The First Complete Chloroplast Genome Sequence of Secale strictum subsp. africanum Stapf (Poaceae), the Putative Ancestor of the Genus Secale

Abstract

Secale strictum ssp. africanum (synonym Secale africanum), a putative ancestor of the genus Secale, has been classified within Secale strictum, although recent phylogenetic studies suggest that it represents a distinct species. This study reports the first complete chloroplast genome of S. africanum, highlighting its structure, genetic composition, and phylogenetic relationships within Secale and related Triticiceae species. Phylogeny reconstruction based on the maximum-likelihood method reveals notable genetic similarity between S. strictum and S. africanum, supporting their genetic and phylogenetic distinction. Here, we assembled the complete, annotated chloroplast genome sequence of Secale strictum ssp. africanum. The genome is 137,068 base pair (bp) long. It is the first complete chloroplast genome that can be used as a reference genome for further analysis. The genome can be accessed on GenBank with the accession number OQ700974. This work sheds light on the evolutionary history of Secale and contributes to our understanding of chloroplast genomics in cereal ancestors, with potential applications in improving cereal crop resilience, advancing breeding strategies, and informing conservation efforts for genetic diversity.

1. Introduction

Secale strictum ssp. africanum Stapf (Poaceae) (synonym Secale africanum) is recognized as one of the oldest lineages within the genus Secale [1]. Despite its long-standing taxonomic significance, the exact classification of S. africanum has been subject to extensive debate due to its morphological and genetic resemblances with S. strictum [2]. Early studies, particularly by Hammer [3,4], suggested a close relationship between S. africanum and S. strictum, often grouping them within the same species complex. However, recent advances in genetic analysis, particularly through single-nucleotide polymorphism (SNP) data [5], have provided evidence of sufficient divergence to consider S. africanum a distinct species. Unlike other species in the genus, S. africanum is endemic to southern Africa [6] and displays the ability to hybridize with other Secale species, an occurrence which suggests potential historical human-mediated dispersal [7].

The unique genetic and evolutionary characteristics of wild Secale species, including S. africanum, hold significant potential in agricultural science. Wild species are invaluable resources in breeding programs, where they are used to obtain distant hybrids with cultivated rye. Such crossbreeding can expand recombinant variability and allows for observations of growth and development of recombinants in heterotic, transgressive, and population-based breeding. The interaction of genomes from different species or subspecies enables the expression of genes with additive and epistatic effects, determining qualitative and quantitative traits [8]. As SNP-based phylogenies are considered to be the most accurate methods of phylogeny reconstruction [9], SNPs identified and extracted from complete chloroplast genome sequences of S. africanum and closely related species have been applied in evolutionary analyses.

This study aims to further clarify the taxonomic placement of S. africanum through chloroplast (cp) genome analysis, which may provide insights into its evolutionary history and genetic divergence within the Secale genus. These findings could have broader implications for understanding the genetic diversity and evolutionary adaptations within this genus, supporting conservation strategies and facilitating the use of S. africanum and related species in crop improvement and breeding programs.

2. Materials and Methods

Seeds of Secale strictum ssp. africanum (introd. no. 6043) were obtained from the Plant Breeding and Acclimatization Institute National Centre for Plant Genetic Resources, Poland. Total DNA was extracted from young sprouts following Doyle and Doyle [10].

The cp genome of S. africanum was sequenced with the use of the DNBseq platform in BGI Shenzhen (Shenzhen, China). After the quality check (FastQC v.0.11.9 tool; Babraham Institute, Cambridge, UK; available online at http://www.bioinformatics.babraham.ac.uk/projects/fastqc), the raw reads were mapped to the reference genome of Secale cereale (NC_021761) [11] in the Geneious v.R7 (Biomatters, Auckland, New Zealand; accessed on 10 June 2022) software with default medium–low sensitivity settings [12]. Reads aligned to the reference cpDNA genome were extracted and used for de novo assembly (K-mer, 23–41; low-coverage cut-off, 5; and minimum contig length, 300; the parameters values were determined experimentally and positively verified in the assembly of the organellar genomes [13,14]).

De novo contigs were extended by mapping raw reads to the generated contigs, reassembling the contigs with mapped reads, and manually scaffolding the extended contigs (minimum sequence overlap of 50 bp and 97% overlap identity). This process was iterated five times. Finally, the reduced sequences were assembled in the circular chloroplast genome. The cp genome was annotated using MFannot v.1.1. (Centre Robert-Cedergren Bio-Informatique et Genomique, University of Montreal, Montreal, QC, Canada) [15] and PlasMapper 2.0 (available online: https://plasmapper.wishartlab.com/; Wishart Laboratory at the University of Alberta, Canada; accessed on 15 June 2022) [16], with default settings. The gene map of the annotated cp genome was developed with the OrganellarGenome DRAW v.1.3.1 tool (available online https://chlorobox.mpimp-golm.mpg.de/OGDraw.html; Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany; accessed on 4 August 2022) [17]. Finally, the phylogenetic position of S. africanum within the Triticodae group was also evaluated. A phylogenetic tree was constructed with the use of the maximum-likelihood (ML) method implemented in IQ-TREE v. 2.2.0.3 (Australian National University, Canberra, Australia) [18] with the GTR + F model selected by ModelFinder v.2 (The University of Hong Kong, Hong Kong) [19]. The ML analysis was performed with 1000 bootstrap replicates. The analysis was based on the alignment of SNP regions identified and extracted by the PhaME v.1.0.4 software (Los Alamos National Laboratory, Los Alamos, NM, USA) [20] from the complete cp genomes of S. africanum and selected representatives of Triticiceae. The option 3 (MinHash distance) of PhaME was used to determine the reference genome from a given set of genomes.

3. Results

The S. africanum cp genome appears as a typical circular, double-stranded molecule with a length of 137,068 bp (GenBank: OQ700974) and an overall GC content of 38.23% (Figure 1). The large single-copy (LSC) region is 81,080 bp long, the short single-copy (SSC) region is 12,818 bp long, and each of the inverted repeat regions (IRs) is 21,585 bp long. The reported cp genome contains 137 genes, including 113 unique genes and 24 genes which are duplicated in the IRs. The group of 113 unique genes features 73 protein-coding genes, 30 tRNA genes, 4 rRNA genes and 5 conserved chloroplast open reading frames (ORFs). The LSC region appears as the most abundant in genes—57 PCGs, 21 tRNA genes and 2 ORFs (ycf3 and ycf4)—whereas there are only 10 PCGs and 1 tRNA gene in the SSC. In the IR, there are four rRNA genes, eight tRNA genes, three ORFs (ycf2, ycf15, and ycf68), and nine PCGs, including ndhH, located on the junction between the IR and the SSC region (Figures S1–S4).

Phylogenetic reconstruction revealed that S. africanum is close to the other Secale cp genomes (Figure 2). Overall, our study provides valuable genetic data for phylogenetic and evolutionary studies of the genus Secale.

4. Discussion

Hybrid rye varieties have advanced breeding by enabling genotype fixation and trait transfer, improving yields but with fluctuating intermediate quality traits [21,22].

Wild Secale species, such as S. strictum and S. vavilovii, offer valuable genetic diversity, including disease resistance and sterilizing cytoplasm, making them important for enhancing variability in rye and triticale breeding [23]. However, limited phylogenetic knowledge slows progress.

Chloroplast genomes, with their small size and slow mutation rate, are crucial for phylogenetic and genetic studies. While largely conserved, structural variations such as gene rearrangements have been observed across families and genera (e.g., [24]).

In this study, the cp genome of S. africanum (Figure S5) was newly assembled and annotated. Its chloroplast genome length, GC content, and gene composition were similar to previously sequenced plastomes of S. cereale (137,051 bp) [11], S. sylvestre (137,116 bp) [25], and S. segetale (137,042 bp) [14], within the size range of angiosperms [26].

This phylogenetic analysis supports the close relationship between S. africanum and S. strictum.

This study reinforces the hypothesis that the restricted distribution of S. africanum in southern Africa may be the result of historical human dispersal rather than an ancient, widespread distribution. Its genetic similarity to S. strictum aligns with theories proposing that the distribution of Secale was influenced by early agricultural activities, possibly explaining why S. africanum is found so far from other Secale taxa.

This work provides the first complete chloroplast genome sequence for Secale strictum ssp. africanum and contributes valuable information regarding its genetic and evolutionary relationship with S. strictum. High genetic similarity, despite geographical isolation, supports the classification of S. africanum as a separate species, while hinting at human activities shaping its current distribution. The data presented here enhance our understanding of Secale evolution and highlight the role of chloroplast genomics in clarifying relationships within cereal ancestors.

However, this study is limited by its focus on chloroplast genomes, which, while informative, provide only part of the picture of genetic divergence and evolutionary history. Future research could integrate nuclear and mitochondrial genome analyses to obtain a more comprehensive understanding of the genetic structure and evolutionary dynamics of S. africanum. Additionally, exploring ecological and environmental factors influencing its restricted distribution could offer insights into its adaptive traits and potential resilience under changing climatic conditions.