Evolutionary divergence in Chenopodium and validation of SNPs in chloroplast rbcL and matk genes by allele-specific PCR for development of Chenopodium quinoa-specific markers

The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbcL and matK gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825, which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbcL gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68% interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the matK gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbcL and matK regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR, designed from rbcL sequences, that could differentiate quinoa from 64 genera including 13 species of the genus Chenopodium. With an estimated age of 10.5-4.1 million years (Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.