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

Abstract

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.

The First Complete Chloroplast Genome Sequence of Mortiño (Vaccinium floribundum) and Comparative Analyses with Other Vaccinium Species

“…Therefore, in relation to other Vaccinium species cp genomes, the V. floribundum cp genome could have undergone a possible expansion/contraction in its IR regions, shifting its IR/SSC-IR/LSC boundaries. This has been reported previously in cp genomes of different genera, such as Litsea [75], Punica [76], Paphiopedilum [77], and Stemona [78]. Additionally, the expansion/contraction of the IR regions could justify the difference in plastome sizes within the Vaccinium genus and contribute to the different gene arrangements of their cp genomes [75][76][77][78][79].…”

“…This has been reported previously in cp genomes of different genera, such as Litsea [75], Punica [76], Paphiopedilum [77], and Stemona [78]. Additionally, the expansion/contraction of the IR regions could justify the difference in plastome sizes within the Vaccinium genus and contribute to the different gene arrangements of their cp genomes [75][76][77][78][79].…”

Reference quality genome sequence of Indian pomegranate cv. ‘Bhagawa’ (Punica granatum L.)

“…expansion, contraction, and rapidly evolving gene families in the Punica clade (De Bie et al, 2006;UniProt Consortium, 2014;Kozlov et al, 2019;Singh et al, 2021). We estimated gene gain and loss rates with a two-lambda model.…”

“…We used the options Diamond to blast, MSA using MAFFT, and tree inference using raxml-ng ( Katoh et al, 2005 ; Buchfink et al, 2015 ). CAFE 4.2.1 was used to identify expansion, contraction, and rapidly evolving gene families in the Punica clade ( De Bie et al, 2006 ; UniProt Consortium, 2014 ; Kozlov et al, 2019 ; Singh et al, 2021 ). We estimated gene gain and loss rates with a two-lambda model.…”

The population genomic analyses of chloroplast genomes shed new insights on the complicated ploidy and evolutionary history in Fragaria

“…Specifically, whether and how HiFi reads could be used to generate high-quality chloroplast genomes is untested. GetOrganelle is an efficient and accurate toolkit for de novo assembly of organelle genomes ( Freudenthal et al., 2020 ; Jin et al., 2020 ; Odago et al., 2021 ; Ruang-Areerate et al., 2021 ; Singh et al., 2021 ; Drown et al., 2022 ; Liu et al., 2022 ; Zhao et al., 2022 ). It can assemble better plastomes using low coverage WGS data compared with NOVOplasty.…”