Rare genetic variation at Zea mays crtRB1 increases β -carotene in maize grain

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Rare genetic variation at Zea mays crtRB1 increases β-carotene in maize grain

Jianbing Yan1–3,13, Catherine Bermudez Kandianis4,5,13, Carlos E Harjes3,12, Ling Bai6, Eun-Ha Kim7,

Xiaohong Yang2, Debra J Skinner4,5, Zhiyuan Fu2, Sharon Mitchell3, Qing Li2, Maria G Salas Fernandez3,8, Maria Zaharieva1, Raman Babu1, Yang Fu2,4,5, Natalia Palacios1, Jiansheng Li2, Dean DellaPenna7, Thomas Brutnell6, Edward S Buckler3,9,10, Marilyn L Warburton1,11 & Torbert Rocheford4,5

Breeding to increase -carotene levels in cereal grains, termed provitamin A biofortification, is an economical approach to address dietary vitamin A deficiency in the developing world. Experimental evidence from association and linkage populations in maize (Zea mays L.) demonstrate that the gene encoding -carotene hydroxylase 1 (crtRB1) underlies a principal quantitative trait locus associated with -carotene concentration and conversion in maize kernels. crtRB1 alleles associated with reduced transcript expression correlate with higher -carotene concentrations. Genetic variation at crtRB1 also affects hydroxylation efficiency among encoded allozymes, as observed by resultant carotenoid profiles in recombinant expression assays. The most favorable crtRB1 alleles, rare in frequency and unique to temperate germplasm, are being introgressed via inexpensive PCR marker-assisted selection into tropical maize germplasm adapted to developing countries, where it is most needed for human health.

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Vitamin A deficiency (VAD) leads to blindness in 250,000–500,000 children each year, with half dying from VAD-related illness within 12 months (data from the World Health Organization (WHO); see URL list in Online Methods section). Improving the micronutrient balance of staple crops such as maize through biofortification is there-fore an economically and socially sound way to address micronutrient malnutrition, including VAD, on a global scale1. Considering factors of portion size, bioavailability and bioconversion, HarvestPlus (HP), a Consultative Group on International Agricultural Research Challenge program designed to use breeding for crop biofortification, projects that daily dietary intake of maize with 15 µg g 1 provitamin A caro-tenoids could greatly alleviate VAD2.

A limited number of carotenoids, including α-carotene, β-carotene (βC) and β-cryptoxanthin (βCX), can be converted to vitamin A through animal metabolism3. A recent study combining informa-tion about carotenoid pathways from model organisms with natural variation for carotenoids in maize grain identified several haplotypes of the gene encoding lycopene epsilon cyclase (lcyε; also known as LOC100280448 and lyce1) that substantially increase the ratio of β- to α-carotenoids in grain4 (Fig. 1a). The favorable lcyε alleles increase the proportion of βC, but a large amount is hydroxylated to βCX and

zeaxanthin (Z), which have 50% and 0% of the provitamin A activity of βC, respectively. Extrapolation from studies of Arabidopsis mutants defective in one or more of four carotenoid hydroxylase genes5,6 indi-cated that natural genetic variation affecting corresponding biochem-ical reactions in maize might positively alter the βC/βCX + Z ratio (Fig. 1a), thus further increasing the provitamin A activity of maize grain. Toward this end, we identified maize orthologs of Arabidopsis β-carotene hydroxylases, and we surveyed and tested naturally occur-ring allelic variation to establish the molecular basis for reduced βC conversion.

RESULTS

crtRB1 is associated with -carotene concentration

Three polymorphisms in Zea mays crtRB1 (also known as HYD3) were significantly associated with carotenoid variation in association panel P1 (Table 1), identified as 5′TE, InDel4, and 3′TE (Fig. 1b; see Online Methods for details). PCR markers for these polymorphisms were assayed in association panels P1, P2 and P3 (Supplementary Fig. 1), which contain different sets of inbred lines with differing crtRB1 allele frequencies. The 5′TE is significantly correlated with β-carotene con-centrations accounting for 32% of phenotypic variation in P1, as well

Beijing, China. 3Institute for Genomic Diversity, Cornell University, Ithaca, New York, USA. 4Department of Crop Sciences, University of Illinois, Urbana, Illinois, USA. 5Department of Agronomy, Purdue University, West Lafayette, Indiana, USA. 6Boyce Thompson Institute, Ithaca, New York, USA. 7Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA. 8Agronomy Department, Iowa State University, Ames, Iowa, USA. 9United States Department of Agriculture–Agricultural Research Service: Plant, Soil and Nutrition Research Unit, Ithaca, New York, USA. 10Department of Plant Breeding and

Genetics, Cornell University, Ithaca, New York, USA. 11United States Department of Agriculture–Agricultural Research Service: Corn Host Plant Resistance Research Unit, Starkville, Mississippi, USA. 12Monsanto, Leesburg, Georgia, USA. 13These authors contributed equally to this work. Correspondence should be addressed to T.R. (torbert@purdue.edu) and M.W. (marilyn.warburton@ars.usda.gov).

Received 23 September 2009; accepted 19 February 2010; published online 21 March 2010; doi:10.1038/ng.551

1International Maize and Wheat Improvement Center (CIMMYT), Texcoco, Mexico. 2National Maize Improvement Center of China, China Agricultural University,

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