Development of Genomic Microsatellite Markers in Carthamus tinctorius L. (Safflower) Using Next Generation Sequencing and Assessment of Their Cross-Species Transferability and Utility for Diversity Analysis

Abstract

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.

Association Mapping for Important Agronomic Traits in Safflower (Carthamus tinctorius L.) Core Collection Using Microsatellite Markers

“…Genomic DNA was extracted from leaf tissues of 10 week-old seedlings of each accession as described by Ambreen et al ( 2015 ). Ninety-three polymorphic SSR markers developed earlier in safflower (Ambreen et al, 2015 ) were used for genotyping the accessions.…”

“…Genomic DNA was extracted from leaf tissues of 10 week-old seedlings of each accession as described by Ambreen et al ( 2015 ). Ninety-three polymorphic SSR markers developed earlier in safflower (Ambreen et al, 2015 ) were used for genotyping the accessions. A three-primers PCR protocol (Perry, 2004 ) was followed for genotyping and the reaction mixture included 50 ng of template DNA, 1.5 mM MgCl 2 , 0.2 mM of each dNTPs, 0.05 μM IR700-labeled M13 primer, 0.05 μM M13-tailed forward primer, 0.05 μM reverse primer and 0.75 units of Taq DNA polymerase (Biotools, Spain).…”

“…A primary requisite for crop improvement is the presence of molecular and phenotypic diversity and identification of genes or quantitative trait loci (QTL) governing traits of agronomic importance. Genetic diversity in safflower has been analyzed using several molecular markers viz ., Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSRs), and Single Nucleotide Polymorphism (SNPs) (Johnson et al, 2007 ; Yang et al, 2007 ; Amini et al, 2008 ; Khan et al, 2009 ; Sehgal et al, 2009 ; Chapman et al, 2010 ; Lee et al, 2014 ; Pearl and Burke, 2014 ; Ambreen et al, 2015 ; Kumar et al, 2015 ). These studies revealed high genetic differentiation among global safflower accessions and validated few “centers of similarity” of safflower that were identified based on morphological traits (Johnson et al, 2007 ; Chapman et al, 2010 ; Pearl and Burke, 2014 ; Kumar et al, 2015 ).…”

The Use of Wild Relatives of Safflower to Increase Genetic Diversity for Fatty Acid Composition and Drought Tolerance

“…and C. oxyacanthus M. Bieb. species are the wild relatives of safflower (Ashri and Knowles, 1960; Majidi and Zadhoush, 2014; Ambreen et al, 2015; Rapson et al, 2015). Cultivated safflower is crossable with both its wild relatives to produce hybrid progenies (Ashri and Knowles, 1960).…”

Morphological Characteristics of Safflower Seedlings Under Compaction Levels in a Clayey Latosol

“…It belongs to the Asteraceae family and is an annual herbaceous diploid (2n = 24) crop that adapts to hot and dry climates, with the ability to survive on minimal surface moisture (Liu et al, 2016). The plant produces edible oil rich in unsaturated fatty acids, and has a high oil content of about 44% (Ambreen et al, 2015).…”