Journal of Cereal Science 97 (2021) 103149 Available online 3 December 2020 0733-5210/© 2020 Published by Elsevier Ltd. Short communication: Finetuning molecular markers for effcient selection of vitamin A-rich tropical maize lines in a molecular breeding scheme Deborah Babalola, Abebe Menkir, Oluyinka Ilesanmi, Queen Obi, Melaku Gedil * International Institute of Tropical Agriculture, Oyo Road, PMB, 5320, Ibadan, Nigeria A R T I C L E INFO Keywords: Biofortifcation Sequence characterization Pro-vitamin A Marker effciency Maize (Zea mays) is an important cereal crop grown in 86% of sub- Saharan Africa (SSA) countries, constituting a staple food for over 50% of the population (Badu-Apraku and Fakorede, 2017). Incidentally, SSA is a region plagued by vitamin A defciency (48%) (Stevens et al., 2015). The genetic variation for carotenoid components and allelic di- versity of carotenoid biosynthesis genes has positioned maize Pro-Vitamin A (PVA) biofortifcation as an effective and sustainable approach to combat Vitamin A defciency. To fast-track this process, molecular markers have been developed based on polymorphisms around three signifcant genes Phytoene synthase1 (psy1), lycopene epsilon cyclase (lcyE) and β-carotene hydroxylase1 (crtRB1)]. However, studies to validate these markers have shown inconsistent predictive ability (Azmach et al., 2013; Yang et al., 2018). The need to explain the factors underlying these inconsistencies and improve the effciencies of the markers for routine utilization in tropical maize biofortifcation programs prompted this study. The approach includes: (a) character- ization of the partial sequence of the three essential genes in tropical maize lines and, (b) to redesign and validate primers based on known polymorphism to select for high PVA. The samples used in this study included 132 yellow maize inbred lines out of which 130 are as described in Azmach et al. (2013), including their carotenoid content. Multiple alignments constructed from nucleotide sequences of each of the three known PVA genes aided new primer design and subsequent sequencing of regions of interest (Fig. 1) on Applied Biosystems 3500 Series Genetic Analyzer system (Applied Bio-system, United States). PCR products were sequenced in both strands. Sequences were analyzed and annotated using CodonCode Aligner (LI-COR, Inc. USA) and CLC Main Workbench sequence analysis software. Entry ID of sequences submitted to GenBank: MH580885, MH580886, MH580887, MH580888, MH580889, MH580890, MH580891, MH580892, MH580893, MH580894, MH580895. In psy1, sequenced region span 58% of the gene (Fig. 1). Identifed polymorphism include a 3bp indel and CCA-repeat ranging from 10 to 15 just before the potential translation start while Fu et al. (2013) identifed at least 9 polymorphisms. Irrespective of their PVA content, favourable haplotype comprising 378bp deletion in 5 ′ UTR and ‘A ′ nucleotide (SNP7) in the 4th exon co-occurred in all samples. This region was also sequenced in 66 additional samples and found to be conserved. The low sequence variation may be due to the selection for the favour- able allele overtime. Multiple functional alleles are present for psy1 based on which PCR-based markers enables high carotenoid content selection in maize. However, the associated total carotenoid content ranged from 6 to 17.278μg/g, which is substantially low for the current breeding target (Orlovskaya et al., 2018; Fu et al., 2013). Sequencing of the whole lcyE gene, including 1755bp upstream and 931bp downstream, identifed 12 InDels and 80 SNPs (Fig. 2), excluding polymorphic sites that only differentiate the reference. Following asso- ciation analysis, there was no statistically signifcant association found below the signifcant thresholds (1.1E04 and 5.4E04 at 1% and 5%, respectively, after Bonferroni multiple test correction-TASSEL user’s guides 2011). At the least p value-9.2E03, several SNPs weakly associ- ated with the ratio of carotenoid in the β-branch to the α-branch. Studies, including GWAS and expression analysis, have pointed out lcyE as in- clusive in genomic regions underlying PVA accumulation in maize (Azmach et al., 2018; Suwarmo et al., 2015). Phylogenetic analysis grouping based on the entire variation across the lcyE gene showed a * Corresponding author. E-mail address: M.Gedil@cgiar.org (M. Gedil). Contents lists available at ScienceDirect Journal of Cereal Science journal homepage: http://www.elsevier.com/locate/jcs https://doi.org/10.1016/j.jcs.2020.103149 Received 23 July 2020; Received in revised form 30 November 2020; Accepted 2 December 2020