be heterozygous at the locus either in self-compatible or self-incompatible genotype. The maximum number of SNPs, a total of 12, were found in GEX1 gene. Four SNPs each were found in genes SRK, BAM2, WDR5a and Alpha1F whereas three SNPs were found in PMZ. No variation was seen in BAM1 and ARC1. SNP locus homozygous in self-compatible and heterozygous in self-incompatible, with the corresponding locus of self-compatible reference genome can be used as potential candidate for developing markers to distinguish them. Such SNPs are identified and recommended for further validation.