Molecular structure, comparative and phylogenetic analysis of the complete chloroplast genome sequences of weedy rye Secale cereale ssp. segetale

Abstract

The complete chloroplast genome of Secale cereale ssp. segetale (Zhuk.) Roshev. (Poaceae: Triticeae) was sequenced and analyzed to better use its genetic resources to enrich rye and wheat breeding. The study was carried out using the following methods: DNA extraction, sequencing, assembly and annotation, comparison with other complete chloroplast genomes of the five Secale species, and multigene phylogeny. As a result of the study, it was determined that the chloroplast genome is 137,042 base pair (bp) long and contains 137 genes, including 113 unique genes and 24 genes which are duplicated in the IRs. Moreover, a total of 29 SSRs were detected in the Secale cereale ssp. segetale chloroplast genome. The phylogenetic analysis showed that Secale cereale ssp. segetale appeared to share the highest degree of similarity with S. cereale and S. strictum. Intraspecific diversity has been observed between the published chloroplast genome sequences of S. cereale ssp. segetale. The genome can be accessed on GenBank with the accession number (OL688773).

Introduction

Secale cereale ssp. segetale is one of the many species of the genus Secale with a previously unknown chloroplast and mitochondrial genome. However, it can be a source of desired genes (e.g., resistance to diseases, high protein content, morphological and biochemical traits) that can enrich rye or wheat breeding^1,2. The lack of knowledge of phylogenetic relationships reduces the progress in rye breeding, which can be enriched with functional features derived from wild rye species³. With new biotic and abiotic stresses and climate change, there is also a need to study wild rye species, which is crucial to improving the yield and quality of this cereal⁴. Therefore, more genetic markers are needed.. One of the way to achieve this is to sequence complete chloroplast genomes. Due to their conservative and non-recombinant nature, chloroplast genomes are a solid tool in genomics and evolutionary research⁵. Certain evolutionary hotspots of the plant plastid genome, such as single nucleotide polymorphisms and insertions/deletions, may provide useful information to elucidate the phylogenetic of taxonomically unresolved plant taxa^6,7. Thus, the availability of complete chloroplast genomes, which include new variable and informational sites, should help explain more precise phylogeny.

To participate in this effort, we have undertaken the sequencing of the complete chloroplast genomes in genus Secale, which are smaller and easier to analyze compared to mitochondrial genomes. So far, only the incomplete S. cereale cpDNA sequences (NC_021761)⁸, three sequences for S. strictum (KY636137, KY636138 and OL979486)⁹ and S. sylvestre (MW557517)¹⁰ are available. The chloroplast genome of S. segetale has recently been published¹¹, however a comprehensive phylogenetic analysis based on whole chloroplast genomes has not been done to date. Therefore, we presume that analysis of the complete chloroplast genome sequences of Secale spp., starting with S. sylvestre¹⁰, will be useful and cost-effective for evolutionary and phylogenetic studies, as it was suggested by our previous studies¹².

In this study, we present the complete chloroplast genome of S. cereale ssp. segetale, which will provide valuable information for genetic studies of Secale species.

Results

Chloroplast genome of Secale cereale ssp. segetale

Sequencing of Secale cereale ssp. segetale chloroplast genome yielded 41 653 350 raw reads, out of which 88 777 were mapped to the reference genome of S. cereale with 97 × average coverage. The S. cereale ssp. segetale cp genome appeared as a typical circular, double-stranded molecule with the length of 137,042 bp (Fig. 1) and overal GC content of 38%. The large single copy (LSC) region is 81,060 bp long, the short single copy (SSC) region is 12,820 bp long, and each of the inverted repeat regions (IR) is 21,581 bp long. Reported cp genome contains 137 genes, including 113 unique genes and 24 genes which are duplicated in the IRs. Group of 113 unique genes features 73 protein-coding genes, 30 tRNA genes, four rRNA genes and five conserved chloroplast open reading frames (ORFs) (Table 1).

Figure 1.

Map of the chloroplast genome of Secale cereale ssp. segetale. The genes inside and outside the circle are transcribed in the clockwise and counterclockwise directions, respectively. Genes belonging to different functional groups are shown in different colors. Tick lines indicate the extent of the inverted repeats (IRa and IRb) that separate the genomes into small single-copy (SSC) and large single-copy (LSC) regions. The innermost darker gray corresponds to GC-content while the lighter gray corresponds to AT content.

Table 1.

Genes present in chloroplast genome of Secale cereale ssp. segetale. Genes list arranged alphabetically.

Category	Group of gene	Name of genes
Photosynthesis	Photosystem I	psaA, psaB, psaC, psaI, psaJ
	Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	Cytochrome complex	petA, petB, petD, petG, petL, petN
	ATP synthase	atpA, atpB, atpE, atpFc, atpH, atpI
	NADH dehydrogenase	ndhAc, ndhBc (× 2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhHb (2x), ndhI, ndhJ, ndhK
	Large subunit of RUBISCO	rbcL
DNA replication and protein synthesis	Ribosomal RNA	rrn4.5 (× 2), rrn5 (× 2), rrn16 (× 2), rrn23 (× 2)
	Small subunit ribosomal proteins	rps2, rps3, rps4, rps7 (× 2), rps8, rps11, rps12e, rps14, rps15(× 2), rps16c, rps18, rps19 (× 2)
	Large subunit ribosomal proteins	rpl2c (× 2), rpl14, rpl16, rpl20, rpl22, rpl23b (× 3), rpl32, rpl33, rpl36
	RNA polymerase subunits	rpoA, rpoB, rpoC1, rpoC2
	Translational initiation factor	infA
	Transfer RNA	trnA-UGC c(× 2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnfM-AUG, trnG-UCCc (× 2), trnH-GUG (× 2), trnI-CAU (× 2), trnI-GAUc (× 2), trnK-UUUc, trnL-CAA (× 2), trnL-CUA, trnL-UAAc, trnM-CAU, trnN-GUU (× 2), trnP-UGG, trnQ-UUG, trnR-ACG (× 2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC (× 2), trnV-UACc, trnW-CCA, trnY-GUA
Other genes	Conserved hypothetical chloroplast ORF	ycf2 (× 2), ycf3ad, ycf4a, ycf15 (× 2), ycf68 (× 2)
	Other proteins	ccsA, cemA, clpP, matK

^aGenes associated with Photosystem I.

^bOne copy of the gene is a pseudogene.

^cGene containing one intron.

^dGene containing two introns.

^eTransspliced gene.

The LSC region appeared as the most abundant in genes—57 PCGs, 21 tRNA genes and two ORFs (ycf3 and ycf4), whereas there are only ten PCGs and one tRNA gene in SSC. In 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 IR and SSC region.

Repeat sequence analysis

A total of 52 repeat sequences structures with length ranging from 30 to 286 bp were revealed in the plastome of Secale cereale ssp. segetale (Table 2). The forward repeats (37) dominated over palindromic (15) repeats. Neither complementary nor reverse repeats were found. Most repeat sequences (69.3%) were detected in the LSC region, followed by IR (28.8%) and SSC regions (1.9%). 50% of these sequences were found within coding regions. The highest number of repeats were found within the sequences of the following genes: rpoC2 (9F), rpl23 (2F and 2P) and rps18 (3F and 1P).

Table 2.

List of repeated sequences in the chloroplast genome of Secale cereale ssp. segetale.

Repeat length	Strat site of repeat A	Location	Repeat A region	Strat site of repeat B	Location	Repeat B region	Repeat type
286	56561	rpl23	LSC	83149	rpl23	IR	P
286	56561	rpl23	LSC	134665	rpl23	IR	F
160	56687	rpl23	LSC	83149	rpl23	IR	P
160	56687	rpl23	LSC	134791	rpl23	IR	F
74	12628	IGS (trnG-UCC–trnfM-CAU)	LSC	12796	IGS (trnfM-CAU–trnG-UCC)	LSC	F
60	101806	IGS (trnN-GUU–rps15)	IR	101806	IGS (trnN-GUU–rps15)	IR	P
60	101806	IGS (trnN-GUU–rps15)	IR	116234	IGS (trnN-GUU–rps15)	IR	F
60	116234	IGS (trnN-GUU–rps15)	IR	116234	IGS (trnN-GUU–rps15)	IR	P
45	56364	IGS (rbcL–rpl23)	LSC	56364	IGS (rbcL–rpl23)	LSC	P
42	41556	IGS (psaA–ycf3)	LSC	41570	IGS (psaA–ycf3)	LSC	F
41	12735	trnfM–CAU	LSC	36344	trnfM-CAU	LSC	F
40	12628	IGS (trnG-UCC–trnfM-CAU)	LSC	36407	IGS (trnfM-CAU–rps14)	LSC	F
40	12796	IGS (trnfM-CAU–trnG-UCC)	LSC	36407	IGS (trnfM-CAU–rps14)	LSC	F
39	12835	IGS (trnfM-CAU–trnG-UCC)	LSC	36447	IGS (trnfM-CAU–rps14)	LSC	F
39	14437	IGS (trnG-UCC–trnT-GGU)	LSC	89975	IGS (rps7–trnV-GAC)	IR	F
39	14437	IGS (trnG-UCC–trnT-GGU)	LSC	128086	IGS (rps7–trnV-GAC)	IR	P
38	38373	psaB	LSC	40597	psaA	LSC	F
36	7542	trnS-GCU	LSC	44850	trnS-GGA	LSC	P
36	43333	I intron ycf3	LSC	90638	IGS (rps7–trnV-GAC)	IR	F
36	43333	I intron ycf3	LSC	127426	IGS (rps7–trnV-GAC)	IR	P
35	12667	IGS (trnG-UCC–trnfM-CAU)	LSC	36447	IGS (trnfM-CAU–rps14)	LSC	F
35	12719	trnfM-CAU	LSC	36328	trnfM-CAU	LSC	F
35	76754	infA	LSC	76772	infA	LSC	F
34	27191	rpoC2	LSC	27212	rpoC2	LSC	F
34	27253	rpoC2	LSC	27328	rpoC2	LSC	F
33	27113	rpoC2	LSC	27164	rpoC2	LSC	F
33	61501	IGS (petA–psbJ)	LSC	61501	IGS (rbcL–rpl23)	LSC	P
32	11271	trnS-UGA	LSC	44857	trnS-GGA	LSC	P
32	12677	IGS (trnG-UCC–trnfM-CAU)	LSC	36457	IGS (trnfM-CAU–rps14)	LSC	F
32	14794	trnT-GGU	LSC	46100	trnT-UGU	LSC	P
32	27072	rpoC2	LSC	27171	rpoC2	LSC	F
32	27219	rpoC2	LSC	27273	rpoC2	LSC	F
32	41556	IGS (psaA–ycf3)	LSC	41584	IGS (psaA–ycf3)	LSC	F
31	8407	IGS (trnS-GCU–psbD)	LSC	8444	IGS (trnS-GCU–psbD)	LSC	F
31	12843	IGS (trnfM-CAU–trnG-UCC)	LSC	36455	IGS (trnfM-CAU–rps14)	LSC	F
31	15484	IGS (trnY-GUA–trnD-GUC)	LSC	33828	intron atpF	LSC	F
31	27054	rpoC2	LSC	27324	rpoC2	LSC	F
31	27064	rpoC2	LSC	27259	rpoC2	LSC	F
31	27241	rpoC2	LSC	27382	rpoC2	LSC	F
31	27316	rpoC2	LSC	27382	rpoC2	LSC	F
31	66279	rps18	LSC	66300	rps18	LSC	F
31	80266	rps3	LSC	80311	rps3	LSC	F
31	101837	IGS (trnN-GUU–rps15)	IR	116265	IGS (trnN-GUU–rps15)	IR	F
31	105588	IGS (ndhF–rpl32)	SSC	105612	IGS (ndhF–rpl32)	SSC	F
30	12870	trnG-UCC	LSC	36314	trnfM-CAU	LSC	F
30	16756	IGS (psbM–petN)	LSC	16756	IGS (psbM–petN)	LSC	P
30	66231	rps18	LSC	66315	rps18	LSC	F
30	66298	rps18	LSC	66319	rps18	LSC	F
30	66677	rps18	LSC	66677	rps18	LSC	P
30	87638	Intron ndhB	IR	87638	Intron ndhB	IR	P
30	87638	Intron ndhB	IR	130432	Intron ndhB	IR	F
30	130432	Intron ndhB	IR	130432	Intron ndhB	IR	P

IGS (trnG-UCC–trnfM-CAU) means spacer between trnG-UCC and trnfM-CAU.

A total of 29 SSRs were detected in the Secale cereale ssp. segetale chloroplast genome (Table 3). The mononucleotide SSRs composed of A/T units were the most common, whereas hexanucleotide SSRs were not detected. 79.3% of SSRs were located within LSC region, 13.8% in IR region while only 6.9% of SSRs were found in SSC region. Most of the SSRs were identified within intergenic spacers (58.6%), while equal proportions (20.7%) were located in the introns and coding sequences.

Table 3.

Distribution of SSR in the Secale cereale ssp. segetale cp genome.

Type	Repeat unit	Length	Start	End	Location	Region
Mononucleotide	A	13	7211	7223	IGS (psbK–psbJ)	LSC
		13	7937	7949	IGS (trnS-GCU–psbD)	LSC
		18	11227	11244	IGS (psbC–trnS-UGA)	LSC
		12	29538	29549	rpoC2	LSC
		12	29945	29956	IGS (rpoC2–rps2)	LSC
		12	33569	33580	intron atpF	LSC
		12	33844	33855	intron atpF	LSC
		13	36192	36204	IGS (trnR-UCU–trnfM-CAU)	LSC
		12	43027	43038	II intron ycf3	LSC
		13	46728	46740	IGS (trnT-UGU–trnL-UAA)	LSC
		12	76716	76727	IGS (rpl36–infA)	LSC
		12	105202	105213	IGS (ndhF–rpl32)	SSC
Trinucleotide	AAT	15	24652	24666	rpoC1	LSC
	AAT	13	47511	47523	IGS (trnL-UAA–trnF-GAA)	LSC
	AAC	12	31552	31563	atpI	LSC
	AAT	12	56494	56505	IGS (rbcL–rpl23)	LSC
	AAG	12	65670	65681	IGS (psaJ–rpl33)	LSC
Tetranucleotide	AAGG	12	42927	42938	II intron ycf3	LSC
	AATG	12	64604	64615	IGS (trnW-CCA–trnP-UGG)	LSC
	AAAG	12	64889	64900	IGS (trnP–UGG-psaJ)	LSC
	AAAG	12	68899	68910	IGS (clpP–psbB)	LSC
	AACG	13	99471	99483	4.5S rRNA	IR
	AAAT	12	108269	108280	ndhD	SSC
	AACG	13	118618	118630	4.5S rRNA	IR
Pentanucleotide	AATAT	18	15656	15673	IGS (trnY-GUA–trnD-GUC)	LSC
	ACCAT	15	43805	43819	I intron ycf3	LSC
	AATAT	18	47154	47171	intron trnL-UAA	LSC
	AATAT	16	101098	101113	IGS (trnN-GUU–rps15)	IR
	AATAT	16	116988	117003	IGS (trnN-GUU-rps15)	IR

IGS IGS (psbK-psbJ) means spacer between psbK and psbJ.

Multigene phylogeny

Phylogeny reconstruction based on sequences of 73 protein-coding genes shared by Secale cereale ssp. segetale and 38 representatives of Pooideae subfamily appeared to be consistent with the systematic position of studied species. The BI and ML tree divided analyzed species into six major clades (Fig. 2). The first cluster contained 23 species representing Triticinae subtribe, four other clades gathered 13 species representing Hordeinae subtribe, whereas the last clad consisted of three Littledalea species (Littledaleeae tribe). Secale cereale ssp. segetale appeared to share the highest degree of similarity with S. cereale and S. cereale ssp. segetale. Mentioned above five Secale species form separate sub-clad within the Triticinae tribe.

Figure 2.

Cladogram illustrating the phylogenetic relationships for Secale cereale ssb. segetale based on complete cp genome sequences. Phylogenetic tree based on sequences of sheared 73 protein-coding genes from five Secale species and 34 other cereal lineages representing Triticodae group within subfamily Pooidae and the cp genome of Oryza sativa as an outgroup, using Bayesian posterior probabilities (PP) and maximum likelihood (ML). Each node has 100% bootstrap support value. The cpDNA sequence obtained in this study is shown in bold.

Comparison with other complete chloroplast genomes of the Secale species

The overall sequence identity of five cp genomes of Secale species was plotted using mVISTA with the annotation of S. cereale ssp. segetale cp genome (obtained by new sequencing in this study) as reference (Fig. 3). The results showed that the Secale cp genomes exhibited a high level of sequence synteny, suggesting a conserved evolutionary pattern. The plastome sequences were fairly conserved across the four data with a few regions with a variation. The sequences of exons were nearly identical throughout the all taxa.

Figure 3.

Percentage of sequence identity between chloroplast genomes of Secale cereale ssp. segetale and other four Secale species using mVISTA program. Gray arrows on the top line show transcriptional direction. The y-axis represents average percent identity between sequences of S. cereale ssp. segetale and other three Secale chloroplast genomes. The x-axis represents the coordinate in the chloroplast genome using S. cereale ssp. segetale as reference. Genome regions are color coded as exon, untranslated regions (UTR), and conserved non-coding sequences (CNS).

Discussion

The task of modern cereal breeding is to obtain new, higher-yielding varieties that have high resistance to pathogens, diseases and abiotic conditions. Unfortunately, progress in rye breeding has been limited, as the varieties used in cultivation have had limited variability due to selection. In addition, attempts to use old varieties have been unsuccessful.

A major advance in rye breeding has been the introduction of hybrid varieties, through which individual genotypes are fixed by continuing self-pollination and transferring monogenic traits into varieties¹³. However, despite the increase in yield, intermediate quality traits are subject to large annual fluctuations. Thus, despite significant increases in grain yield and decreases in protein content in the experiments, increases in grain yield did not significantly positively or negatively affect intermediate quality traits⁴.

A number of taxa in the genus Secale may represent a potential source of genetic variability in rye breeding³. Species such as Secale strictum and Secale vavilovii may be sources of new genetic variability, with resistance to ear fusariosis and septoria leaf blotch), while Secale vavilovii may also be a source of sterilizing cytoplasm (source of sterilising cytoplasm). Wild rye species and subspecies provide excellent starting material for studies aimed at expanding recombination variability in cultivated rye and triticale (× Triticosecale Wittmark). Because of their genetic distinctiveness and high trait expression, they represent a valuable source of genes in which our cultivars are deficient¹⁴. An example is the study of the efficiency of crossing the wild species Secale vavilovii and the rye subspecies Secale cereale ssp. afghanicum, Secale cereale ssp. ancestrale, Secale cereale ssp. dighoricum, Secale cereale ssp. segetale with the crop species Secale cereale ssp. cereale, and the resulting F1 crosses may be a potential source of variation in common rye³. Unfortunately, the lack of knowledge of phylogenetic relationships reduces the progress in rye breeding.

For understanding plant origin and evolution chloroplast genome sequences are very useful. With maternally inherited traits, a genome of relatively small size and a slow mutation rate of the genome¹⁵, analysis of the phylogenetic relationships of multiple chloroplast DNA can help understand plant phylogeny, population genetic analysis and taxonomic status at the molecular level¹⁶.

Although cp genomes of angiosperm plants are generally conservative in terms of sequence and number of genes¹⁷, levels of structural variation have been observed in the genome that vary across families and genera, such as gene duplication and large-scale rearrangements of genes, introns and IR domains (e.g.^18,19).

The S. cereale ssp. segetale cp genome appeared as a typical circular, double-stranded molecule (Fig. 1) and overal GC content, which is similar to previously sequenced plastomes of S. cereale (137,051 bp; NCBI LC645358), S. sylvestre (137 116 bp)¹⁰ or within the size range of angiosperms²⁰.

The results obtained by Du et al.¹¹ are similar to ours. The size of the genome, the lengths of the LSC, SSC and IR sequences differ slightly. In contrast, larger differences are seen in the number of genes. The genome we analyzed contains 73 protein-coding genes (82 in¹¹), 30 tRNA genes (41 in¹¹) four rRNA genes (8 in¹¹) and five conserved chloroplast open reading frames (ORFs)(lack of information in¹¹).

It is difficult to say where the above-mentioned differences came from. The rich interspecific genetic diversity of S. S. cereale ssp. segetale has been previously reported (e.g.²¹). Significant differences were found between and within populations of S. c. ssp. segetale. A high degree of genetic variability has also been described using chromosomal markers^22,23. These results deserve attention and further research.

The polymorphisms found in S. c. ssp. segetale chloroplast genome sequences can be used e.g. to elucidate evolutionary histories such as the origin of Secale species or accessions at the inter- and, thanks to the research described in this manuscript, intra-species level. Furthermore, the polymorphic sites promote practical applications for molecular analysis to protect S. c. ssp. segetale accession²⁴ and, potentially in the long term, the rye breeding industry. Unfortunately, the analyses of the genome previously published by Du et al. do not include many details, in addition to those mentioned above, which does not allow for a more detailed analysis.

Certain regions of the plastome are predisposed to indel and substitution mutations. Comparative studies of the plastome show the evolution of, among other, tandem repeats and their role in generating substitutions and indels^25,26. Once the composition of repeat sequences in the plastome is determined, it is possible to predict microstructural changes by analyzing the correlation between repeats, indels and substitutions. In addition to the paucity of genomic resources, the phylogeny of the genus Secale is enigmatic (e.g.^27,28). Therefore, it is important to fully explore the polymorphic regions of Secale chloroplasts in an evolutionary context.

For the total of 52 repeat sequence structures revealed in the Secale cereale ssp. segetale plastome, the vast majority were detected in the LSC region (Table 2). The highest number of repeats was found within the sequences of the rpoC2, rpl23 and rps18 genes. Regardless of its function the rpoC2, gene encoding the β-subunit of plastid RNA polymerase is a relatively rapidly evolving chloroplast sequence²⁹. Analogically, rpl23 gene and its pseudogene which are observed in the grass family belong to highly polymorphic genes considered as a hotspots of illegitimate recombination in cp genomes³⁰.

Chloroplast SSRs identification not only serves as a one of cp genome characteristics but also represent ideal molecular tools with various applications like investigation of domestication history, sites of origin or genetic diversity and relationships between wild and cultivated species^31,32. In 2016, Hagenblad et al.³³ analyzed the genetic diversity of 76 accessions of wild, feral and cultivated rye based on SNP polymorphisms. They performed an analysis of five chloroplast SSRs, derived from Lolium and wheat. Discriminant analysis of principal components (DAPC) of cpSSR data indicated very large genetic variation within the genus Secale and did not reflect taxonomic groups, except for S. strictum and S. africanum, which formed a separate cluster.

CpSSRs are mainly distributed within intergenic spacers of Secale plastomes; similar distribution preferences of cpSSRs have been reported in Avena spp., Pseudoroegneria libanotica and Salvia miltiorrhiza^34–36.

Phylogenetic analysis has shown that Secale cereale ssp. segetale appeared to share the highest degree of similarity with S. cereale and S. cereale ssp. segetale. The five Secale species form separate sub-clad within the Triticinae tribe, which confirms previous phylogenetic data of the genus Secale (e.g.³⁷).

The results showed that the Secale cp genomes exhibited a high level of sequence synteny, suggesting a conserved evolutionary pattern. The plastome sequences were fairly conserved across the four data with a few regions with a variation. The sequences of exons were nearly identical throughout the all taxa.

Conclusions

Here we assembled the complete, annotated chloroplast genome sequence of Secale cereale ssp. segetale. The genome is 137 042 base pair (bp) long and contains 137 genes, including 113 unique genes and 24 genes which are duplicated in the IRs. The phylogenetic analysis showed that Secale cereale ssp. segetale appeared to share the highest degree of similarity with S. cereale and S. strictum. Intraspecific diversity has been observed between the published chloroplast genome sequences of S. cereale ssp. segetale. The cp genome will provide a series of resources for evolutionary and genetic studies about species of rye. The assembled genome sequences and annotation information have been deposited in GenBank under the accession number OL688773.

Material and methods

DNA extraction, sequencing, assembly and annotation

Seeds of Secale cereale ssp. segetale introd. no. 1782/94 were obtained from the Botanical Garden of the Polish Academy of Sciences in Warsaw. Total DNA was extracted from young sprouts following Doyle and Doyle³⁸.

The chloroplast (cp) genome of Scecale cereale ssp. segetale was sequenced with the use of DNBseq platform in BGI Shenzhen (China). After the quality check (FastQC tool 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) in Geneious v.R7 software with default medium–low sensitivity settings³⁹. Reads aligned to the reference cpDNA genome were extracted and used for de novo assembly (K-mer—23–41, low coverage cut-off—5, minimum contig length—300). 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 chloroplast genome was annotated using MFannot⁴⁰ and PlasMapper⁴¹ with manual adjustments. The gene map of the annotated cp genome was developed with the OrganellarGenome DRAW tool⁴².

Repeat sequence analysis

The chloroplast simple sequence repeats (SSRs) were detected using Phobos v.3.3.12⁴³. Only perfect SSRs with a motif size of one to six nucleotide units were considered, the following thresholds for chloroplast SSRs identification were used: ≥ 12 repeat units for mononucleotide SSRs, ≥ 6 repeat units for dinucleotide SSRs, ≥ 4 repeat units for trinucleotide SSRs, and ≥ 3 repeat units for tetra-, penta- and hexanucleotide SSRs⁴⁴. Analysis of long genomic repeats, i.e. forward (F), reverse (R), palindromic (P) and complementary (C) sequences, was performed using REPuter software⁴⁵ with the following settings: (1) hamming distance of 3, (2) sequence identity ≥ 90%, and (3) minimum repeat size ≥ 30 bp. A single IR region was used to eliminate the influence of doubled IR regions.

Multigene phylogeny

The phylogenetic position of Scecale cereale ssp. segetale within Triticodae group was also evaluated. For that purpose 73 concatenated protein-coding gene sequences shared with other 38 Pooideae species were used. The cpDNA of Oryza sativa was used as an outgroup (Table 4). For phylogeny reconstruction Bayesian Inference (BI) method was used. The best-fit model of sequence evolution was identified in MEGA v.7⁴⁶, and the GTR + G + I model was selected. The BI analysis was performed in MrBayes v.3.2.6⁴⁷. Parameter settings were previously described by Androsiuk et al.⁴⁸.

Table 4.

List of species used in phylogenetic studies. Species names arranged alphabetically.

Species	Accession number
Oryza sativa	NC_008155
Aegilops bicornis	NC_024831
Aegilops comosa	NC_046697
Aegilops cylindrica	NC_023096
Aegilops geniculata	NC_023097
Aegilops kotschyi	NC_024832
Aegilops longissima	NC_024830
Aegilops searsii	NC_024815
Aegilops sharonensis	NC_024816
Aegilops speltoides	NC_022135
Aegilops tauschii	NC_022133
Aegilops umbellulata	NC_046696
Australopyrum retrofractum	NC_043840
Connorochloa tenuis	NC_037165
Elymus dahuricus	NC_049159
Elymus kamoji	NC_051511
Elymus trachycaulus	NC_050404
Hordeum bogdanii	NC_043839
Hordeum jubatum	NC_027476
Hordeum vulgare subsp. Spontaneum	NC_042692
Hordeum vulgare subsp. vulgare	NC_008590
Kengyilia melanthera	NC_042706
Leymus chinensis	NC_044900
Littledalea alaica	NC_037519
Littledalea przevalskyi	NC_037497
Littledalea racemosa	NC_036350
Psathyrostachys huashanica	NC_045871
Psathyrostachys juncea	NC_043838
Secale cereale	NC_021761
Secale cereale subsp. segetale	LC645358
Secale cereale subsp. segetale	LC645358
Secale strictum	KY636137
Secale sylvestre	MW557517
Triticum aestivum	NC_002762
Triticum macha	NC_025955
Triticum monococcum	NC_021760
Triticum timopheevii	NC_024764
Triticum turgidum	NC_024814
Triticum urartu	KJ614411
Triticum zhukovskyi	NC_046698

For multigene phylogeny maximum likelihood (ML) analyses was conducted using RAxML-NG⁴⁹ under three different strategies. (1) One of the IR regions was removed from all chloroplast genomes to reduce overrepresentation of duplicated sequences then we run RAxML-NG on the unpartitioned alignment under GTR + I + G substitution model as a single partition; (2) The same data was partitioned by gene, exon, intron and intergenic spacer regions and allowed separate base frequencies, α-shape parameters, and evolutionary rates to be estimated for each; (3) we inferred the best-fitting partitioning strategy with PartitionFinder2⁵⁰ for the alignment. The best fitting nucleotide substitution models were inferred with jModelTest2⁵¹. Phylogenetic trees were visualized and edited with FigTree 1.4.4⁵². Support for the ML tree branches was calculated by the non-parametric bootstrap method with 1000 replicates.

Comparison with other complete chloroplast genomes of the Secale species

The percentage of sequence identity among complete chloroplast genomes of the five Secale: S. cereale ssp. segetale (OL688773), S. cereale ssp. segetale (LC645358), S. cereale (NC_021761), S. strictum (KY636137), and S. sylvestre (MW557517) was comparatively analyzed and plotted using the program mVISTA⁵³, with alignment algorithm of LAGAN⁵⁴, a cut-off of 70% identity, and annotation of S. cereale ssp. segetale (OL688773) as reference.

Ethics approval and consent to participate

Authors confirm that the use of plants in the present study complies with international, national and/or institutional guidelines.

Author contributions

L.S. study conception and design, L.S. and R.G. conducted experiments, P.A. and L.S. drafted the manuscript, bioinformatic analyses were performed by P.A., Ł.P., J.P.J. and D.C.-L.

Funding

This work was supported by a grant from the Institute of Biology, University of Szczecin, Poland.

Data availability

The genome can be accessed on GenBank with the accession number (OL688773).

Competing interests

The authors declare no competing interests.