ABSTRACT
Pomegranate is an important fruit crop for ensuring livelihood and nutrition security in fragile semi-arid regions of the globe having limited irrigation resources. This is a high-value, nutritionally rich, and export-oriented agri-commodity that ensures high returns on investment to growers across the world. Although it is a valuable fruit crop, it has received only a limited genomics research outcome. To fast-track the pomegranate improvement program, de novo whole-genome sequencing of the main Indian cultivar 'Bhagawa' was initiated by the Indian Council of Agricultural Research-National Research Center on Pomegranate (ICAR-NRCP). We have demonstrated that a combination of commercially available technologies from Illumina, PacBio, 10X Genomics, and BioNano Genomics could be used efficiently for sequencing and reference-grade de novo assembly of the pomegranate genome. The research led to a final reference-quality genome assembly for 'Bhagawa' of 346.08 Mb in 342 scaffolds and an average N50 of 16.12 Mb and N90 of 1088.62 Kb. This assembly covered more than 98% of the estimated pomegranate genome size, 352.54 Mb. The LTR assembly index (LAI) value of 10 and 93.68% Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness score over the 1,440 ortholog genes of the completed pomegranate genome indicates the quality of the assembled pomegranate genome. Furthermore, 29,435 gene models were discovered with a mean transcript length of 2,954 bp and a mean coding sequence length 1,090 bp. Four transcript data samples of pomegranate tissues were mapped over the assembled 'Bhagawa' genome up to 95% significant matches, indicating the high quality of the assembled genome. We have compared the 'Bhagawa' genome with the genomes of the pomegranate cultivars 'Dabenzi' and 'Taishanhong.' We have also performed whole-genome phylogenetic analysis using Computational Analysis of Gene Family Evolution (CAFE) and found that Eucalyptus grandis and pomegranate diverged 64 (60-70) million years ago. About 1,573 protein-coding resistance genes identified in the 'Bhagawa' genome were classified into 32 domains. In all, 314 copies of miRNA belonging to 26 different families were identified in the 'Bhagawa' genome. The reference-quality genome assembly of 'Bhagawa' is certainly a significant genomic resource for accelerated pomegranate improvement.
ABSTRACT
This study reports genome wide characterization and development of first set of microsatellite markers through in silico analysis of eight sequenced Xanthomonas axonopodis pv. punicae strains available in the public database. SSR survey resulted in identification of ~ 4638 perfect SSRs, with mean marker frequency 901 SSRs/Mb and densitiy of 11,006 bp/Mb aross the eight genomes. Frequency distribution graphs revealed hexa-nucleotide repeats were more prominent fowllowed by tri-, tetra-, di- and penta-nucleotides in the analysed genomes. We desinged 2927 SSR primers that are specific to the strain LMG 859 and ePCR confirmed on seven other Xap genomes. This resulted in identification of 542 informative SSRs that are producing single amplicons, from which 66 primers were successfully validated through wet lab experiments on eight Xap isolates of pomegranate. Furthermore, utility of these SSRs were demostrated by analysing molecular diversity among 22 Xap isolates using 20 Xap_SSR primers. SSRs revealed moderate genetic diversity among Xap isolates (61%) and grouped 11 isolates that are repersenting six different states into one cluster. This proved the earlier evidence of wider spread of ST3 type Xap acoss India using Multi locus Sequence Typing (MLST) technique. In summary, Xap_SSR will serve as powerful genomics tools that would helps in monitoring of population dynamics, taxonomy, epidomology and quarantine aspects in bacterial blight pathogen through development of microsatellite based Multilocus Variable number of Tandem repeat analysis (MLVA) in future. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03209-z.
ABSTRACT
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.
ABSTRACT
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.
