DNA sequence diversity and the origin of cultivated safflower (Carthamus tinctorius L.; Asteraceae)

  • Mark A Chapman1Email author and

    Affiliated with

    • John M Burke1

      Affiliated with

      BMC Plant Biology20077:60

      DOI: 10.1186/1471-2229-7-60

      Received: 16 May 2007

      Accepted: 06 November 2007

      Published: 06 November 2007

      Abstract

      Background

      Safflower (Carthamus tinctorius L.) is a diploid oilseed crop whose origin is largely unknown. Safflower is widely believed to have been domesticated over 4,000 years ago somewhere in the Fertile Crescent. Previous hypotheses regarding the origin of safflower have focused primarily on two other species from sect. CarthamusC. oxyacanthus and C. palaestinus – as the most likely progenitors, although some attention has been paid to a third species (C. persicus) as a possible candidate. Here, we describe the results of a phylogenetic analysis of the entire section using data from seven nuclear genes.

      Results

      Single gene phylogenetic analyses indicated some reticulation or incomplete lineage sorting. However, the analysis of the combined dataset revealed a close relationship between safflower and C. palaestinus. In contrast, C. oxyacanthus and C. persicus appear to be more distantly related to safflower.

      Conclusion

      Based on our results, we conclude that safflower is most likely derived from the wild species Carthamus palaestinus. As expected, safflower exhibits somewhat reduced nucleotide diversity as compared to its progenitor, consistent with the occurrence of a population genetic bottleneck during domestication. The results of this research set the stage for an investigation of the genetics of safflower domestication.

      Background

      Safflower (Carthamus tinctorius L.) is a thistle-like, self-compatible, annual, diploid (2n = 24) herbaceous crop that thrives in hot, dry climates, and is capable of surviving on minimal surface moisture. It is believed to have been domesticated somewhere in the Fertile Crescent region over 4,000 years ago [1]. Following its initial domestication, safflower cultivation is thought to have expanded to both the east and west [2], with Knowles [3] ultimately recognizing seven "centers of similarity" (the Far East, India-Pakistan, the Middle East, Egypt, Sudan, Ethiopia and Europe). Safflower lines native to each 'center' are remarkably similar in height, branching, spines, flower color and head size; however, consistent morphological differences are maintained between the centers.

      For centuries, safflower was grown on a local scale for its flowers, which served as a source of dye (carthamine) for textiles and food coloring, as well as for use in religious ceremonies [4]. Floral extracts were also used to flavor foods, and have historically been valued for their numerous medicinal properties. Cultivation of safflower in the New World commenced in 1899, and commercial production of safflower as an oilseed crop began in the 1950s [5]. More recently, there has been growing interest in safflower for its potential as a large-scale production platform for plant-made pharmaceuticals [6, 7].

      To date, phylogenetic investigations of Carthamus species have focused on either delimiting the sections within the genus [e.g. [8, 9]], or on the development of DNA fingerprinting methodologies for investigating relationships amongst safflower cultivars [e.g. [10]]. Relationships between closely-related species within each section are, however, only poorly understood, and details surrounding the origin and early evolution of safflower are lacking. What we currently know is that safflower belongs to a group of closely related diploid species (sect. Carthamus; all 2n = 24 chromosomes [11]) whose ranges extend from central Turkey, Lebanon, and Israel in the west to northwestern India in the east. In addition to C. tinctorius, this section is composed of C. curdicus Hanelt, C. gypsicola Iljin, C. oxyacanthus Bieb. (= C. oxyacantha M. Bieb.), C. palaestinus Eig, and C. persicus Desf. ex Willd. (= C. flavescens Spreng) [8, 12]. However, these species all exhibit some degree of cross-compatibility with one another [reviewed in [12, 13]] and thus reproductive isolation alone cannot be used to delimit the species. Carthamus curdicus and C. palaestinus exhibit restricted geographical distributions (Northern Iran and Southern Israel, respectively), whereas C. persicus, C. gypsicola and C. oxyacanthus are more widely distributed throughout the Middle East [12].

      Hypotheses regarding the origin of safflower have focused on C. oxyacanthus or C. palaestinus as the most likely progenitors, although C. persicus has also been suggested as a possible progenitor [14]. Here we report on levels of nucleotide diversity within and among species of sect. Carthamus, and investigate the origin of cultivated safflower using data derived from seven nuclear genes.

      Results

      DNA sequence diversity

      Sequence data were collected from all seven gene regions for each of the 23 individuals surveyed (Tables 1 and 2), encompassing all species within sect. Carthamus. All sequences have been deposited in the Genbank Data Library and are available under accession nos. EF483951–EF483974, EF483983–EF484014, EF519712–EF519729, EF519732–EF519751, EF519754–EF519770, EF519774–EF519792, EF519795–EF519811, EF519815–EF519834 and EF519838–EF519857. Excluding indels, sequence lengths varied from 365 to 621 base pairs (bp) per locus, and all sequences included both exons and introns (Table 3). Thus, we were able to analyze 3239 bp of aligned sequence per individual with 1317 bp (40%) coming from exons and 1922 bp (60%) coming from introns. Across taxa, the number of indel polymorphisms per locus varied from four to thirteen, with a total of 53 indels in the data set. All indels were excluded from the analyses of nucleotide polymorphism.
      Table 1

      List of accessions surveyed, including information on the source of each sample.

      Species

      Code

      Cultivar/Accession

      PI/herbariuma

      Originb

      C. tinctorius L.

      saffW

      W6 6730

      PI 576995

      China*

      C. tinctorius L.

      saffL

      LESAF 494

      PI 603207

      Canada

      C. tinctorius L.

      saffE

      ENANA

      PI 610263

      Spain*

      C. tinctorius L.

      saffU

      USB

      PI 560163

      USA

      C. tinctorius L.

      saffAZ

      ARIZ SAFF COMP III

      PI 572418

      USA

      C. tinctorius L.

      saffAC

      AC SUNSET

      PI 592391

      Canada

      C. tinctorius L.

      saff1063

      BJ-1063

      PI 250601

      India*

      C. tinctorius L.

      saff673

      BJ-673

      PI 193473

      Ethiopia*

      C. tinctorius L.

      saff2701

      BJ-2701

      PI 253762

      Iraq*

      C. tinctorius L.

      saff1067

      BJ-1067

      PI 250606

      Egypt*

      C. tinctorius L.

      saffTS

      TOZI SPINY

      PI 271070

      Sudan*

      C. curdicus Hanelt

      curd

      Hanelt

      W 12361

      Iraq

      C. gypsicola Iljin

      gypA

      UZ99a

      -

      Uzbekistan

      C. gypsicola Iljin

      gypB

      UZ99b

      -

      Uzbekistan

      C. oxyacanthus Bieb.

      oxy2

      K-2

      PI 426428

      Pakistan

      C. oxyacanthus Bieb.

      oxy1076

      K-1076

      PI 426185

      Afghanistan

      C. oxyacanthus Bieb.

      oxy604

      K-604

      PI 426467

      Pakistan

      C. palaestinus Eig

      palBJ

      BJ-1964

      PI 235663

      Israel

      C. palaestinus Eig

      pal96

      Ashri1917

      GAT 3796

      Israel

      C. palaestinus Eig

      pal97

      Ashri1642

      GAT 3797

      Israel

      C. palaestinus Eig

      pal98

      Ashri

      GAT 3798

      Israel

      C. persicus Willd.

      perG

      Garcia-Jacas 2002

      -

      Turkey

      C. persicus Willd.

      per00

      Aydem 157

      GAT 3800

      Turkey

      a Numbers refer to the USDA accession (PI), Vienna Museum of Natural History (W) or Gatersleben herbarium accession (GAT). No number indicates a private collection. '-' indicates missing voucher specimens. More seeds are, however, available from these collections.

      b Asterisks refers to the seven accessions of safflower from the seven so-called "centers of similarity" (ref. [3]; see text for details)

      Table 2

      Summary of genes surveyed and primer sequences employed.

      Locus

      Functional Annotation via BLAST

      Primer Sequences (Forward/Reverse)

      A19

      At2g21330

      5'-CTAGAGAACACSGARGCTAACCG

       

      Putative fructose bisphosphate aldolase

      5'-TGGCGAAACGRGCACCYTGTTGG

      A25

      At2g45740

      5'-TTGCATGSTCTTATCAGTCC

       

      Similar to putative peroxisomal membrane protein PEX11-1

      5'-GAAGABCCCATCCARCAGAAGAG

      A25a

      -

      Same as A25 Forward

        

      5'-TCTCTCTCATGACACCATGTAAA

      A25b

      -

      5'-GCTCCACAGATCAGGCATTT

        

      Same as A25 Reverse

      A27

      At3g19900

      5'-CTTGCAWTGAATGTCATGTGGAAG

       

      Unknown protein

      5'-GCTCCCCARCATTTCA

      A39

      At2g28315

      5'-ACTAGTTGGCATYTRATGGTAACA

       

      Putative glucose-6-phosphate/phosphate-tranlocator

      5'-GCCRACAAAATTGAGCTGAAGATC

      A39a

      -

      Same as A39 Forward

        

      5'-TCATGGACCAGAAATGAYGTT

      A39b

      -

      5'-AGCACCCTTCCYTACTGCAT

        

      Same as A39 Reverse

      B7

      At4g27700

      5'-AGAAAAYARCTTTGTGATTCTTGATG

       

      Contains Rhodanese Homology Domain

      5'-GAWGARCAAGCTACTATRATCTTTG

      B12

      At3g55800

      5'-CAAGTGGCTGCAGCCATGGG

       

      Sedoheptulose-bisphosphatase precursor

      5'-ACATCRGGMACCATTCCWCCGGTGT

      B27

      At4g33250

      5'-AAGGCTCTTATGGCHATGCC

       

      Similar to eukaryotic translation initiation factor 3 subunit 11

      5'-CGGTTYTTRGCWGCTTCATCCCARAACTG

      B27a

      -

      Same as B27 Forward

        

      5'-TGATGCAAAATAGTTTGTTGGAA

      B27b

      -

      5'-TGGTGMTYCTTTTCCAATTC

        

      Same as B27 Reverse

      Primer names followed by 'a' or 'b' were designed to amplify a given locus in two parts. Functional annotations were taken from the Genbank record for each Arabidopsis locus. For the unknown protein a BLASTn search failed to yield a putative function

      Table 3

      Details regarding gene regions analyzed.

      Locus

      Aligned Lengtha

      No. Indels

      N(var)b

      A19

      365

      4

      34

      A25

      486

      6

      42

      A27

      395

      13

      50

      A39

      578

      4

      49

      B7

      386

      9

      51

      B12

      408

      7

      57

      B27

      621

      10

      64

      Combined

      3239

      53

      267

      a Alignment size (bp) after removing primers, indels, and ambiguous regions

      b Number of variable characters

      Single nucleotide polymorphisms (SNPs) were considerably more common than indels. A total of 220 SNPs were found across the full data set, resulting in an average of 1 SNP per 15 bp of sequence. Considering just the 11 safflower individuals, there were 34 SNPs, corresponding to an average of 1 SNP per 95 bp of sequence. Estimates of nucleotide diversity for C. tinctorius, C. palaestinus and C. oxyacanthus are presented in Table 4.
      Table 4

      Estimates of nucleotide variability and Tajima's D.

      Locus

      Taxon

      θW

      πTot

      πSil

      Tajima's D a

      A19

      C. tinctorius

      0.0041

      0.0046

      0.0087

      0.34

       

      C. palaestinus

      0.0078

      0.0095

      0.0180

      1.03

       

      C. oxyacanthus

      0.0013

      0.0016

      0

      0.85

      A25

      C. tinctorius

      0.0071

      0.0102

      0.0109

      1.44

       

      C. palaestinus

      0.0059

      0.0066

      0.0089

      0.52

       

      C. oxyacanthus

      0.0047

      0.0036

      0.0036

      -1.30

      A27

      C. tinctorius

      0.0023

      0.0014

      0.0006

      -0.95

       

      C. palaestinus

      0

      0

      0

      n/a

       

      C. oxyacanthus

      0.0063

      0.0077

      0.0087

      1.22

      A39

      C. tinctorius

      0

      0

      0

      n/a

       

      C. palaestinus

      0.0012

      0.0008

      0.0010

      -1.05

       

      C. oxyacanthus

      0.0070

      0.0057

      0.0084

      -1.07

      B7

      C. tinctorius

      0.0016

      0.0010

      0.0009

      -0.84

       

      C. palaestinus

      0.0054

      0.0071

      0.0112

      1.43

       

      C. oxyacanthus

      0.0012

      0.0014

      0.0025

      0.85

      B12

      C. tinctorius

      0.0062

      0.0102

      0.0175

      2.20

       

      C. palaestinus

      0.0067

      0.0075

      0.0129

      0.53

       

      C. oxyacanthus

      0.0277

      0.0354

      0.0601

      1.76

      B27

      C. tinctorius

      0.0020

      0.0013

      0.0014

      -1.04

       

      C. palaestinus

      0.0035

      0.0044

      0.0048

      1.18

       

      C. oxyacanthus

      0.0229

      0.0184

      0.0202

      -1.42

      Average

      C. tinctorius

      0.0033

      0.0041

      0.0057

      0.19

       

      C. palaestinus

      0.0044

      0.0051

      0.0081

      0.61

       

      C. oxyacanthus

      0.0101

      0.0105

      0.0148

      0.13

      a None of the estimates of Tajima's D were significant

      While diversity varied across loci, C. tinctorius generally harbored the lowest levels of diversity with Watterson's θ (θW) ranging from 0 to 0.0071 (mean = 0.0033), total nucleotide diversity (πTot) ranging from 0.0008 to 0.0102 (mean = 0.0041), and silent-site diversity (πSil) ranging from 0.0006 to 0.0175 (mean = 0.0057). Carthamus oxyacanthus, on the other hand, exhibited the highest levels of diversity, with θW ranging from 0.0012 to 0.0277 (mean = 0.0101), πTot ranging from 0.0014 to 0.0354 (mean = 0.0105), and πSil ranging from 0 to 0.0601 (mean = 0.0148). Carthamus palaestinus was intermediate to the other two species with θW ranging from 0 to 0.0078 (mean = 0.0044), πTot ranging from 0 to 0.0095 (mean = 0.0051), and πSil ranging from 0 to 0.0180 (mean = 0.0081).

      Because of the relatively small amount of exonic sequence included in each gene fragment (188 bp on average), individual estimates of synonymous and non-synonymous diversity must be viewed with caution. Averaging across loci, however, revealed that our estimates of non-synonymous variability are substantially lower than our estimates of synonymous variability, suggesting that diversity at these loci is primarily governed by purifying selection (data not shown). After correcting for multiple comparisons, none of the Tajima's D estimates were significantly different from zero (Table 4).

      Phylogenetic relationships

      Comparison of the NJ trees produced from the single gene analyses suggests that reticulate evolution and/or incomplete lineage sorting has occurred in Carthamus sect. Carthamus (Fig. 1). While a number of overall similarities in tree topology are evident from these analyses, there are several instances in which the phylogenetic position of an individual varies depending on the gene analyzed. In some cases, individuals harbored divergent alleles at one or more loci, possibly indicating that contemporary hybridization has played an active role in the evolution of sect. Carthamus. Of particular note are the positions of the C. gypsicola alleles which are sometimes found in divergent clades (e.g., for genes A19 and B12). Some C. oxyacanthus alleles show a similar pattern (e.g., genes A39 and B27). When comparing the seven trees for the individual loci, however, some patterns begin to emerge. Overall, C. oxyacanthus is most often found to be relatively distantly related to C. tinctorius, and frequently associated with alleles from C. gypsicola. Of particular note is the close relationship between individuals of C. tinctorius and C. palaestinus, suggesting that the species most closely related to safflower (and hence its most likely progenitor) is C. palaestinus (Fig. 1).
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-7-60/MediaObjects/12870_2007_Article_201_Fig1_HTML.jpg
      Figure 1

      Phylogenetic relationships among species ofCarthamussect.Carthamusbased on single-gene analyses. Neighbor-Joining trees were generated for each individual gene. Species names and accession codes are given in Table 1. Accession names followed by -1 or -2 denote alleles for a given locus. Alleles followed by a * were determined using haplotype subtraction by maximum likelihood; the remainder of the alleles were determined by cloning.

      Despite the occurrence of some incongruities between loci, the NJ, ML and Bayesian trees based on the combined data (which are nearly identical to each other in topology; Fig. 2 and data not shown) are in overall agreement with our interpretations of the single gene analyses. Carthamus oxyacanthus is resolved as the species most distantly related to safflower, with high ML bootstrap and Bayesian posterior probabilities. Within C. oxyacanthus the two lines from Pakistan are more closely related to each other than they are to the line from Afghanistan. Carthamus persicus appears to be paraphyletic, perhaps due to recent gene flow. As predicted from the individual gene trees, C. palaestinus is the most closely related species to safflower, and we conclude that this species is the most likely progenitor of safflower. In the Bayesian analysis, all four C. palaestinus individuals are found in a well-supported clade along with all of the safflower individuals. Similarly, in the ML analysis, three of the four individuals of C. palaestinus are found in such a clade (87% BS), with the fourth resolving at the base of this clade along with individuals of C. curdicus and C. persicus. Some relationships can also be resolved among safflower individuals; for example, saffAZ and saffAC form a well-supported clade at the base of the safflower/C. palaestinus group. Relationships between the other cultivars are, however, poorly-supported.
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-7-60/MediaObjects/12870_2007_Article_201_Fig2_HTML.jpg
      Figure 2

      Phylogenetic relationships among species ofCarthamussect.Carthamusbased on a combined analysis of seven nuclear genes. Maximum likelihood (A) and Bayesian (B) trees generated for the combined dataset. Bootstrap values (> 75%) for the ML tree and posterior probabilities (> 0.90) for the Bayesian tree are given alongside branches. Species names and accession codes are given in Table 1.

      Discussion

      Origin of safflower

      A close relationship between members of sect. Carthamus has been proposed based on data from crossing studies [reviewed in [12, 13]], the identification of natural hybrids amongst some species within the section [1, 14], and phylogenetic analyses involving some members of the section [9]. Prior to the present investigation, however, the phylogenetic relationships amongst all species within sect. Carthamus had not been investigated, and the identity of the progenitor of safflower had only been hypothesized. While Ashri & Knowles [14] proposed that safflower was derived from hybridization between C. oxyacanthus and C. persicus, this hypothesis is clearly not supported by our results. Rather, Carthamus palaestinus and safflower are found in the same clade indicating a close relationship between these species. We thus propose that C. palaestinus, which is native to the deserts of southern Israel and western Iraq, is the wild progenitor of safflower. Safflower and C. palaestinus share a self-compatible breeding system [12]; thus, the near absence of heterozygous loci in these species (Fig. 1) is unsurprising. Carthamus persicus and some populations of C. oxyacanthus are self-incompatible, and this is evident from the presence of much more heterozygosity in these species (Fig. 1). The cause of the non-monophyly of C. persicus remains unknown due to the small sample sizes necessarily employed here; however the retention of ancestral polymorphism and/or contemporary gene flow (C. persicus is self-incompatible) could be responsible.

      As noted in the Introduction, Knowles [3] recognized seven distinct "centers of similarity" of safflower, including the Far East, India-Pakistan, the Middle East, Egypt, Sudan, Ethiopia and Europe. Interestingly, our data provide little support for the distinctiveness of safflower accessions from these disparate geographic locales. Indeed, while there is a small amount of phylogenetic structure apparent within safflower, it appears that most of the accessions surveyed herein are highly similar at a genetic level. Moreover, much of the substructuring within safflower is not well-supported in either the Bayesian or ML analyses. The exceptions to this are a pair of accessions from the USA and Canada (saffAZ and saffAC, respectively), and possibly one accession from Egypt (saff1067). Considering what we know about the history of safflower cultivation, the North American accessions (saffAZ, saffAC, and saffU) are presumably recent introductions, and from our data it appears that they may be derived from relatively divergent ancestral stocks (Fig. 2). Further investigation of the 'seven centers' hypothesis, will require the development and application of more variable markers to a much more robust sampling of the available safflower germplasm. While a recent investigation of safflower cultivars using RAPDs, ISSRs and AFLPs revealed some genetic structuring within the species [10], the authors did not address the question of whether or not the seven morphological centers of diversity correspond to genetic subgroups within safflower.

      Levels of nucleotide diversity

      The domestication of plant species is typically accompanied by a reduction in genetic diversity resulting from the population genetic bottleneck that occurs during domestication. Although results vary across species, crops generally harbor ca. two-thirds of the diversity that is present in their wild progenitors [15]. We found a similar value here, with a 20–30% (depending on the measure) reduction in nucleotide diversity in safflower as compared to C. palaestinus (Table 4). Because θW is roughly proportional to heterozygosity, we can further conclude that a randomly selected pair of safflower sequences will differ at an average of 1 out of every 303 bp (i.e., 1/0.0033 ≅ 303). This makes safflower considerably less diverse than crops such as maize (1 out of every 105 bp [16]) and sunflower (1 out of every 140 bp [17]) but far more diverse than crops such as soybean (1 out of every 1030 bp [18]).

      Conclusion

      Insights into the origin of crop plants and knowledge of the identities of their progenitors are of great value in both basic and applied research programs. For example, the comparative analysis of crop plants and their wild progenitors can shed light on the genetic mechanisms underlying organismal evolution [19, 20]. Similarly, comparative analyses of this sort can be a powerful tool for identifying genes underlying agronomically-important traits [2123]. The identification of C. palaestinus as the wild progenitor of safflower opens the door for such analyses within the genus Carthamus. Moreover, because safflower is a modern-day oilseed crop and a member of the same family as cultivated sunflower, which has been the subject of a great deal of recent study [24, 25], the initiation of such work in safflower would create a comparative framework for studying the evolution of oilseed crops within the Asteraceae.

      Methods

      Plant materials and DNA extraction

      Tissue for DNA extraction was either obtained from live plants grown from seed or from herbarium specimens (Table 1). Seeds were obtained from archived collections held at the USDA Western Regional Plant Introduction Station in Pullman, WA. These included 11 accessions of C. tinctorius L., three accessions of C. oxyacanthus and one accession of C. palaestinus. In addition Dr. R. Vilatersana (Institut Botànic de Barcelona) kindly provided seeds of C. gypsicola and C. persicus. Herbarium specimens of C. palaestinus (three accessions) and C. persicus (one accession) were provided by the IPK Gatersleben Herbarium (GAT), and C. curdicus (one accession) was provided by the Vienna Museum of Natural History (Herbarium W). All species are presumed to be diploid based on prior investigations [reviewed in [11]].

      For the live plants, seeds were clipped with a razor blade and germinated on damp filter paper in Petri dishes (48 hours dark/48 hours light). Seedlings were then planted in soil and grown in the greenhouse. Total genomic DNA was then isolated from 100 mg of leaf tissue using the DNeasy plant mini kit (Qiagen, Valencia, CA). For the herbarium extractions, tissue lysis and extraction followed the same protocol as the fresh leaf tissue except that only ca. 20 mg of leaf tissue was used.

      Locus selection and sequencing

      The seven nuclear genes used in this study (Table 2) were selected from a set of universal markers that were recently developed for use in the Asteraceae [26]. The loci selected for inclusion in this study all produced a single amplicon that could be sequenced directly (only those individuals that were heterozygous for insertions/deletions were cloned). Three of the loci (A25, A39, and B27; Table 2) did not amplify well in the herbarium material, presumably due to DNA degradation; internal primers were thus designed to amplify these loci in two overlapping segments that were later aligned into a single contig. For A39 the first portion still could not be amplified in the herbarium specimens, and hence was scored as missing data for those individuals.

      PCR was performed in a 20 μl total volume containing 20 ng of template DNA, 30 mM Tricine pH 8.4-KOH, 50 mM KCl, 2 mM MgCl2, 100 μM of each dNTP, 0.2 μM of each primer, and 2 units of Taq polymerase. Thermal cycling followed a 'touchdown' protocol, with a final annealing temperature of 50° or 55°C, as follows: (1) initial denaturing step of 3 minutes at 95°C, (2) ten cycles of 30 s denaturation at 94°C, 30 s annealing at 60° or 65°C (annealing temperature was reduced by one degree per cycle), 45 s extension time at 72°C, (3) 30 cycles of 30 s at 94°C, 30 s at 50° or 55°C, 45 s at 72°C, and (4) a final extension of 20 m at 72°C. Following PCR amplification, the presence of amplicons was confirmed via agarose gel electrophoresis.

      To prepare for DNA sequencing, 10 μl of each PCR product was incubated at 37°C for 45 m with 4 units of Exonuclease I and 0.8 units of Shrimp Alkaline Phosphatase (USB, Cleveland, OH). Enzymes were subsequently denatured by heating to 80°C for 15 minutes. Purified PCR amplicons (0.5 – 2 μl depending on approximate concentration) were then sequenced with the primers used for the initial PCR. DyeNamic (Amersham, Piscataway, NJ) or BigDye v3.1 (Applied Biosystems, Foster City, CA) chemistry was used for the sequencing following the manufacturers' protocols with minor modifications. Unincorporated dyes were removed from the sequencing reactions via Sephadex (Amersham) clean-up and sequences were resolved on a Basestation (MJ Research, San Francisco, CA) or ABI 3730xl (Applied Biosystems) automated DNA sequencer. For individuals that were heterozygous for indels at a particular locus (as evidenced by the initial sequencing chromatogram), the unpurified PCR product was cloned using the pDrive (Qiagen) or TOPO TA (Invitrogen, Carlsbad, CA) cloning vectors following the manufacturers' protocols. In order to protect against Taq errors, PCR products from five positive clones per cloning reaction were then prepared and sequenced as above, except that the T7 and M13 universal vector primers were used.

      Data analyses

      DNA sequences were edited using Chromas 2.12 (Technelysium, Helensvale, Australia). Heterozygous bases from uncloned PCR products were detected by the presence of double peaks and coded following the conventions of the International Union of Biochemistry and Molecular Biology. From these, haplotypes were resolved using the maximum likelihood algorithm PL-EM [27] within the HapAnalyzer software [28]. Sequences were aligned using Clustal W2 [29] with the default settings, followed by manual adjustments. Indels were scored as additional characters using GapCoder [30], although regions that could not be aligned unambiguously and length variants at simple-sequence repeats were excluded from the analysis. Heterozygotes were common, and alleles were kept separate for the individual gene phylogenetic analyses. For the combined dataset, however, the phase of alleles across loci could not be reliably determined. As such, pairs of cloned alleles were collapsed into a single genotype for each gene and then the seven genes were concatenated for each individual.

      Estimates of nucleotide diversity (π and θ, calculated on a per-site basis) as well as Tajima's D [31] were obtained for the three taxa with three or more samples (C. tinctorius, C. palaestinus, and C. oxyacanthus) using the software package DnaSP 4.00.5 [32, 33]. Neighbor-Joining trees were generated separately for each locus and for the combined dataset using PAUP* ver. 4.0b [34]. The combined dataset was also subjected to Maximum likelihood (ML) and Bayesian analyses. ML analysis was carried out using PHYML v2.4.4 [35] under the HKY+Γ model of molecular evolution with four substitution rate classes with 500 bootstrap replicates. Bayesian analysis was conducted with MrBayes [36] as implemented in the Geneious package (v3.0.6; Biomatters Ltd., Auckland, New Zealand). MCMC analysis was run with four chains simultaneously for 1,100,000 generations, subsampling every 200 generations. Samples prior to the generation 100,000 were treated as burn-in and discarded.

      Declarations

      Acknowledgements

      We would like to thank J. Burger and four anonymous reviewers for comments on an earlier version of the manuscript, R. Vilatersana for seeds and K. Pistrick (IPK Gatersleben) and E. Vitek (Vienna Museum of Natural History) for sending herbarium specimens. This work was supported in part by grants to JMB from the National Science Foundation (DBI-0332411) and the United States Department of Agriculture (03-35300-13104).

      Authors’ Affiliations

      (1)
      Department of Plant Biology, Miller Plant Sciences Building, University of Georgia

      References

      1. Knowles PF, Ashri A: Safflower: Carthamus tinctorius (Compositae). Evolution of Crop Plants 2nd Edition (Edited by: Smartt J, Simmonds NW). Harlow, UK , Longman 1995, 47–50.
      2. Smith JR: Safflower. Champaign, IL. , AOCS Press 1996.View Article
      3. Knowles PF: Centers of plant diversity and conservation of crop germplasm - Safflower. Economic Botany 1969,23(4):324–329.
      4. Weiss EA: Castor, Sesame and Safflower. New York, NY , Barnes and Noble, Inc. 1971.
      5. Knowles PF: Safflower. Advances in Agronomy 1958, 10:289–323.View Article
      6. Lacey DJ, Wellner N, Beaudoin F, Napier JA, Shewry PR: Secondary structure of oleosins in oil bodies isolated from seeds of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.). Biochemical Journal 1998, 334:469–477.PubMed
      7. Markley N, Nykiforuk C, Boothe J, Moloney M: Producing proteins using transgenic oilbody-oleosin technology. BioPharm International, June 2006. http://​www.​sembiosys.​ca/​Docs/​Biopharma.​pdf. [http://​www.​sembiosys.​ca/​Docs/​Biopharma.​pdf]
      8. Vilatersana R, Garnatje T, Susanna A, Garcia-Jacas N: Taxonomic problems in Carthamus (Asteraceae): RAPD markers and sectional classification. Botanical Journal of the Linnean Society 2005,147(3):375–383.View Article
      9. Vilatersana R, Susanna A, Garcia-Jacas N, Garnatje T: Generic delimitation and phylogeny of the Carduncellus-Carthamus complex (Asteraceae) based on ITS sequences. Plant Systematics and Evolution 2000, 221:89–105.View Article
      10. Sehgal D, Raina SN: Genotyping safflower (Carthamus tinctorius) cultivars by DNA fingerprints. Euphytica 2005,146(1–2):67–76.View Article
      11. Garnatje T, Garcia S, Vilatersana R, Valles J: Genome size variation in the genus Carthamus (Asteraceae, Cardueae): Systematic implications and additive changes during allopolyploidization. Annals of Botany 2006,97(3):461–467.View ArticlePubMed
      12. Lopez-Gonzalez G: Acerca de la clasificacion natural del genero Carthamus L., s.l. Anales del Jardin Botanico de Madrid 1989, 47:11–34.
      13. McPherson MA, Good AG, Topinka AKC, Hall LM: Theoretical hybridization potential of transgenic safflower (Carthamus tinctorius L.) with weedy relatives in the New World. Canadian Journal of Plant Science 2004,84(3):923–934.
      14. Ashri A, Knowles PF: Cytogenetics of Safflower (Carthamus L.) species and their hybrids. Agronomy Journal 1960,52(1):11–17.View Article
      15. Buckler IV ES, Thornsberry JM, Kresovich S: Molecular diversity, structure and domestication of grasses. Genetic Research, Camb 2001, 77:213–218.
      16. Tenaillon MI, Sawkins MC, Long AD, Gaut RL, Doebley JF, Gaut BS: Patterns of DNA sequence polymorphism along chromosome 1 of maize (Zea mays ssp. mays L.). PNAS 2001, 98:9161–9166.View ArticlePubMed
      17. Liu AZ, Burke JM: Patterns of nucleotide diversity in wild and cultivated sunflower. Genetics 2006,173(1):321–330.View ArticlePubMed
      18. Zhu YL, Song QJ, Hyten DL, Van T C.P., Matukumalli LK, Grimm DR, Hyatt SM, Fickus EW, Young ND, Cregan PB: Single-Nucleotide Polymorphisms in Soybean. Genetics 2003, 163:1123–1134.PubMed
      19. Doebley J, Stec A: Genetic analysis of the morphological differences between maize and teosinte. Genetics 1991, 129:285–295.PubMed
      20. Matsuoka Y: Origin matters: Lessons from the search for the wild ancestor of maize. Breeding Science 2005,55(4):383–390.View Article
      21. Doebley J, Stec A, Hubbard L: The evolution of apical dominance in maize. Nature 1997,386(6624):485–488.View ArticlePubMed
      22. Frary A, Nesbitt TC, Frary A, Grandillo S, van der Knaap E, Cong B, Liu J, Meller J, Elber R, Alpert KB, Tanksley SD: fw2.2: a quantitative trait locus key to the evolution of tomato fruit size. Science 2000, 289:85–88.View ArticlePubMed
      23. Li CB, Zhou AL, Sang T: Rice domestication by reducing shattering. Science 2006,311(5769):1936–1939.View ArticlePubMed
      24. Burke JM, Knapp SJ, Rieseberg LH: Genetic consequences of selection during the evolution of cultivated sunflower. Genetics 2005,171(4):1933–1940.View ArticlePubMed
      25. Burke JM, Tang S, Knapp SJ, Rieseberg LH: Genetic analysis of sunflower domestication. Genetics 2002,161(3):1257–1267.PubMed
      26. Chapman MA, Chang J, Weisman D, Kesseli RV, Burke JM: Universal markers for comparative mapping and phylogenetic analysis in the Asteraceae (Compositae). Theor Appl Genet 2007,115(6):747–755.View ArticlePubMed
      27. Qin ZHS, Niu TH, Liu JS: Partition-ligation-expectation-maximization algorithm for haplotype inference with single-nucleotide polymorphisms. American Journal of Human Genetics 2002,71(5):1242–1247.View ArticlePubMed
      28. Jung HY, Park JS, Park YJ, Kim YJ, Kimm K, Koh I: HapAnalyzer: Minimum Haplotype Analysis System for Association Studies. Genomics and Informatics 2004,2(2):107–109.
      29. Thompson JD, Higgins DG, Gibson TJ: Clustal-W - Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 1994,22(22):4673–4680.View ArticlePubMed
      30. Young ND, Healy J: GapCoder automates the use of indel characters in phylogenetic analysis. BMC Bioinformatics 2003, 4:Art. 6.View Article
      31. Tajima F: Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 1989,123(585–595):585–595.PubMed
      32. Rozas J, Rozas R: DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 1999,15(2):174–175.View ArticlePubMed
      33. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 2003,19(18):2496–2497.View ArticlePubMed
      34. Swofford DL: PAUP*. Phylogenetic analysis using parsimony (* and other methods). Vers. 4.0 Edition Sunderland, MA , Sinauer 2001.
      35. Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 2003,52(5):696–704.View ArticlePubMed
      36. Huelsenbeck JP, Ronquist F: MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 2001,17(8):754–755.View ArticlePubMed

      Copyright

      © Chapman and Burke. 2007

      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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