Exploring Genetic Diversity: Optimizing Simple Sequence Repeat (SSR) Markers in Crotalaria for Enhanced Precision in...

Crotalaria is a plant genus with more than 700 species of shrubs and herbs. Despite its potential economic importance, Crotalaria has received limited research attention; hence, there is limited information on its genetic diversity. Hence, there is need to establish its genetic diversity as a foundation for its conservation and breeding. The current study aimed to optimize and validate simple sequence repeat (SSR) markers polymerase chain reaction-conditions for the assessment of genetic diversity in Crotalaria. The genomic DNA of 31 Crotalaria accessions was extracted from 2-week-old leaves using a modified CTAB protocol and Quick-DNA Plant/Seed Kits (Zymo Research Corp) were used for recalcitrant samples. The samples were then amplified using the 29 SSR markers under the optimized conditions. The polymorphism information content (PIC) of the polymorphic markers was calculated to determine their effectiveness. This study determined that the optimal concentrations of dNTPs, MgCl₂, and primers as 2.5, 2, and 5 mM, respectively, and the quantity of the DNA template was 1 μL, and the quantity of Taq was 0.125 μL in a 25 μL reaction mixture. The mean PIC value was 0.233, which shows that the markers were slightly informative. The marker PC004 was the most informative marker with the highest PIC value (0.605) and it detected the largest number of alleles despite being a hexanucleotide motif repeat. Its uniqueness augments its potential use in the assessment of genetic diversity. This study implies that the SSR markers designed and optimized for the study are significant for genetic diversity and population structure analysis of Crotalaria species and molecular verification of Crotalaria genotypes as well as other related genera. Besides, the results of the study form a basis for genetic improvement of Crotalaria.