Safflower (Carthamus tinctorius L.) is a dryland oilseed crop yielding high quality edible oil. Previous studies have described significant phenotypic variability in the crop and used geographical distribution and phenotypic trait values to develop core collections. However, the molecular diversity component was lacking in the earlier collections thereby limiting their utility in breeding programs. The present study evaluated the phenotypic variability for 12 agronomically important traits during two growing seasons (2011–12 and 2012–13) in a global reference collection of 531 safflower accessions, assessed earlier by our group for genetic diversity and population structure using AFLP markers. Significant phenotypic variation was observed for all the agronomic traits in the representative collection. Cluster analysis of phenotypic data grouped the accessions into five major clusters. Accessions from the Indian Subcontinent and America harbored maximal phenotypic variability with unique characters for a few traits. MANOVA analysis indicated significant interaction between genotypes and environment for both the seasons. Initially, six independent core collections (CC1–CC6) were developed using molecular marker and phenotypic data for two seasons through POWERCORE and MSTRAT. These collections captured the entire range of trait variability but failed to include complete genetic diversity represented in 19 clusters reported earlier through Bayesian analysis of population structure (BAPS). Therefore, we merged the three POWERCORE core collections (CC1–CC3) to generate a composite core collection, CartC1 and three MSTRAT core collections (CC4–CC6) to generate another composite core collection, CartC2. The mean difference percentage, variance difference percentage, variable rate of coefficient of variance percentage, coincidence rate of range percentage, Shannon's diversity index, and Nei's gene diversity for CartC1 were 11.2, 43.7, 132.4, 93.4, 0.47, and 0.306, respectively while the corresponding values for CartC2 were 9.3, 58.8, 124.6, 95.8, 0.46, and 0.301. Each composite core collection represented the complete range of phenotypic and genetic variability of the crop including 19 BAPS clusters. This is the first report describing development of core collections in safflower using molecular marker data with phenotypic values and geographical distribution. These core collections will facilitate identification of genetic determinants of trait variability and effective utilization of the prevalent diversity in crop improvement programs.

Carthamus tinctorius L. (safflower) is an important oilseed crop producing seed oil rich in unsaturated fatty acids. Scarcity of identified marker-trait associations is a major limitation toward development of successful marker-assisted breeding programs in safflower. In the present study, a safflower panel (CartAP) comprising 124 accessions derived from two core collections was assayed for its suitability for association mapping. Genotyping of CartAP using microsatellite markers revealed significant genetic diversity indicated by Shannon information index (H = 0.7537) and Nei's expected heterozygosity (I = 0.4432). In Principal Coordinate Analysis, the CartAP accessions were distributed homogeneously in all quadrants indicating their diverse nature. Distance-based Neighbor Joining analysis did not delineate the CartAP accessions in consonance with their geographical origin. Bayesian analysis of population structure of CartAP demonstrated the unstructured nature of the association panel. Kinship analysis at population (Gij) and individual level (Fij) revealed absence of or weak relatedness between the CartAP accessions. The above parameters established the suitability of CartAP for association mapping. We performed association mapping using phenotypic data for eight traits of agronomic value (viz., seed oil content, oleic acid, linoleic acid, plant height, number of primary branches, number of capitula per plant, 100-seed weight and days to 50% flowering) available for two growing seasons (2011–2012 and 2012–2013) through General Linear Model and Mixed Linear Model. Our study identified ninety-six significant marker-trait associations (MTAs; P < 0.05) of which, several MTAs with correlation coefficient (R2) > 10% were consistently represented in both models and in both seasons for traits viz., oil content, oleic acid content, linoleic acid content and number of primary branches. Several MTAs with high R2-values were detected either in a majority or in some environments (models and/or seasons). Many MTAs were also common between traits (viz., oleic/linoleic acid content; plant height/days to 50% flowering; number of primary branches/number of capitula per plant) that showed positive or negative correlation in their phenotypic values. The marker-trait associations identified in this study will facilitate marker-assisted breeding and identification of genetic determinants of trait variability.

Carthamus tinctorius L. (safflower) is an important oilseed crop that is cultivated in several countries. The present study investigates the genetic diversity and population structure of 531 safflower accessions from 43 countries representing all safflower growing regions of the world. Diversity analysis was performed using ten informative EcoRI/ MseI amplified fragment length polymorphism primer pairs that were identified by screening 150 primer combinations. The selected primer pairs generated 381 fragments of which 157 were polymorphic among the analyzed accessions. The genetic diversity indices obtained for the entire collection (I= 0.4536, H=0.2955) indicated high levels of molecular variability. The distance-based, neighbor-joining method classified the accessions into six clusters with internal subgroupings that were in consonance with 19 clusters obtained using Bayesian model-based BAPS analysis. Clusters obtained through STRUCTURE analysis (at K=4) could not be correlated with their geographically diverse origins, while BAPS analysis (at K=19) revealed geographical delineation with low admixture levels among most of the studied accessions. Accessions from Far East and Egypt clustered in distinct groups, indicating conserved nature of their gene pools. The Near East and Iran-Afghanistan regions were collectively found to harbor maximum diversity in accordance with earlier reports. Accessions from the Indian subcontinent showed substantial diversity that was previously undetected. The American accessions showed low molecular variability in contrast to earlier studies. Genetic sub-structuring within gene pools and interrelationships between accessions belonging to different regional pools was also observed. To the best of our knowledge, this is the first comprehensive study of existing genetic variability in a large collection of safflower germplasm with a global distribution, which provides a more accurate representation of genetic structuring in the crop. This information will facilitate selection of elite genotypes for broadening the genetic base of various breeding programs in safflower.

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BackgroundSafflower (Carthamus tinctorius L.), an Asteraceae member, yields high quality edible oil rich in unsaturated fatty acids and is resilient to dry conditions. The crop holds tremendous potential for improvement through concerted molecular breeding programs due to the availability of significant genetic and phenotypic diversity. Genomic resources that could facilitate such breeding programs remain largely underdeveloped in the crop. The present study was initiated to develop a large set of novel microsatellite markers for safflower using next generation sequencing.Principal FindingsLow throughput genome sequencing of safflower was performed using Illumina paired end technology providing ~3.5X coverage of the genome. Analysis of sequencing data allowed identification of 23,067 regions harboring perfect microsatellite loci. The safflower genome was found to be rich in dinucleotide repeats followed by tri-, tetra-, penta- and hexa-nucleotides. Primer pairs were designed for 5,716 novel microsatellite sequences with repeat length ≥ 20 bases and optimal flanking regions. A subset of 325 microsatellite loci was tested for amplification, of which 294 loci produced robust amplification. The validated primers were used for assessment of 23 safflower accessions belonging to diverse agro-climatic zones of the world leading to identification of 93 polymorphic primers (31.6%). The numbers of observed alleles at each locus ranged from two to four and mean polymorphism information content was found to be 0.3075. The polymorphic primers were tested for cross-species transferability on nine wild relatives of cultivated safflower. All primers except one showed amplification in at least two wild species while 25 primers amplified across all the nine species. The UPGMA dendrogram clustered C. tinctorius accessions and wild species separately into two major groups. The proposed progenitor species of safflower, C. oxyacantha and C. palaestinus were genetically closer to cultivated safflower and formed a distinct cluster. The cluster analysis also distinguished diploid and tetraploid wild species of safflower.ConclusionNext generation sequencing of safflower genome generated a large set of microsatellite markers. The novel markers developed in this study will add to the existing repertoire of markers and can be used for diversity analysis, synteny studies, construction of linkage maps and marker-assisted selection.

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Genetic diversity provides the foundation for plant breeding and genetic research. Over 3000 rice genomes were recently sequenced as part of the 3K Rice Genome (3KRG) Project. We added four additional Indian rice accessions to create a panel of 3004 accessions. However, such a large collection of germplasm is difficult to preserve and evaluate. The construction of core and mini-core collections is an efficient method for the management of genetic resources. In this study, we developed a mini-core comprising 520 accessions that captured most of the SNPs and represented all of the phenotypes and geographic regions from the original panel. The mini-core was validated using different statistical analyses and contained representatives from all major rice groups, including japonica , indica , aus / boro , and aromatic/basmati. Genome-wide association analyses of the mini-core panel efficiently reproduced the marker–trait associations identified in the original panel. Haplotype analysis validated the utility of the mini-core panel. In the current era with many ongoing large-scale sequencing projects, such a strategy for mini-core design should be useful in many crops. The rice mini-core collection developed in this study would be valuable for agronomic trait evaluation and useful for rice improvement via marker-assisted molecular breeding.

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