Investigation of Morpho-agronomic Performance, Genetic Diversity and Similarity Centers Exploration in International Safflower Panel

Abstract

Safflower has tremendous potential for various purposes; still area under safflower cultivation is limited. Safflower remains underutilized or neglected crop species due to low seed oil content, spininess and susceptibility to different diseases and pests attack. To reduce all such hampering factors and improve safflower productivity, we need an extensive investigation of the genetic diversity at different levels to identify germplasm containing novel alleles. During current Ph.D thesis, an international safflower panel was investigated for its morpho-agronomic performance conducting field experiments at two diverse locations (Pakistan and Turkey). Genetic diversity, population structure, and similarity centers were explored utilizing three molecular marker systems (iPBS-retrotransposon, ISSR and silicoDArT), while marker-trait associations were identified with silicoDArT markers. Safflower accessions provided by Plant Genetic Resources Institute, Pakistan (17 accessions) and Central Research Institute for Field Crop, Turkey (20 accessions) were also included along with international safflower panel (94 accessions) in iPBS retrotransposon and ISSR studies. The planned aspects were analyzed in a systematic manner to achieve these goals. The first study was conducted focusing the importance of the genetic diversity for crop improvement. The genetic variability that existed among and within populations for desirable agronomic traits can be used to develop elite cultivars. A total of 94 safflower accessions from 26 different countries were used in this study to evaluate morpho-agronomic performance, pattern of similarity centers and identification of best performing accessions by conducting two field experiments in two different geographical locations (Pakistan and Turkey) using augmented design. Genetic diversity for important yield and yield related traits was described including seed yield per plant (ranged from 4.86 to 51.02g), capitulum diameter (ranged from 17.30 to 28.30mm), branches per plant (ranged from 5.10 to 17.30) and capitula per plant (ranged from 8.70 to 80.40), and showed a good level of variation along with other studied traits in the current evaluation. Using the principal component analysis, it was observed that days to flower initiation, days to 50% flowering, days to flower completion, seed yield per plant, capitula per plant, branches per plant, seeds per capitulum and capitulum diameter were the major contributors to the observed genetic variability in the evaluated safflower panel. Seed yield per plant reflected a significant viii and positive correlation with capitula per plant, branches per plant and capitulum diameter, and these traits can be suggested as a selection criterion in safflower breeding programs. The constellation plot and multivariate analysis was in agreement with the patterns of seven similarity centers based on seed yield per plant, capitula per plant, capitulum diameter, and branches per plant. The second study was aimed to investigate the genetic diversity and population structure of 131 safflower accessions using 13 iPBS-retrotransposon markers. A total of 295 iPBS bands were observed among which 275 (93.22%) were found polymorphic. Mean Polymorphism information content (0.48) and diversity parameters including mean effective number of alleles (1.33), mean Shannon’s information index (0.33), overall gene diversity (0.19), Fstatistic (0.21), and inbreeding coefficient (1.00) reflected the presence of sufficient amount of genetic diversity in the studied plant materials. Analysis of molecular variance (AMOVA) showed that more than 40% of genetic variation was derived from populations. Model-based structure, principal coordinate analysis (PCoA) and unweighted pair group method with arithmetic means (UPGMA) algorithms clustered the 131 safflower accessions into four main populations A, B, C, D and an unclassified population, with no meaningful geographical origin. Most diverse accessions originated from Asian countries including; Afghanistan, Pakistan, China, Turkey, and India. Four accessions; Turkey3, Afghanistan4, Afghanistan2, and Pakistan24 were found most genetically distant. The findings of this study are most probably supported by the seven similarity centers hypothesis of safflower. The third study was conducted to investigate genetic diversity, population structure and similarity centers pattern for 131 safflower accessions using 12 ISSR markers. A sum of 201 ISSR bands were obtained among which 188 (93.844%) were found polymorphic. Mean Polymorphism information content (0.448) and diversity parameters including mean effective number of alleles (1.655), mean Shannon’s information index (0.557), mean expected heterozygosity (0.354), and mean overall gene diversity (0.377) showed a good level of genetic diversity in the studied safflower materials. Model-based structure, unweighted pair-group method with arithmetic means (UPGMA), and principal coordinate analysis (PCoA) clustered all accessions into three main populations; A, B, C and an unclassified population. Accessions originated from Asian countries like Pakistan and Israel were found most ix diverse. Three accessions; Pakistan11, Israel1, and Pakistan26 were found most genetically distant. Analysis of molecular variance (AMOVA) revealed highly significant differentiation among the identified populations, and population × country combinations. The results presented in this work most probably supported the hypothesis of seven similarity centers of safflower. The fourth study was performed to explore genetic diversity, similarity centers pattern, and marker trait associations of the 94 safflower accessions with DArTseq generated silicoDArT markers. Mean Diversity parameters including; observed number of alleles (1.99), effective number of alleles (1.54), Shannon’s information index (0.48), expected heterozygosity (0.32), and unbiased expected heterozygosity (0.32) for the entire population reflected the presence of sufficient amount of genetic diversity in the international safflower panel using 12232 silicoDArT markers. Analysis of molecular variance (AMOVA) revealed that most of the variations (91%) in world safflower panel are due to differences within country groups. Model-based structure, Neighbor Joining algorithms, and principal coordinate analysis (PCoA) clustered the 94 safflower accessions into two populations representing meaningful heterotic groups for breeding purposes. Asian countries including; Egypt, India and Turkey exhibited the most diverse accessions in the available safflower panel. Three accessions; Egypt-5, Egypt-2, and India-2 were found most genetically distant. The 51.17% kinship coefficient ranged from -0.4 to 0, while 4.99% of the kinship coefficient ranged from 0.6 to 1, respectively in the international safflower panel. Current results supported the hypothesis of seven similarity centers for safflower throughout the world. Our study identified five significant marker-trait associations for traits viz., capitula per plant, 100-seed weight, plant height, seeds per capitulum, and seed yield per plant. This is a pioneering study involving the comprehensive investigation of genetic diversity and similarity centers pattern of safflower at morpho-agronomic and molecular level. A new selection criteria was devised that can be implement to select best performing accessions in safflower breeding programs. Genetic diversity and population structure with iPBS-retrotransposon and marker-trait associations with silicoDArT markers were elucidated for the first time in safflower upto the best of our knowledge .We envisage that this study will be very helpful for global safflower x breeding community in order to develop cultivars with higher morpho-agronomic performance.