Abstract
Cassava is a widely grown staple in Sub-Saharan Africa and consumed as a cheap source of calories, but the crop is deficient in micronutrients including pro-vitamin A carotenoids. This challenge is currently being addressed through biofortification breeding that relies on phenotypic selection. Gene-based markers linked to pro-vitamin A content variation are expected to increase the rate of genetic gain for this critical trait. We sequenced four candidate carotenoid genes from 167 cassava accessions representing the diversity of elite breeder lines from IITA. Total carotenoid content was determined using spectrophotometer and total β-carotene was quantified by high-performance liquid chromatography. Storage root yellowness due to carotenoid pigmentation was assessed. We carried out candidate gene association analysis that accounts for population structure and kinship using genome-wide single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing. Significant SNPs were used to design competitive allele-specific PCR assays and validated on the larger population for potential use in marker-assisted selection breeding. Candidate gene sequencing of the genes β-carotene hydroxylase (crtRB), phytoene synthase (PSY2), lycopene epsilon cyclase (lcyE), and lycopene beta cyclase (lcyB) yielded a total of 37 SNPs. Total carotenoid content, total β-carotene, and color parameters were significantly associated with markers in the PSY2 gene. The SNPs from lcyE were significantly associated with color while those of lcyB and crtRB were not significantly associated with carotenoids or color parameters. These validated and breeder-friendly markers have potential to enhance the efficiency of selection for high β-carotene cassava, thus accelerating genetic gain.
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Acknowledgements
We acknowledge the support of Ruth Uwugiaren and Tessema Gezahegn in the laboratory work and Andrew Ikpan and the staff of the Cassava Breeding Unit of IITA for conducting field trials. The Next Generation Cassava Breeding project (www.nextgencassava.org) is appreciated for providing the genome-wide information on the SNPs of the population used in this study. This research was part of a PhD project supported by the HarvestPlus Project and the CGIAR Research Program on Roots, Tubers, and Bananas (CRP-RTB).
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LU conducted the field and laboratory experiments, carried out statistical analysis, and prepared the draft manuscript. MG supervised the laboratory work and revised the manuscript. EP and PK contributed in securing funds for the project and revised the manuscript. AA contributed to supervision of the work and revised the manuscript. CN contributed to the laboratory and field work and revised the manuscript. IR conceived and led the project, took part in statistical analysis, and contributed to drafting the manuscript.
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The datasets generated during and/or analyzed during the current study are available at www.cassavabase.org, ftp://ftp.cassavabase.org/manuscripts/MolecularBreeding_Udoh_et_al_2017.zip.
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Electronic supplementary material
Supplementary Table 1
Sequences of PSY2 and lcyE SNP used for competitive allele specific PCR marker development (DOCX 51.5 KB).
Supplementary Table 2
Summary of the sequenced candidate genes, uncovered SNPs and resultant amino acid changes (DOCX 84.5 KB).
Supplementary Fig. 1
Simplified diagram of the carotenoid biosynthetic pathway in plants, adapted from (Ampomah-Dwamena et al. 2009). The candidate genes sequenced in the present study are denoted in red fonts. PSY2, phytoene synthase; PDS, phytoene desaturase; Z-ISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerase; crtRB, β-carotene hydroxylase; lcyE, lycopene epsilon cyclase; lcyB, lycopene beta cyclase; crtRB, β-carotene hydroxylase (PDF 34.5 KB).
Supplementary Fig. 2
Distribution of carotenoid estimates. (a) yellow color intensity as estimated using chromameter b* reading; (b) carotenoids quantified using iCheck Fluoro; and (c) visually scored root yellowness using color chart scores (PDF 42.3 KB).
Supplementary Fig. 3
Alignment of the three cassava Phytoene synthase genes from the reference genome compared with PSY2 from re-sequencing of other white- and yellow-root cassava clones as well as other species (PDF 950 KB).
Supplementary Fig. 4
Pattern of linkage disequillibrium as measured by squared correlation coefficient (r2) of alleles occurring between and within the candidate genes in this study (lcyE, lcyB, crtRB and PSY2). Note the extensive LD in lcyB (PDF 96.7 KB).
Supplementary Fig. 5
Comparison of various association mapping models using Q-Q plot of observed against expected p-values. a) BLUPs of Total carotenoids estimated by spectrophotometer, b) total β-carotene (TBC), c) Total carotenoids estimated by iCheck Fluoro and d) chromameter b*. The naïve and P model show the most inflated p-value (PDF 98.0 KB).
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Udoh, L.I., Gedil, M., Parkes, E.Y. et al. Candidate gene sequencing and validation of SNP markers linked to carotenoid content in cassava (Manihot esculenta Crantz). Mol Breeding 37, 123 (2017). https://doi.org/10.1007/s11032-017-0718-5
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DOI: https://doi.org/10.1007/s11032-017-0718-5