Morphological, Biochemical, and Molecular Diversity of an Indian Ex Situ Collection of Pomegranate (Punica granatum L.).

Pomegranate (Punica granatum, L.) is a fruit tree that is increasingly popular worldwide due to the health-related properties of the fruit juice. While several studies highlighted the rich phytochemical diversity, few efforts have been devoted to an integrative understanding of the level of diversity of this species. This study investigated the diversity of 40 pomegranate accessions in an Indian ex situ collection by using twenty-nine morphological traits, six biochemical parameters, and twenty-nine Simple Sequence Repeats (SSR) markers. Among the evaluated traits, fruit volume (23.34% CV), fruit weight (21.12% CV), and fruit color (*a) (22.69 % CV) largely contributed to the morphological classification. Based on Mahalanobis D2 distance and Tocher's clustering, the 40 pomegranate accessions were grouped into eight clusters, partly consistent with their origin. Specifically, cultivars introduced from foreign countries were present in distinct clusters. The SSR marker analysis generated 66 alleles. The observed heterozygosity values ranged from 0.05 to 0.63, with a mean value of 0.30. Maximum molecular genetic dissimilarity was observed between 'IC-318720' and 'Gul-e-Shah Red' (0.30). The neighbor-joining dendrogram separated wild accessions from cultivated varieties. The combination of morphological, biochemical, and molecular characterization allowed for comprehensively characterizing the pomegranate diversity and provided information on the relationships between the different aspects of the diversity. This work also suggests that the origin of the accessions is an important factor of discrimination and that the level of admixture between local and foreign material is currently limited.

Corrigendum: Chloroplast genome sequencing, comparative analysis, and discovery of unique cytoplasmic variants in pomegranate (Punica granatum L.).

[This corrects the article DOI: 10.3389/fgene.2021.704075.].

In Vitro and In Planta Antagonistic Effect of Endophytic Bacteria on Blight Causing Xanthomonas axonopodis pv. punicae: A Destructive Pathogen of Pomegranate

Pomegranate bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a highly destructive disease. In the absence of host resistance to the disease, we aimed to evaluate the biocontrol potential of endophytic bacteria against Xap. Thus, in this study, we isolated endophytes from pomegranate plants, identified them on the basis of 16S rDNA sequencing, tested them against Xap, and estimated the endophyte-mediated host defense response. The population of isolated endophytes ranged from 3 × 106 to 8 × 107 CFU/g tissue. Furthermore, 26 isolates were evaluated for their biocontrol activity against Xap, and all the tested isolates significantly reduced the in vitro growth of Xap (15.65% ± 1.25% to 56.35% ± 2.66%) as compared to control. These isolates could reduce fuscan, an uncharacterized factor of Xap involved in its aggressiveness. Lower blight incidence (11.6%) and severity (6.1%) were recorded in plants sprayed with endophytes 8 days ahead of Xap spray (Set-III) as compared to control plants which were not exposed to endophytes (77.33 and 50%, respectively%) during in vivo evaluation. Moreover, significantly high phenolic and chlorophyll contents were estimated in endophyte-treated plants as compared to control. The promising isolates mostly belonged to the genera Bacillus, Burkholderia, and Lysinibacillus, and they were deposited to the National Agriculturally Important Microbial Culture Collection, India.

Chloroplast Genome Sequencing, Comparative Analysis, and Discovery of Unique Cytoplasmic Variants in Pomegranate (Punica granatum L.).

Here we report on comprehensive chloroplast (cp) genome analysis of 16 pomegranate (Punica granatum L.) genotypes representing commercial cultivars, ornamental and wild types, through large-scale sequencing and assembling using next-generation sequencing (NGS) technology. Comparative genome analysis revealed that the size of cp genomes varied from 158,593 bp (in wild, "1201" and "1181") to 158,662 bp (cultivar, "Gul-e-Shah Red") among the genotypes, with characteristic quadripartite structures separated by a pair of inverted repeats (IRs). The higher conservation for the total number of coding and non-coding genes (rRNA and tRNA) and their sizes, and IRs (IR-A and IR-B) were observed across all the cp genomes. Interestingly, high variations were observed in sizes of large single copy (LSC, 88,976 to 89,044 bp) and small single copy (SSC, 18,682 to 18,684 bp) regions. Although, the structural organization of newly assembled cp genomes were comparable to that of previously reported cp genomes of pomegranate ("Helow," "Tunisia," and "Bhagawa"), the striking differences were observed with the Lagerstroemia lines, viz., Lagerstroemia intermedia (NC_0346620) and Lagerstroemia speciosa (NC_031414), which clearly confirmed previous findings. Furthermore, phylogenetic analysis also revealed that members outside the genus Punica were clubbed into a separate clade. The contraction and expansion analysis revealed that the structural variations in IRs, LSC, and SSC have significantly accounted for the evolution of cp genomes of Punica and L. intermedia over the periods. Microsatellite survey across cp genomes resulted in the identification of a total of 233 to 234 SSRs, with majority of them being mono- (A/T or C/G, 164-165 numbers), followed by di- (AT/AT or AG/CT, 54), tri- (6), tetra- (8), and pentanucleotides (1). Furthermore, the comparative structural variant analyses across cp genomes resulted in the identification of many varietal specific SNP/indel markers. In summary, our study has offered a successful development of large-scale cp genomics resources to leverage future genetic, taxonomical, and phylogenetic studies in pomegranate.

Comparative transcriptome profiling of pomegranate genotypes having resistance and susceptible reaction to Xanthomonas axonopodis pv. punicae.

Pomegranate (Punica granatum L.) is an important fruit crop, rich in fiber, vitamins, antioxidants, minerals and source of different biologically active compounds. The bacterial blight caused by Xanthomonas axonopodispv. punicae is a serious threat to the crop leading to 60-80% yield loss under epiphytotic conditions. In this work, we have generated comparative transcriptome profile to mark the gene expression signatures during resistance and susceptible interactions. We analyzed leaf and fruits samples of moderately resistant genotype (IC 524207) and susceptible variety (Bhagawa) of pomegranate at three progressive infection stages upon inoculation with the pathogen. RNA-Seq with the Illumina HiSeq 2500 platform revealed 1,88,337 non-redundant (nr) transcript sequences from raw sequencing data, for a total of 34,626 unigenes with size >2 kb. Moreover, 85.3% unigenes were annotated in at least one of the seven databases examined. Comparative analysis of gene-expression signatures in resistant and susceptible varieties showed that the genes known to be involved in defense mechanism in plants were up-regulated in resistant variety. Gene Ontology (GO) analysis successfully annotated 90,485 pomegranate unigenes, of which 68,464 were assigned to biological, 78,107 unigenes molecular function and 44,414 to cellular components. Significantly enriched GO terms in DEGs were related to oxidations reduction biological process, protein binding and oxidoreductase activity. This transcriptome data on pomegranate could help in understanding resistance and susceptibility nature of cultivars and further detailed fine mapping and functional validation of identified candidate gene would provide scope for resistance breeding programme in pomegranate.

Assessment of genetic diversity and population structure in pomegranate (Punica granatum L.) using hypervariable SSR markers.

The present study investigates the genetic diversity and population structure among 42 diverse pomegranate genotypes using a set of twenty one class I hypervariable SSR markers (> 24 bp), which were reported earlier from the analysis of cv. Dabenzi genome. The study material comprised 16 indigenous and 13 exotic cultivars, and 13 wild accessions. A total of 66 alleles (Na) were detected with an average of 3.14 alleles per marker. The average values of polymorphic information content (PIC), observed heterozygosity (Ho) and Shannon's gene diversity index (I) were 0.44, 0.21 and 0.95, respectively suggesting moderate genetic diversity. The pairwise genetic distance ranged from 0.07 to 0.80 with a mean value of 0.53. Population structure analysis divided all the genotypes into four subpopulations (SP1, SP2, SP3 and SP4). Interestingly, the results of phylogenetic and principal component analyses coincided with the results of structure analysis and the grouping of genotypes followed the geographical origins. AMOVA revealed that 25% of the variation was attributed to differences among populations, whereas 75% within the subpopulations with significant F ST value 0.25 (p < 0.001), indicating a high level of genetic differentiations or low level of gene flow. Based on the F ST values, pomegranate genotypes belonging to SP4 (indigenous cultivars) followed by SP1 (exotic lines) exhibited higher gene diversity and genetic differentiations within and among populations. These genetic relationships based on SSR markers could be harnessed in future genetic improvement of pomegranate through informed hybridization programs.

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.

Targeted metabolite profiling to gain chemometric insight into Indian pomegranate cultivars and elite germplasm.

BACKGROUND: Pomegranate fruit is an excellent source of bioactive polyphenolics, known to contribute significantly to human health. India is the largest producer of pomegranate in the world and produces the finest quality fruit with highly desirable consumer traits such as soft seeds, low acidity, and attractive fruit and aril color. Knowledge of the extent of variation in key metabolites (sugars, organic acids, phenolics, and anthocyanins) is key to selecting superior genotypes for germplasm improvement. Relevant information with respect to Indian genotypes is scarce. The present study therefore aims to evaluate quantitatively important metabolites in some cultivars and elite germplasm of pomegranate in India. RESULTS: Identification and quantification of primary and secondary metabolites such as sugars, organic acids, vitamin C, polyphenolics, and anthocyanins were conducted using a liquid chromatography - mass spectrometry (LC-MS) platform. Fructose and citric acid were the predominant sugar and organic acid, respectively. Wild genotypes had significantly higher concentrations of organic acids, antioxidant activity, and phenolics, namely punicalagin, ellagic acid, sinapic, and ferulic acid. CONCLUSION: Cyanidin and delphinidin derivatives of anthocyanins were more abundant in red aril commercial genotypes. Results suggest that wild-sour accessions represent a rich source of polyphenolics that can be utilized in future breeding programs to breed healthier varieties, food supplements, and pharmaceutical products. © 2019 Society of Chemical Industry.