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.

Complete chloroplast genome sequencing and comparative analysis reveals changes to the chloroplast genome after allopolyploidization in Cucumis

Allopolyploids undergo ‘genomic shock’ leading to large genetic and epigenetic modifications. Previous studies mainly focused on nuclear changes, while little is known about the inheritance and changes of organelle genome in allopolyploidization. The synthetic allotetraploid Cucumis ×hytivus, which is generated via hybridization between C. hystrix and C. sativus, is a useful model system to study cytonuclear variation. Here, we report the chloroplast genome of allotetraploid C. ×hytivus and its diploid parents via sequencing and comparative analysis. The obtained chloroplast genomes ranged in length from 154,673 to 155,760 bp, and their gene contents, gene orders, and GC contents were similar to each other. Comparative genome analysis support chloroplast maternally inheritance. However, we identified 51 Indels and 292 SNPs genetic variation of chloroplast genome in allopolyploid C. ×hytivus relative to its female parent C. hystrix. Nine intergenic regions with rich variation were identified through comparative chloroplast genome analysis in the Cucumis subgenus. The phylogenetic network based on the chloroplast genome sequences clarified the evolution and taxonomic position of the synthetic allotetraploid C. ×hytivus. The results of this study provide us with an insight into the changes of organelle genome after allopolyploidization, and a new understanding of the cytonuclear evolution.

Differentiation of Hedyotis diffusa and Common Adulterants Based on Chloroplast Genome Sequencing and DNA Barcoding Markers

Chinese herbal tea, also known as Liang Cha or cooling beverage, is popular in South China. It is regarded as a quick-fix remedy to relieve minor health problems. Hedyotis diffusa Willd. (colloquially Baihuasheshecao) is a common ingredient of cooling beverages. H. diffusa is also used to treat cancer and bacterial infections. Owing to the high demand for H. diffusa, two common adulterants, Hedyotis brachypoda (DC.) Sivar and Biju (colloquially Nidingjingcao) and Hedyotis corymbosa (L.) Lam. (colloquially Shuixiancao), are commonly encountered in the market. Owing to the close similarity of their morphological characteristics, it is difficult to differentiate them. Here, we sequenced the complete chloroplast genomes of the three species of Hedyotis using next-generation sequencing (NGS). By comparing the complete chloroplast genomes, we found that they are closely related in the subfamily Rubioideae. We also discovered that there are significant differences in the number and repeating motifs of microsatellites and complex repeats and revealed three divergent hotspots, rps16-trnQ intergenic spacer, ndhD and ycf1. By using these species-specific sequences, we propose new DNA barcoding markers for the authentication of H. diffusa and its two common adulterants.

Construction of complete chloroplast genome of the endemic species Paphiopedilum delenatii Guillaumin (1924) of Vietnam

Chloroplasts and mitochondria are organelles that have their own genome in a cell. The chloroplast genome provides information on the evolutionary relationship and species identification, valuable markers for transgenic plants, and cloning plants, etc. The application of Next Generation Sequencing has improved the chloroplast genome sequencing. However, the assembly process of chloroplast genome is quite complicated due to the need of different complex bioinformatics tools, high configuration computer and laborous. Here we configured the process of assembling the chloroplast genome of Paphiopedilum delenatii. The assembled chloroplast genome was 160,955 bp in length, including a large and a small single copy region (LSC, SSC) separated by a pair of inverted repeats (IR). Total genes were 130 genes, GC content is 35.6%. Genome data was mapped and registered in GenBank under accession number MK463585. The optimal parameters for genome assembling were recommended. This study not only provided information for conservation of the Vietnam endemic Paphiopedilum delenatii species but also supported the genome assemble researches which could be applied on other subjects.