Green-fruited Solanum habrochaites lacks fruit-specific carotenogenesis due to metabolic and structural blocks.

Members of the tomato clade exhibit a wide diversity in fruit color, but the mechanisms governing inter-species diversity of coloration are largely unknown. The carotenoid profiles, carotenogenic gene expression and proteome profiles of green-fruited Solanum habrochaites (SH), orange-fruited S. galapagense, and red-fruited S. pimpinellifolium were compared with cultivated tomato [S. lycopersicum cv. Ailsa Craig (SL)] to decipher the molecular basis of coloration diversity. Green-fruited SH, though it showed normal expression of chromoplast-specific phytoene synthase1 and lycopene ß-cyclase genes akin to orange/red-fruited species, failed to accumulate lycopene and ß-carotene. The SH phytoene synthase1 cDNA encoded an enzymatically active protein, whereas the lycopene ß-cyclase cDNA was barely active. Consistent with its green-fruited nature, SH's fruits retained chloroplast structure and PSII activity, and had impaired chlorophyll degradation with high pheophorbide a levels. Comparison of the fruit proteomes with SL revealed retention of the proteome complement related to photosynthesis in SH. Targeted peptide monitoring revealed a low abundance of key carotenogenic and sequestration proteins in SH compared with tomato. The green-fruitedness of SH appears to stem from blocks at several critical steps regulating fruit-specific carotenogenesis namely the absence of chloroplast to chromoplast transformation, block in carotenoid biosynthesis, and a dearth of carotenoid sequestering proteins.

Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

Fluorescent dye labeled DNA size standards for molecular mass detection in visible/infrared range.

BACKGROUND: Targeting Induced Local Lesions in Genomes (TILLING) is a high throughput reverse genetics tool which detects mismatches (single point mutations or small indels) in large number of individuals of mutagenized populations. Currently, TILLING is intensively used for genomics assisted molecular breeding of several crop plants for desired traits. Most commonly used platform for mutation detection is Li-COR DNA Analyzer, where PCR amplified products treated with single strand mismatch specific nuclease are resolved on denaturing gels. The molecular size of any cut product can be easily estimated by comparing with IR dye labeled markers of known sizes. Similar fluorescent dye labeled size markers are also used for several genotyping experiments. Currently, commercially available size standards are expensive and are restricted up to only 700 bp which renders estimation of products of sizes greater than 700 bases inaccurate. FINDINGS: A simple protocol was developed for labeling 5' end of multiple DNA size markers with fluorescent dyes. This method involves cloning a pool of different size markers of DNA in a plasmid vector. PCR amplification of plasmid using IR dye labeled universal primers generates 5' fluorescent labeled products of various sizes. The size of products constituting the ladder can be customized as per the need. The generated size markers can be used without any further purification and were found to be stable up to one year at -20°C. CONCLUSIONS: A simple method was developed for generating fluorescent dye labeled size standards. This method can be customized to generate different size standards as per experimental needs. The protocol described can also be adapted for developing labeled size standards for detection on platforms other than Li-COR i.e. other than infra red range of the spectrum.