Comparative analysis of 84 chloroplast genomes of Tylosema esculentum reveals two distinct cytotypes

Abstract

Tylosema esculentum (marama bean) is an important orphan legume from southern Africa that has long been considered to have the potential to be domesticated as a crop. The chloroplast genomes of 84 marama samples collected from various geographical locations in Namibia and Pretoria were compared in this study. The cp genomes were analyzed for diversity, including SNPs, indels, structural alterations, and heteroplasmy. The marama cp genomes ranged in length from 161,537 bp to 161,580 bp and contained the same sets of genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The genes rpoC2 and rpoB, and the intergenic spacers trnT-trnL and ndhG-ndhI were found to be more diverse than other regions of the marama plastome. 15 haplotypes were found to be divided into two groups, differing at 122 loci and at a 230 bp inversion. One type appears to have greater variability within the major genome present, and variations amongst individuals with this type of chloroplast genome seems to be distributed within specific geographic regions but with very limited sampling for some regions. However, deep sequencing has identified that within most of the individuals, both types of chloroplast genomes are present, albeit one is generally at a very low frequency. The inheritance of this complex of chloroplast genomes appears to be fairly constant, providing a conundrum of how the two genomes co-exist and are propagated through generations. The possible consequences for adaptation to the harsh environment in which T. esculentum survives are considered. The results pave the way for marama variety identification, as well as for understanding the origin and evolution of the bean.

Comparative Analysis of Tylosema esculentum Mitochondrial DNA Revealed Two Distinct Genome Structures

“…However, the mitogenomes of the other 39 individuals were very different from the reference, with a large number of sequence and structural differences, termed type 2 (Table S1). This is consistent with the previously published study of T. esculentum chloroplast genomes, where these 84 individuals were found to contain two distinct germplasms (Li and Cullis, 2023). These two cytotypes actually differ not only in the chloroplast genome but also in the mitochondrial genome.…”

“…It has been confirmed that these minor alleles are not from homologous sequences in the nuclear genome or the chloroplast genome, suggesting the presence of two different mitogenomes in the same individual. It was previously reported that heteroplasmy also exists in the chloroplast genome of A11, with a minor genome frequency reaching 11% (Jin and Cullis, 2023). This means that in the chloroplasts and mitochondria of A11, both major and minor genomes exist, and the frequency of the minor genome is higher than that of the other studied plants.…”

“…The double-stranded DNA was quantified by the Invitrogen TM Qubit TM 3.0 Fluorometer after mixing 5 μl DNA with 195 μl working solution, and 200 ng DNA was electrophoresed on a 1.5% agarose TBE gel at 40 V for 24 hours. The plant materials included another 84 marama individuals, 44 of which were plants grown in different geographical locations in Namibia and South Africa, and the remaining 40 were progeny plants grown from seeds collected there, as described in the previous study (Table S1) (Li and Cullis, 2023). The WGS Illumina reads of these 84 individuals are available and stored in the NCBI SRA database (PRJNA779273).…”

“…2,184,632 PacBio HiFi reads with a total length of 21.6 G bases were generated for Sample 4 and 498 Mb reads for Sample 32. In addition, whole-genome sequencing of DNA from fresh young leaves or embryonic axis of germinating seeds from the 84 samples collected in Namibia and South Africa was performed using the Illumina platform, as described in the previous study (Li and Cullis, 2023).…”

“…The previous comparative analysis of 84 T. esculentum chloroplast genomes has found two distinct germplasms. The two types of chloroplast genomes are different from each other at 122 loci and at a 230 bp inversion (Li and Cullis, 2023). Among many of these loci, heteroplasmy, the existence of two or more different alleles, could be seen, albeit one generally at a frequency below 2%.…”