ASSESSMENT OF GENETIC DIVERSITY AND OUTCROSSING IN SAFFLOWER (Carthamus tinctorius L.) VARIETIES USING SSR MARKERS

Safflower (Carthamus tinctorius L.) is a member of Asteraceae family originated in the Middle Eastern region. The cultivated safflower is a diploid with 24 chromosomes and genome size of about 1.4 Gb. Understanding of genetic diversity in the safflower germplasm accessions is critical for conservation, maintenance and selection of appropriate accessions for use in breeding activities. In this study, genetic diversity and relatedness of 30 Mexican safflower varieties were compared with 30 public bred Indian cultivars using 50 SSR markers. The genetic diversity measures viz., allele number, major allele frequency, observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC) and the population parameters viz., population structure, genetic admixture, analysis of molecular variance (AMOVA) and fixation index (Fst) were analyzed across 60 genotypes. The allele number ranged from 1 to 10 with the mean of 2.7 per locus. The major allele frequency ranged from 0.386 to 1 with the mean of 0.74. The observed heterozygosity (Ho) ranged from 0 to 1 with the mean of 0.088. The expected heterozygosity (He) ranged from 0 to 0.768 with the mean of 0.332. The PIC values for each SSR primer pair ranged from 0 to 0.751 with the mean of 0.286. The low average number of alleles, high major allele frequency and low average PIC values across 60 accessions suggested that low level of SSR polymorphism in the safflower cultivars. Neighbor joining (NJ) tree based on pair-wise simple matching coefficients clearly placed Indian and Mexican accessions in two distinct clusters. NJ tree also revealed several subgroups within Mexico and India groups. The model based STRUCTURE analysis also showed two subpopulations (K=2) clearly thus grouping the Indian and Mexican cultivars into distinct clusters. However, it was unclear to what extent the two varietal groups were genetically differentiated. Partitioning of variation through hierarchical AMOVA showed that about 40% of variation is explained between populations and 60% between individuals within population. The pair-wise Fst estimate between Mexico and India groups was 0.39639. These results indicated that both India and Mexico varietal groups are highly differentiated and a substantial genetic diversity exists between them, which can be used for generating new variability for improving agronomic traits in safflower breeding programmes. In this study, an effort was also made to check outcrossing between nine safflower genotypes grown under honeybee excluded and protected net condition using SSR markers. Previously generated genotypic data of 60 genotypes were used to identify a single or a combination of SSR markers which could uniquely differentiate one genotype from the other. Five hundred plant progenies of the variety A1 (of the seeds collected from protected net field) grown in grow-out test (GOT) plot were genotyped using A1 specific SSR primer pair ct-266. The ct-266 allelic pattern of progenies was examined to check if any progeny that showed heterozygous allelic pattern (carrying A1 allele + allele from any parental genotype grown in the outcross block). It was observed that 496 progenies had the allelic pattern of A1. Only four progenies had allelic pattern that was different from A1; the pattern was not heterozygous but was similar to that of two parental genotypes; hence, these plants were considered as contaminants. Thus, the genotyping results showed that there was no evidence of outcrossing between A1 and other safflower genotypes under honeybee excluded protected net condition. Genotyping of 500 A1 progenies (of the seeds collected from open field) was also done using A1 specific SSR primer pair ct-266. All the 500 plants showed the allelic pattern of A1 suggesting that outcrossing did not happen between A1 and other genotypes even under open field conditions. The results have strengthened the observations of safflower breeders that out-crossing may not happen in safflower in the absence of honeybees. However, the results are only indicative and not conclusive because of limited progeny size and genotypes used. Therefore, the findings need further validation using larger progeny across genotypes. LIST OF ABBREVIATIONS AND SYMBOLS RFLP : Restriction Fragment Length Polymorphism RAPD : Randomly Amplified Polymorphic DNA SSR : Simple Sequence Repeats STS : Sequence Tagged Sites SCAR : Sequence Characterised Amplified Regions CAPS : Cleaved Amplified Polymorphic Sequences ISSR : Inter Simple Sequence Repeats AFLP : Amplified Fragment Length Polymorphism ANGRAU : Acharya N.G. Ranga Agricultural University AMOVA : Analysis of Molecular Variance CTAB : Cetyl Trimethyl Ammonium Bromide EDTA : Ethylene Diamine Tetra Acetic Acid TRIS : Tris (hydroxyl methyl) amino methane PVP : Polyvinylpyrrolidone TAE : Tris Acetate EDTA TBE : Tris Boric acid EDTA PCR : Polymerase Chain Reaction dNTP : Deoxynucleotide Triphosphate Taq : Thermus aquaticus PIC : Polymorphism Information Content Cm : Centi meter et al. : and others g : Gram mm : Milli meter viz. : Namely μl : Micro litres μg : Micro gram % : Percent UV : Ultra violet H2O : Water mM : Micro molar ng : Nano gram oC : Degree Celsius bp : Base Pairs PAGE : Poly Acrylamide Gel Electrophoresis W/v : Weight per volume W/w : Weight per weight V : Volts Fst : Fisher’s statistic DOR : Directorate of Oilseeds Research GOT : Grow-out Test ICRISAT : International Crops Research Institute for Semi-Arid Tropics AICRP : All India Coordinated Crop Research Project