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.

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.

Validation of QTLs for plant ideotype, earliness and growth habit traits in pigeonpea (Cajanus cajan Millsp.).

Pigeonpea productivity is greatly constrained by poor plant ideotype of existing Indian cultivars. Enhancing pigeonpea yield demands a renewed focus on restructuring the ideal plant type by using more efficient approaches like genomic tools. Therefore, the present study aims to identify and validate a set of QTLs/gene(s) presumably associated with various plant ideotype traits in pigeonpea. A total of 133 pigeonpea germplasms were evaluated along with four checks in the augmented design for various ideotype traits i.e. initiation of flowering (IF), days to 50% flowering (DFF), days to maturity (DM), plant height (PH), primary branches (PB), seeds per pod (SP) and pod length (PL). We observed significant genetic diversity in the germplasm lines for these traits. The genetic control of IF, DFF, DM and PH renders these traits suitable for detection of marker trait associations. By using residual maximum likelihood algorithm, we obtained appropriate variance-covariance structures for modeling heterogeneity, correlation of genetic effects and non-genetic residual effects. The estimates of genetic correlations indicated a strong association among earliness traits. The best linear unbiased prediction values were calculated for individual traits, and association analysis was performed in a panel of 95 diverse genotypes with 19 genic SSRs. Out of five QTL-flanking SSRs used here for validation, only ASSR295 could show significant association with FDR and Bonferroni corrections, and accounted for 15.4% IF, 14.2% DFF and 16.2% DM of phenotypic variance (PV). Remaining SSR markers (ASSR1486, ASSR206 and ASSR408) could not qualify false discovery rate (FDR) and Bonferroni criteria, hence declared as false positives. Additionally, we identified two highly significant SSR markers, ASSR8 and ASSR390 on LG 1 and LG 2, respectively. The SSR marker ASSR8 explained up to 22 and 11% PV for earliness traits and PB respectively, whereas ASSR390 controlled up to 17% PV for earliness traits. The validation and identification of new QTLs in pigeonpea across diverse genetic backgrounds brightens the prospects for marker-assisted selection to improve yield gains in pigeonpea.

Identification and validation of SSR markers for Xanthomonas axonopodis pv. punicae an incitant of bacterial blight of pomegranate.

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.

Characterization of Alternaria and Colletotrichum Species Associated with Pomegranate (Punica granatum L.) in Maharashtra State of India

Fungal pathogens are a major constraint affecting the quality of pomegranate production around the world. Among them, Alternaria and Colletotrichum species cause leaf spot, fruit spot or heart rot (black rot), and fruit rot (anthracnose) or calyx end rot, respectively. Accurate identification of disease-causing fungal species is essential for developing suitable management practices. Therefore, characterization of Alternaria and Colletotrichum isolates representing different geographical regions, predominantly Maharashtra-the Indian hub of pomegranate production and export-was carried out. Fungal isolates could not be identified based on morphological characteristics alone, hence were subjected to multi-gene phylogeny for their accurate identification. Based on a maximum likelihood phylogenetic tree, Alternaria isolates were identified as within the A. alternata species complex and as A. burnsii, while Colletotrichum isolates showed genetic closeness to various species within the C. gloeosporioides species complex. Thus, the current study reports for the first time that, in India, the fruit rots of pomegranate are caused by multiple species and not a single species of Alternaria and Colletotrichum alone. Since different species have different epidemiology and sensitivity toward the commercially available and routinely applied fungicides, the precise knowledge of the diverse species infecting pomegranate, as provided by the current study, is the first step towards devising better management strategies.

Association mapping to discover significant marker-trait associations for resistance against fusarium wilt variant 2 in pigeonpea [Cajanus cajan (L.) Millspaugh] using SSR markers.

Pigeonpea production is severely constrained by wilt disease caused by Fusarium udum. In the current study, we discover the putative genomic regions that control resistance response to variant 2 of fusarium wilt using association mapping approach. The association panel comprised of 89 diverse pigeonpea genotypes including seven varieties, three landraces and 79 germplasm lines. The panel was screened rigorously for 3 consecutive years (2013-14, 2014-15 and 2015-2016) against variant 2 in a wilt-sick field. A total of 65 pigeonpea specific hypervariable SSR markers (HASSRs) were screened representing seven linkage groups and 29 scaffolds of the pigeonpea genome. A total of 181 alleles were detected, with average values of gene diversity and polymorphism information content (PIC) of 0.55 and 0.47, respectively. Further analysis using model based (STRUCTURE) and distance based (clustering) approaches separated the entire pigeonpea collection into two distinct subgroups (K = 2). The marker trait associations (MTAs) were established based on three-year wilt incidence data and SSR dataset using a unified mixed linear model. Consequently, six SSR markers were identified, which were significantly associated with wilt resistance and explained up to 6% phenotypic variance (PV) across the years. Among these SSRs, HASSR18 was found to be the most stable and significant, accounting for 5-6% PV across the years. To the best of our knowledge, this is the first report of identification of favourable alleles for resistance to variant 2 of Fusarium udum in pigeonpea using association mapping. The SSR markers identified here will greatly facilitate marker assisted resistance breeding against fusarium wilt in pigeonpea.

New Hypervariable SSR Markers for Diversity Analysis, Hybrid Purity Testing and Trait Mapping in Pigeonpea [Cajanus cajan (L.) Millspaugh].

Draft genome sequence in pigeonpea offers unprecedented opportunities for genomics assisted crop improvement via enabling access to genome-wide genetic markers. In the present study, 421 hypervariable simple sequence repeat (SSR) markers from the pigeonpea genome were screened on a panel of eight pigeonpea genotypes yielding marker validation and polymorphism percentages of 95.24 and 54.11%, respectively. The SSR marker assay uncovered a total of 570 alleles with three as an average number of alleles per marker. Similarly, the mean values for gene diversity and PIC were 0.44 and 0.37, respectively. The number of polymorphic markers ranged from 39 to 89 for different parental combinations. Further, 60 of these SSRs were assayed on 94 genotypes, and model based clustering using STRUCTURE resulted in the identification of the two subpopulations (K = 2). This remained in close agreement with the clustering patterns inferred from genetic distance (GD)-based approaches i.e., dendrogram, factorial and principal coordinate analysis (PCoA). The AMOVA accounted majority of the genetic variation within groups (89%) in comparison to the variation existing between the groups (11%). A subset of these markers was implicated for hybrid purity testing. We also demonstrated utility of these SSR markers in trait mapping through association and bi-parental linkage analyses. The general linear (GLM) and mixed linear (MLM) models both detected a single SSR marker (CcGM03681) with R2 = 16.4 as associated with the resistance to Fusarium wilt variant 2. Similarly, by using SSR data in a segregating backcross population, the corresponding restorer-of-fertility (Rf) locus was putatively mapped at 39 cM with the marker CcGM08896. However, The marker-trait associations (MTAs) detected here represent a very preliminary type and hence demand deeper investigations for conclusive evidence. Given their ability to reveal polymorphism in simple agarose gels, the hypervariable SSRs are valuable genomic resource for pigeonpea research community, particularly in South Asia and East Africa where pigeonpea is primarily grown.