Chloroplast Genomes and Phylogenetic Analysis of Three Carthamus (Asteraceae) Species.

The genus Carthamus Linnaeus, which belongs to the tribe Cardueae in the Asteraceae family, originated in the Mediterranean region and consists of approximately 20 species worldwide. Understanding the phylogeny of the Carthamus is crucial for the cultivation of C. tinctorius. Although chloroplast genomes are widely used for species identification and evolutionary studies, there have been limited investigations on the chloroplast genomes of Carthamus species. In this study, we assembled the chloroplast genomes of C. persicus, C. tinctorius × C. persicus, and C. lanatus and combined them with the five chloroplast genomes of C. tinctorius for comparative genomic analysis. The sizes of the chloroplast genomes of C. lanatus, C. persicus, and C. tinctorius × C. persicus were 152,602 bp, 153,177 bp, and 153,177 bp, respectively. Comparative analysis showed that the chloroplast genome structures of the four Carthamus species were highly conserved. Additionally, the phylogenomic analysis demonstrated that the plastid genome and angiosperms353 dataset significantly improved the phylogenetic support of Carthamus species. This analysis supported Carthamus as a monophyletic taxon and its internal division into the sect. Carthamus and sect. Atractylis. The Carthamus was closely related to Carduncellus, Femeniasia, Phonus, and Centaurea. In conclusion, this study not only expands our understanding of the cp genomes of Carthamus species but also provides support for more comprehensive phylogenetic studies of Carthamus.

Assessment of genetic diversity and population genetic structure of Carthamus species and Iranian cultivar collection using developed SSR markers.

This experiment was conducted to assess genetic relationships among safflower genotypes from different geographical regions of Iran and other countries using newly developed simple sequence repeat (SSR) markers. By enrichment method, 32 primer pairs were designed of which 18 pairs were able to detect polymorphism in 105 safflower cultivars from Carthamus tinctorius, C. oxyacanthus, C. lanatus, C. glaucus, C. boissieri and C. dentatus. The selected SSR primers amplified a total of 59 alleles with an average of 3.27 alleles per locus among the cultivars and the average values of gene diversity, heterozygosity and PIC were 0.45, 0.37 and 0.39, respectively. Neighbour-joining cluster analysis based on Nei's genetic distance categorized populations of Carthamus in six major clusters; all wild accessions were grouped differently from cultivated genotypes. Cluster analysis significantly distinguished C. oxyacanthus genotypes in different categories: centre (Arak), northeast (Azarbaiejan), east (Kermanshah) and southeast (Shiraz, Chaharmahal and Kohgiluyeh). The presence of C. boissieri and C. glaucus in one cluster appeared to be in close relationship between each other, indicating a common ancestor. The results revealed that C. dentatus discriminated from the species with n = 10 chromosomes, C. boissieri and C. glaucus assigned in to separate subsection. In summary, this study has shown that domesticated and wild genotypes were clustered into two major groups indicating these markers as appropriate tools to amassment genetic diversity and genome mapping.

Differential spreading of HinfI satellite DNA variants during radiation in Centaureinae.

BACKGROUND AND AIMS: Subtribe Centaureinae appears to be an excellent model group in which to analyse satellite DNA and assess the influence that the biology and/or the evolution of different lineages have had on the evolution of this class of repetitive DNA. Phylogenetic analyses of Centaureinae support two main phases of radiation, leading to two major groups of genera of different ages. Furthermore, different modes of evolution are observed in different lineages, reflected by morphology and DNA sequences. METHODS: The sequences of 502 repeat units of the HinfI satellite DNA family from 38 species belonging to ten genera of Centaureinae were isolated and compared. A phylogenetic reconstruction was carried out by maximum likelihood and Bayesian inference. KEY RESULTS: Up to eight different HinfI subfamilies were found, based on the presence of a set of diagnostic positions given by a specific mutation shared by all the sequences of one group. Subfamilies V-VIII were mostly found in older genera (first phase of radiation in the subtribe, late Oligocene-Miocene), although some copies of these types of repeats were also found in some species of the derived genera. Subfamilies I-IV spread mostly in species of the derived clade (second phase of radiation, Pliocene to Pleistocene), although repeats of these subfamilies exist in older species. Phylogenetic trees did not group the repeats by taxonomic affinity, but sequences were grouped by subfamily provenance. Concerted evolution was observed in HinfI subfamilies spread in older genera, whereas no genetic differentiation was found between species, and several subfamilies even coexist within the same species, in recently radiated groups or in groups with a history of recurrent hybridization of lineages. CONCLUSIONS: The results suggest that the eight HinfI subfamilies were present in the common ancestor of Centaureinae and that each spread differentially in different genera during the two main phases of radiation following the library model of satellite DNA evolution. Additionally, differential speciation pathways gave rise to differential patterns of sequence evolution in different lineages. Thus, the evolutionary history of each group of Centaureinae is reflected in HinfI satellite DNA evolution. The data reinforce the value of satellite DNA sequences as markers of evolutionary processes.

Species-genomic relationships among the tribasic diploid and polyploid Carthamus taxa based on physical mapping of active and inactive 18S-5.8S-26S and 5S ribosomal RNA gene families, and the two tandemly repeated DNA sequences.

In the genus Carthamus (2n=20, 22, 24, 44, 64; x=10, 11, 12), most of the homologues within and between the chromosome complements are difficult to be identified. In the present work, we used fluorescent in situ hybridisation (FISH) to determine the chromosome distribution of the two rRNA gene families, and the two isolated repeated DNA sequences in the 14 Carthamus taxa. The distinctive variability in the distribution, number and signal intensity of hybridisation sites for 18S-26S and 5S rDNA loci could generally distinguish the 14 Carthamus taxa. Active 18S-26S rDNA sites were generally associated with NOR loci on the nucleolar chromosomes. The two A genome taxa, C. glaucus ssp. anatolicus and C. boissieri with 2n=20, and the two botanical varieties of B genome C. tinctorius (2n=24) had diagnostic FISH patterns. The present results support the origin of C. tinctorius from C. palaestinus. FISH patterns of C. arborescens vis-à-vis the other taxa indicate a clear division of Carthamus taxa into two distinct lineages. Comparative distribution and intensity pattern of 18S-26S rDNA sites could distinguish each of the tetraploid and hexaploid taxa. The present results indicate that C. boissieri (2n=20) is one of the genome donors for C. lanatus and C. lanatus ssp. lanatus (2n=44), and C. lanatus is one of the progenitors for the hexaploid (2n=64) taxa. The association of pCtKpnI-2 repeated sequence with rRNA gene cluster (orphon) in 2-10 nucleolar and non-nucleolar chromosomes and the consistent occurrence of pCtKpnI-1 repeated sequence at the subtelomeric region in all the taxa analysed indicate some functional role of these sequences.

Sequence analysis of KpnI repeat sequences to revisit the phylogeny of the Genus Carthamus L.

Repetitive DNA sequences constitute a significant proportion of eukaryotic genomes. Knowledge about the distribution of repetitive DNA sequences is necessary in order to gain insights into the organization, evolution and behavior of eukaryotic genomes. Therefore, we used two repetitive DNA sequences pCtKpnI-I and pCtKpnI-II, earlier reported in Carthamus tinctorius L. to study the phylogeny and to revise the taxonomic status of the taxa belonging to the genus. The study unraveled two major lines within the genus Carthamus; one line included all the diploid taxa (2n = 24) and the other line comprised the taxa with 2n = 20 and the polyploid taxa (2n = 44 and 64). The results of the present study will prove useful in molecular breeding for improving some targeted agronomic traits in genus Carthamus.

Development of molecular markers and linkage maps for the Carthamus species C. tinctorius and C. oxyacanthus.

A set of SSR and RFLP markers for safflower (Carthamus tinctorius) and jeweled distaff thistle (C. oxyacanthus) was generated from cDNA and genomic libraries and by mining public and proprietary sequence databases. In total, 1412 PCR-based markers and 75 RFLP markers were screened and polymorphic loci were mapped in an intraspecific F2 population of C. tinctorius and an interspecific BC1 population of C. tinctorius x C. oxyacanthus. The two populations shared one common parent and the resulting linkage maps could be compared for synteny. The level of polymorphism was low in both populations and only 8.2% and 13.7% of the analyzed markers could be mapped in the intraspecific and interspecific maps, respectively. The two maps showed significant colinearity of markers in several regions and an apparent translocation or inversion event on one linkage group. Noticeable segregation distortion was found on one linkage group of the C. tinctorius map and dense clustering of loci occurred on several linkage groups of the C. oxyacanthus map. The two maps represent the first major linkage analysis of Carthamus species. The molecular tools will be useful for a variety of genetic and genomic applications in safflower and its related species and have been used in our laboratory to map a flower color gene in C. tinctorius.

Nuclear DNA assay in solving issues related to ancestry of the domesticated diploid safflower (Carthamus tinctorius L.) and the polyploid (Carthamus) taxa, and phylogenetic and genomic relationships in the genus Carthamus L. (Asteraceae).

The multipronged nuclear DNA assay by random amplified polymorphic DNA (RAPD) fingerprinting, ribosomal DNA repeat unit length polymorphism, internal transcribed sequence (ITS) RFLP, and comparative sequence analysis of ITS and external transcribed sequence (ETS) regions of the 29 accessions belonging to 18 Carthamus taxa including five unverified species was undertaken to obtain new information on (1) interrelationships among botanical varieties of cultivated safflower, C. tinctorius, and phylogenetic relationships (2) among the safflower and its close relatives and (3) that of Carthamus species and subspecies. The root tip cells of the 12 accessions contained 24 chromosomes followed by 64, 44 and 20 chromosomes in 9, 6 and 2 accessions, respectively. Barring C. lanatus, the accessions within each taxon had the same zygotic number. The present results strongly support the view that the wild C. palaestinus (2n=24) and the cultivated C. tinctorius (2n=24) are closely related. With few exceptions, all DNA based dendrograms support three lineages within the genus. One lineage is constituted by C. arborescens (2n=24) alone. The present data indicates that because of unique composition of its nuclear genome vis-à-vis other Carthamus taxa, C. arborescens should be placed in a separate subgenus. The two remaining lineages, constituted by the taxa with 2n=24, and the taxa with 2n=20, 2n=44 and 2n=64, respectively should be given the rank of two taxonomic sections in the other subgenus. The present study also demonstrates that none of the present taxa with 2n=24 have contributed to the origin of polyploid taxa. Carthamus boisserii (2n=20) and C. glaucus ssp. anatolicus (2n=20) are more likely to be one of the diploid progenitor of C. lanatus ssp. creticus (2n=64), C. lanatus (2n=44), C. lanatus ssp. lanatus (2n=44) and C. lanatus ssp. montanus (2n=44), and C. lanatus ssp. turkestanicus (2n=64), respectively. Within Lanatus species complex, constituted by C. lanatus, C. lanatus ssp. lanatus, C. lanatus ssp. montanus, C. lanatus ssp. turkestanicus and C. lanatus ssp. creticus, high proportion of autapomorphic characters and low number of synapomorphies in the ITS and ETS sequences suggest a relatively recent diversification of the taxa within the species complex. Carthamus lanatus ssp. creticus (2n=64) and C. lanatus ssp. turkestanicus (2n=64) within the complex deserve species rank. This analysis provided evolutionary relatedness of the five unverified taxa with the known Carthamus taxa.

Phylogenetic analysis of Carthamus species based on the nucleotide sequence of the nuclear SACPD gene and chloroplast trnL-trnF IGS region.

To clarify the phylogenetic relationships of Carthamus species, we performed sequence analysis of the nuclear stearoyl acyl carrier protein desaturase (SACPD) gene and the chloroplast intergenic spacer region between leucine and phenylalanine tRNA genes (trnL-trnF IGS) in 13 taxa of Carthamus. The previous division of the genus into 4 taxonomic sections and allocation of particular genomes to various taxa on the basis of morphological, cytogenetic, and biosystematic analyses is not supported by the present study. Our results provide evidence of the occurrence of 5 nuclear genomes (A, B, C, X, and Y) and 3 cytoplasm types (A, B, and C) in the genus Carthamus. The cultivated safflower, C. tinctorius (2n = 24), has the B genome and type B cytoplasm. Both of these are not present in the polyploid taxa. This contradicts the earlier view that one of the genomes involved in the origin of the polyploid taxa of Carthamus is the B genome. Comparison with an outgroup species (Cirsium japonicum) indicated that C. arborescens is the most primitive species in the genus. Carthamus palaestinus is genetically closest to the cultivated safflower.

Molecular evidence for hybrid origins of the invasive polyploids Carthamus creticus and C. turkestanicus (Cardueae, Asteraceae).

The hexaploids Carthamus creticus and C. turkestanicus are noxious weeds with wide but non-overlapping Mediterranean distributions, and C. creticus, together with another polyploid, C. lanatus, have also invaded similar climatic regions in North and South America, South Africa and Australia. Here we infer their origins using sequences of the plastid intergenic spacer trnH-psbA and the intron trnK and three introns of nuclear low-copy genes of the RNA Polymerase family (RPD2 and the duplicated RPC2), as well as RAPD markers (random amplified polymorphic DNA). Phylogenetic analyses of the nuclear introns and additivity analysis of the RAPD markers support the hypotheses that the two hexaploids are allopolyploids sharing a tetraploid progenitor lineage represented by the broadly Mediterranean C. lanatus, combined with different diploid progenitor lineages consistent with the different geographic distributions of the hexaploids. Whereas C. leucocaulos from the south-eastern Greek Islands represents the diploid progenitor lineage of the western C. creticus, the Irano-Turanian C. glaucus represents the diploid progenitor lineage of the eastern C. turkestanicus. The plastid data suggest that the diploid lineages served as the maternal progenitors of the hexaploids.