Multiresidue analysis of pesticides in four different pomegranate cultivars: Investigating matrix effect variability by GC-MS/MS and LC-MS/MS.

Matrix effect (ME) is unavoidable in multiresidue pesticide analysis, even when using highly advanced instruments, and differences in MEs can affect residue analytical accuracy due to pomegranate cultivar composition variations. However, literature to support this claim is limited.The study used GC-MS/MS and LC-MS/MS to investigate four different Indian pomegranate cultivar extracts and their MEs on multi-class pesticides.The whole fruit and arils of all cultivarswere tested for 22 GC-amenable and 21 LC-amenable pesticides. Principal component analysis of the data confirmed that each cultivar had unique MEs for each pesticide.The majority of pesticides showed acute variations in recovery rates with 95% confidence, while GC-MS/MS-amenablepesticides showed more variation. Although extrapolative dilution reduced the influence of MEs on analytical accuracy, a generalized matrix-matching for all cultivars was not possible to achieve.To reduce the variability in MEs, it is recommended that a cultivar-specific matrix-matched standard should be used.

Genome wide identification, characterization and validation of novel miRNA-based SSR markers in pomegranate (Punica granatum L.).

A total of 17,439 mature miRNAs (~ 21 nt) earlier generated through RNA seq in the pomegranate were used for in silico analysis. After complexity reduction, a total of 1922 representative mature miRNAs were selected and used as query sequences against pomegranate genome to retrieve 2540 homologous contigs with flanking regions (~ 800). By using pre-miRNA prediction web server, a total of 1028 true contigs harbouring pri-miRNAs encoding 1162 pre-miRNAs were identified. Survey of these sequences for SSRs yielded a total of 1358 and 238 SSRs specific to pri-miRNA and pre-miRNAs, respectively. Of these, primer pairs were designed for 897 pri-miRNA and 168 pre-miRNA SSRs. In pri-miRNA sequences, hexa-nucleotides repeats were found to be most abundant (44.18%) followed by mono- (18.41%) and di-nucleotide (17.01%), which is also observed in pre-miRNA sequences. Further, a set of 51 randomly selected pre-miRNA-SSRs was examined for marker polymorphism. The experimental validation of these markers on eight pomegranate genotypes demonstrated 92.15% polymorphism. Utility of these functional markers was confirmed via examination of genetic diversity of 18 pomegranate genotypes using 15 miRNA-SSRs. Further, potential application of miRNA-SSRs for discovery of trait specific candidate genes was showed by validating 51 mature miRNA against publically available 2047 EST sequences of pomegranate by target and network analysis. In summary, the current study offers novel functional molecular markers for pomegranate genetic improvement.

Genetic diversity and association mapping of bacterial blight and other horticulturally important traits with microsatellite markers in pomegranate from India.

This genetic diversity study aimed to estimate the population structure and explore the use of association mapping strategies to identify linked markers for bacterial resistance, growth and fruit quality in pomegranate collections from India. In total, 88 accessions including 37 cultivated types were investigated. A total of 112 alleles were amplified by use of 44 publicly available microsatellites for estimating molecular genetic diversity and population structure. Neighbor-joining analysis, model-based population structure and principal component analysis corroborated the genetic relationships among wild-type and cultivated pomegranate collections from India. Our study placed all 88 germplasm into four clusters. We identified a cultivated clade of pomegranates in close proximity to Daru types of wild-type pomegranates that grow naturally near the foothills of the Himalayas. Admixture analysis sorted various lineages of cultivated pomegranates to their respective ancestral forms. We identified four linked markers for fruit weight, titratable acidity and bacterial blight severity. PGCT001 was found associated with both fruit weight and bacterial blight, and the association with fruit weight during both seasons analyzed was significant after Bonferroni correction. This research demonstrates effectiveness of microsatellites to resolve population structure among the wild and cultivar collection of pomegranates and future use for association mapping studies.