Phylogenomic investigation of safflower (Carthamus tinctorius) and related species using genotyping-by-sequencing (GBS)

doi:10.21203/rs.3.rs-1745176/v1

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Phylogenomic investigation of safflower (Carthamus tinctorius) and related species using genotyping-by-sequencing (GBS)

https://doi.org/10.21203/rs.3.rs-1745176/v1

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published 17 Apr, 2023

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Safflower (Carthamus tinctorius) is a source of high-quality edible oil growing in moisture-limited environments. Despite its economic importance, the relationships to close wild species in Carthamus and the presence and relationships of ecotypes within safflower are still not fully clarified. Here we use genotyping-by-sequencing to identify the wild progenitor of C. tinctorius, infer phylogenetic relationship within series Carthamus and identify groups of closely related lineages within cultivated safflower. Phylogenetic and population genetic analyses found C. palaestinus to be the closest relative and single progenitor of C. tinctorius, which confirms the Levant as the area of domestication of the crop. Flow cytometry showed all analyzed samples of C. oxyacantha, C. palaestinus and C. tinctorius to be diploid (2n = 2x = 24) with 2C genome sizes of 2.4 to 2.7 pg. Analyses of a set of 114 worldwide distributed safflower accessions arrived at two to five genetic groups, which showed, however, no correlation with the geographic origins of these accessions. From this, we conclude that the trade of safflower seeds resulted in multiple introductions of genotypes from the Levant to other areas with suitable climate conditions for the plant, as well as exchange of genotypes among these areas.

Domestication

Ecotypes

Genotyping-by-sequencing (GBS)

Phylogeny

Safflower

Wild progenitor

Safflower (Carthamus tinctorius L., Asteraceae) is used to produce an orange dye that is sometimes used as a substitute for saffron and as an oilseed crop that grows in hot and dry climates tolerating saline soils [1]. Safflower was domesticated in the Fertile Crescent over 4,000 years ago [1,2], most probably for its use as colorant, but is nowadays cultivated in suitable areas of Asia, northeastern Africa and Europe [3,4] with a total cultivation area of 1.1 million hectares resulting in seed yield of approximately 1 million tons per year [5]. Safflower’s high-quality edible oil contains higher amounts of unsaturated fatty acids like oleic and linoleic acids than other seed oils. Despite the growing commercial importance of safflower, genetic diversity and the parental origin of this species are not fully clarified mainly due to the low variability of up-to-now applied markers. Among the species of Carthamus, C. oxyacantha and C. palaestinus – both with 2n = 24 chromosomes like C. tinctorius – have been reported as the most likely wild ancestors of safflower [3,6-9]. Wild relatives together with landraces carry valuable characteristics for crop breeding, such as biotic and abiotic stress resistances [10]. Thus, hybridization of safflower with its closest relatives could be used for the introgression of traits into the crop and can play a significant role in future breeding of cultivars. Some species of Carthamus including C. lanatus, C. palaestinus and C. oxyacantha have been reported to have the potential to cross with safflower and produce fertile offspring [6,7,11]. Due to partly low reproducibility of applied markers and conflict between published data sets [3,9,12-17], we here use genome-wide single-nucleotide polymorphisms (SNP) obtained through genotyping-by-sequencing [18] (GBS) on a diverse set of C. tinctorius accessions and six wild Carthamus species for a high-resolution phylogeny. The objectives of our study are (i) to infer phylogenetic relationships of Carthamus species closely related to safflower, i.e. section Carthamus, (ii) to identify the closest wild relative(s) of safflower to enable breeding for the increase of genetic diversity regarding fatty acid content, drought tolerance and other agro-morphological traits, (iii) to look into genetic diversity of safflower accessions, and (iv) to see if the three to ten postulated morphology and geography-defined groups [9,19-24] are genetically distinct and can be recovered using genomic data.

Phylogenetic inference

In an initial phylogenetic analysis, we included six Carthamus species, i.e. C. boissieri, C. glaucus, C. lanatus (with three subspecies), C. oxyacantha, C. palaestinus and C. tenuis together with a small but geographically diverse subset of C. tinctorius to infer the closest relative of safflower. The GBS-derived data matrix consisted of 108,985 aligned characters of which 3468 were parsimony informative. We used neighbor-joining (NJ), Bayesian phylogenetic inference (BI) and maximum parsimony (MP) analyses which resulted in identical (BI, MP) or compatible (BI, MP, NJ) tree topologies. The MP analysis retained 2484 equally parsimonious trees with a consistency index (CI) of 0.68 and a retention index (RI) of 0.95. The strict consensus of these trees, summarizing MP (bootstrap, bs) and BI (posterior probabilities, pp) support values, is provided in Fig. 1.

This unrooted tree consists of two major clades, one including the 24-chromosome species (chromosome numbers according to Vilatersana et al. [25]) C. oxyacantha, C. palaestinus, and C. tinctorius together with 2n = 20 C. glaucus, while clade two harbors 2n = 20 C. tenuis and C. boisseri, together with the 2n = 44 allotetraploid C. lanatus. Carthamus tinctorius groups within C. palaestinus, indicating the latter to be the closest relative of safflower and probably the wild progenitor of the crop. The two Iranian C. oxyacantha accessions from Isfahan and Kashan are separated from the other members of this species and fall within the C. palaestinus/C. tinctorius clade, indicating introgression from these species. Support values for the branches separating the species are generally very high throughout the tree (Fig. 1).

Since, C. tinctorius was found in a clade with C. oxyacantha, C. palaestinus and C. glaucus, we included these wild species in our analysis of 114 safflower accessions. The alignment for this dataset consisted of 435,024 characters, which resulted in four MP trees (CI = 0.30, RI = 0.51) that were rooted with the six non-introgressed C. oxyacantha accessions. In this tree (Fig. 2) C. palaestinus groups within C. tinctorius with 100% bootstrap support for the clade unifying these species. Within C. tinctorius bootstrap support values >75% occur only for a few and rather small clades of safflower accessions. Although C. tinctorius accession formed five larger groups (Fig. 2), no clear geographic pattern was found, i.e. in most clades, safflower accessions from diverse geographical regions were united.

Population assignment analysis and Principal Component Analysis (PCA)

In addition to tree-based estimations of relationships of C. tinctorius we also used Bayesian population assignment and principal component analyses. Carthamus oxyacantha, C. palaestinus, C. glaucus and C. tinctorius individuals were subjected to Structure-like analyses in Lea. Evanno’s Δk suggested K = 3 as the best fitting category number that was used for the final analysis (Fig. 3). The analysis confirmed our tree-based assumption that two of the C. oxyacantha accessions (from Isfahan and Kashan, Iran) were introgressed by genotypes from the C. palaestinus/C. tinctorius group and in addition identified also a third accession (from Arak, Iran) showing traces of gene flow. Carthamus palaestinus (from Israel) has the greatest similarity with C. tinctorius. Carthamus glaucus (from Lebanon) is clearly distinct from the former two species although some allelic overlap occurs with one Russian and two Iranian safflower accessions (Fig. 3) and two individuals of C. glaucus share alleles with C. palaestinus/C. tinctorius.

Also PCA resulted in three groups of populations (Fig. 4). The first two principal components explained 42.8% of the total molecular variation. PCA results were similar to those of the phylogenetic trees and Lea plots as they grouped C. tinctorius and C. palaestinus together while C. oxyacantha and C. glaucus were clearly separated from them along the first and second axis, respectively.

Analysis of genome size

Flow cytometry was used to measure genome sizes and infer ploidy levels of C. tinctorius, C. oxyacantha, and C. palaestinus individuals (Table 1). We found that all measured samples of C. oxyacantha, C. palaestinus and C. tinctorius represent diploid (2n = 2x = 24) 2C genome sizes ranging from 2.4 pg to 2.7 pg.

Inferring phylogenetic relationships among closely related species is a challenge because of inter-locus phylogenetic discordance and/or the difficulty of obtaining variable markers. Despite various phylogenetic studies carried out in Carthamus, phylogenetic relationships within Carthamus are poorly understood and the closest wild relative of cultivated safflower remained elusive for a long time [26] due to limited taxon sampling and/or low variability of the applied markers [12,27-29]. Still, studies published during the last decades mostly identified C. palaestinus from the southwestern Fertile Crescent as the possible wild ancestor [3,8,12], which fits geographically with the earliest archeological remains of safflower that were identified in Syria [2]. Also, intra-species relationships within safflower are not well understood. Based on morphological traits like flower color, inflorescence size, plant height and leaf shape, and their geographical distribution, between five and ten centers of diversity were postulated for safflower, all harboring region-specific ecotypes [1,9,19,20,26]. Here we performed phylogenetic and population genetic analyses based on genome-wide distributed SNPs that were obtained through GBS to clarify the phylogenetic relationships of the relatives of C. tinctorius and to see if described ecotypes of safflower are genetically distinct. The GBS approach is known to have the potential for better resolved relationships within and among closely related species in comparison to earlier fragment size analyses or sequence comparisons of single nuclear or chloroplast DNA regions [31-34].

Our phylogenetic analyses of GBS data representing seven Carthamus species resulted in a tree (Fig. 1) where C. boisseri, C. lanatus and C. tenuis form one clade that is sister to the clade of C. glaucus, C. oxyacantha, C. palaestinus and C. tinctorius. Support values for the clades in the tree are generally very high and C. boisseri, C. lanatus, C. oxyacantha, and C. tenuis result as monophyletic species. In contrast, C. tinctorius groups within C. palaestinus, and both together within a paraphyletic C. glaucus. Within the C. palaestinus/C. tinctorius clade also two C. oxyacantha accessions group, which we assumed to be hybrids between C. oxyacantha and C. palaestinus or C. tinctorius, a result that was confirmed by Structure-like analysis (Fig. 3). Also in C. glaucus two individuals show allelic similarities with C. tinctorius (Fig. 3), which could indicate gene flow and explains that two clades of a paraphyletic C. glaucus were recovered in the phylogenetic trees of which the introgressed individuals are sister to C. palaestinus/C. tinctorius. Our data indicate that hybridization between the species closely related with C. tinctorius is still possible on the homoploid level, as the C. oxyacantha individual from Isfahan (Iran) was measured with the same genome size as other individuals from this species (Table 1). The two major clades we obtained in our phylogenetic tree essentially reflect the two sections Carthamus and Atractylis of Bowles et al. [8] although in our analysis C. glaucus groups with the sect. Carthamus species while in their analysis as well as in Vilatersana et al. [25] and Tarıkahya-Hacıoglu et al. [35] C. glaucus is a member of the sect. Atractylis taxa. This inconsistency is however not unique to our analysis. Already in Sasanuma et al. [29], C. glaucus is grouping with C. tinctorius while Sehgal et al. [12] and Mehrotra et al. [30] retrieved C. glaucus accessions in both clades of Carthamus. Unfortunately, we relied on gene bank passport data and had no additional seeds available when we obtained the GBS results so that we could not check chromosome numbers for our C. glaucus individuals to be sure that they are indeed belonging to the 2n = 20-chromosome taxa. As in all before-mentioned studies C. glaucus materials from gene banks were used instead of freshly collected plants from the wild, it might be worthwhile to re-determine gene bank-derived C. glaucus accessions in future analyses to be sure about the species affiliation of these lineages.

In our second GBS dataset we included a diverse set of 114 C. tinctorius accessions together with the sect. Carthamus species of our initial analysis but excluded the two obviously introgressed C. oxyacantha accessions. While C. glaucus appears again paraphyletic in the resulting phylogenetic tree (Fig. 2), the individuals of C. palaestinus fall within the safflower accessions occurring in two of their different clades. This result shows that the genomic diversity of C. tinctorius cannot be separated from C. palaestinus and is a strong indication that the latter is the wild progenitor of safflower, as its genetic diversity is well within the range of C. tinctorius. This result is supported by the population assignment analysis where C. palaestinus has the highest similarity with C. tinctorius, and confirms earlier findings regarding the ancestor of safflower [3,9,12].

Within safflower we found no clear subgroups. Carthamus tinctorius in the tree provided in Fig. 2 has four groups of genetically similar accessions, however, only two of the groups are monophyletic and none of them shows a clear geographic pattern. Expectedly, bootstrap values within this species are low, often not even reaching 50%. We interpret this result as an indication of strong trading connections among the early centers of safflower cultivation in Eurasia followed by multiple independent introductions of safflower germplasm from these centers into regions like the Americas and Australia where safflower cultivation started only during the last century. In addition to the mere introduction of foreign germplasm to new areas also gene flow between newly attained and older local cultivars can further blur phylogeographic patterns. This general picture does not change when C. palaestinus, C. oxyacantha and C. glaucus individuals with traces of safflower introgression (Fig. 3) are removed from the dataset: In this case the resulting clades and subclades in the phylogenetic analysis again lack geographic structure (see Supplementary Fig. S1 online). This result is in strong contrast to the morphology-defined geographic groups of Kupcov [19], Hanelt [26], Ashri [20] and Knowles and Ashri [1], which all postulated the presence of certain ecotypes typical for specific geographic regions, although they did not arrive at a conclusion about the number of such safflower ecotypes. This discrepancy can be explained either by the quantitative nature of the used morphological traits that are (i) controlled by very few genetic loci with the respective allelic composition not reflected in the GBS data we obtained, and/or (ii) these traits might have an environmental and ecological component that influences characters like flower color or plant height but genetic inheritance might be low. Still, in contrast to our results here Chapman et al. [9], using 24 nuclear microsatellite markers, found five weakly supported groups with a possible geographic correlation. In contrast to our dataset they obtained these groups by restricting their analysis to Ashri’s [20] set of assumed autochthonous accessions, i.e. sets of materials that were not influenced by introductions during the last century. For the safflower accessions currently stored in gene banks we conclude that neither geographic origin nor certain morphological traits are usable indicators of genetic similarity or diversity. For breeding approaches, it is therefore necessary to either use molecular analyses to identify rather diverse genotypes or use gene bank passport data to identify accessions with the preferred or extreme agronomic traits.

Using GBS that combines next-generation sequencing with a genomic complexity reduction approach, we were easily able to discern all analyzed safflower accessions and could resolve their phylogenetic relationships. However, within C. tinctorius we could not recover any geographically defined groups/ecotypes that were postulated earlier based on morphological character combinations thought to prevail in specific regions. This we explain with seed exchange among the areas where safflower is commercially grown, which might have blurred an eventually formerly existing geographic pattern of locally evolved genotypes.

In contrast to the intra-species structure in C. tinctorius our GBS analysis recovered species relationships in C. sections Carthamus and Atractylis with high support values. We identified C. palaestinus as closest relative and likely progenitor of safflower. Carthamus glaucus resulted as a paraphyletic species that harbors C. tinctorius/C. palaestinus, but its phylogenetic position in our trees casts some doubts about the species affiliation of the used material. The other analyzed species appear to be monophyletic.

Taxon sampling

One hundred sixty-seven individuals (see Supplementary Table S1 online) including 114 cultivated C. tinctorius and 53 individuals of wild species of Carthamus, i.e. C. lanatus (18), C. oxyacantha (8), C. glaucus (6), C. palaestinus (7), C. tenuis (6), and C. boissieri (8), were included in GBS-based phylogenetic analyses. A set of eight diverse individuals of C. tinctorius was used together with individuals of C. palaestinus, C. oxyacantha and C. glaucus for population structure analyses.

The analyzed materials were obtained as seeds from the gene bank collections of RTIPP-SBUK (Research and Technology Institute of Plant Production, Shahid-Bahonar University, Kerman, Iran), IPK (Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany) and NPGS (National Plant Germplasm System, U.S. Department of Agriculture, USA) and grown at least to a four-leaf stage before harvesting leaves for DNA extraction. Detailed information such as passport data and observation records for each accession can be found in the online GRIN (Germplasm Resources Information Network) database of NPGS (http://www.ars-grin.gov/npgs/) and Genebank Information System of the IPK Gatersleben (https://gbis.ipk-gatersleben.de/). Collection and use of plant materials was conducted in accordance with institutional, national and international regulations. Seed exchange followed the Nagoya rules of access and benefit sharing through standard material transfer agreements.

DNA extraction and genotyping-by-sequencing (GBS)

Genomic DNA was extracted from young leaves with a DNeasy Plant kit (QIAGEN). To obtain genome-wide SNPs, we used a two-enzyme GBS protocol that uses the restriction enzymes PstI (NEB Inc.) and MspI (NEB Inc.) to digest 200 ng of genomic DNA. For the library preparation and individual barcoding, the protocol of Wendler et al. [36] was followed. The individual libraries were sequenced in multiplex on an Illumina HiSeq 2500 (100 bp single-end reads) at the sequencing facilities of Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany.

GBS data preparation and filtering

Quality assessment of all raw sequence samples was performed using fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Sequences were de-multiplexed allowing for one base mismatch in barcodes, and the restriction site and barcode were trimmed from all GBS sequence reads using cutadapt [37]. Sequence data were filtered and clustered using the ipyrad bioinformatics pipeline [38]. We conducted two runs of ipyrad: (i) one with a small set of C. tinctorius accession but including seven wild Carthamus species, and (ii) using 114 individuals from C. tinctorius plus its closest three relatives according to the prior analysis. The minimal number of samples to possess a certain locus was set to 90% of the individuals. The clustering threshold of reads within and between individuals was set to 0.85 and 0.90 in the first and second dataset, respectively. For the other parameters the default settings were used. The assembly of the GBS data was done de novo.

Phylogenomic analyses

After removing samples with a too low number of reads, we used MrBayes 3.2.6 [39] and Paup* 4a169 [40] to infer phylogenetic relationships within our set of Carthamus species and accessions. NJ, based on GTR distances, and MP trees were calculated in Paup* to analyze the aligned sequence data matrices. In MP gaps were treated as missing data and we used a heuristic search with TBR branch swapping with initially 250 random addition sequences (RAS) restricting the number of collected trees to 50 per replicate. The resulting shortest trees were then used as starting trees in a second heuristic search without setting a limit on tree numbers. Bootstrap support values were obtained by 500 bootstrap re-samples using the same settings as before except that we excluded RAS. Paup* was also used to infer the model of sequence evolution.

For BI two times four chains were run for 5 million generations for the dataset including the set of different Carthamus species, specifying the model of sequence evolution as GTR+Γ. We sampled a tree every 1000 generations and summarized the trees in MrBayes. Converging log-likelihoods, potential scale reduction factors for each parameter and inspection of tabulated model parameters in MrBayes suggested that stationary had been reached. The first 25% of trees were discarded as burn-in. For the large dataset including all C. tinctorius accessions, phylogenetic inference calculations in MrBayes were run for more than three weeks without converging chains when we cancelled this analysis.

Population structure analysis

The dataset of SNPs generated from ipyrad was used as input for the analysis of population structure for C. oxyacantha, C. palaestinus, C. glaucus and eight C. tinctorious cultivars in the R package LEA [41]. The number of subpopulations (K) was assumed to range from one to seven. An admixture analysis [42] using sparse nonnegative matrix factorization (snmf) was used for the estimation of population genetic structure. In addition, a Principal Components Analysis (PCA) was conducted in ipyrad on the same dataset.

Analysis of genome size

To obtain ploidy levels we used flow cytometry to measure genome sizes of Carthamus species [43]. For this, we collected a leaf from each of three individuals from eight C. tinctorius and four C. oxyacantha accessions and three leaves from one C. palaestinus individual. Afterwards the leaves were transported to the lab and genome sizes were measured on a CyFlow flow cytometer (Sysmex Partec GmbH) using propidium iodide (PI) as staining reagent (Sigma-Aldrich) according to Jakob et al. [44]. The tomato cultivar ‘Stupicke’ was used as the size standard (2C DNA content = 1.96 pg [45]).

Acknowledgements

The authors thank C. Koch, B. Kränzlin and P. Oswald for technical assistance in the greenhouses or lab, S. König and A. Himmelbach for GBS library preparation and NG sequencing, and the gene banks of IPK, RTIPP-SBUK and USDA for Carthamus germplasm. Financial support by IPK is acknowledged.

Author contributions

Designed study: S.S.N., Z.N., F.R.B. Coordinated study: Z.N., S.S.N., F.R.B. Provided data or materials: G.M.N. Performed experiments: S.S.N., Z.N. Analyzed data: Z.N., S.S.N., F.R.B., R.H. The initial manuscript was written by F.R.B., S.S.N., Z.N. All authors contributed to and approved the final version of the manuscript.

Data availability statement

All sequence data are available through the NCBI nucleotide database under accession numbers XXXXXX–YYYYYY (We are in the process of sequence submission; numbers will be added before final manuscript submission)

Additional information

Competing interest

The authors declare no competing interests.

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Table 1. Genome sizes of C. tinctorius, C. oxyacantha, and C. palaestinus individuals measured by flow cytometry against tomato cv. ‘Stupicke’ (2C = 1.96 pg) as size standard.

Accession	Species	Genome size pg/2C (± SD)
C_tin_TN79548_Hungary	C. tinctorius	2.50 (±0.03)
C_tin_CW74_USA	C. tinctorius	2.59 (±0.05)
C_tin_CART156_Pakistan	C. tinctorius	2.49 (±0.05)
C_tin_CART86_Tunisia	C. tinctorius	2.46 (±0.02)
C_tin_CART118_India	C. tinctorius	2.44 (±0.06)
C_tin_CART227_Germany	C. tinctorius	2.50 (±0.02)
C_tin_K101_Iran	C. tinctorius	2.54 (±0.03)
C_tin_Goldasht_Iran	C. tinctorius	2.68 (±0.09)
C_oxy_IR_Aligoodarz	C. oxyacantha	2.54 (±0.02)
C_oxy_IR_Azerbaijan	C. oxyacantha	2.53 (±0.04)
C_oxy_IR_Hamedan	C. oxyacantha	2.40 (±0.07)
C_oxy_IR_Isfahan	C. oxyacantha	2.42 (±0.05)
C_pal_PI235663_1	C. palaestinus	2.60 (±0.01)

No competing interests reported.

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Phylogenomic investigation of safflower (Carthamus tinctorius) and related species using genotyping-by-sequencing (GBS)

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