Eco-TILLING for natural single nucleotide polymorphism towards genomics-assisted mutation breeding in pineapple (Ananas comosus (L.) Merr.)

Pineapple (Ananas comosus (L.) Merr.) is self-incompatible or seeds can be produced only by hybridization between distantly related varieties. Existing varieties of pineapple are generally hybrids thus inherently heterozygous and heterogenous. By Eco TILLING (Ecotype-Target Induced Local Lesions IN Genomes), the rich natural single nucleotide polymorphism (SNP) expected for a highly heterozygous genome can be mined. The natural SNPs between varieties can also be detected by pooling their genomic DNA samples and subjecting the DNA admixture to Eco TILLING. Using gene-specific the DNA markers, SNP mining can be targeted at specific genome sequence of the gene. The present study is aimed to map the base-positions of natural SNPs in two pineapple hybrid parental varieties at specific gene regions. Based on the published genome database of pineapple, DNA markers were designed for three fruit quality genes coded as LCY18, PSY09 and PDS13. These gene-specific markers were used to screen for natural SNPs in 15 individual plants each of 'Sweet 16' and 'Clone 74' pineapple varieties. Eco TILLING was carried out using individual plant DNA samples, pooled of these DNA samples per variety, and admixture of the variety DNA pools. Following the Eco TILLING protocol established in the laboratory, the likely base positions of the SNPs inherent in each variety and between the two pineapple varieties were screened using the Advanced Analytical Technologies Inc. Fragment Analyzer sup TM at LCY18 locus, numerous SNPs were identified in individual and pooled DNA samples. Three district SNPs were characterized in 'Sweet 16' while two in 'Clone 74', respectively. On the other hand, Eco TILLING has revealed that PSY09 locus is conserved in both varieties. No SNP was detected in each and between pineapple varieties, whether screened using individual or pooled DNA samples. Three inherent SNPs were identified in some plants of 'Sweet 16' at PDS13 locus, while sequence conservation was again revealed by Eco TILLING in 'Clone 74'. The Eco TILLING protocol using the specific Fragment Analyzer is now optimized. The technology is precise in detecting point mutations in inherent heterozygous genome sequence, or in DNA heteroduplexes formed upon DNA pooling of different varieties. More pineapple genes will be screened for natural SNPs and in other hybrid parental varieties of pineapple. The Eco TILLING protocol will be used to mine for induced SNPs at target gene regions in a large population of pineapple mutants.