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BackgroundCharacterization of genetic variations in maize has been challenging, mainly due to deterioration of collinearity between individual genomes in the species. An international consortium of maize research groups combined resources to develop the maize haplotype version 3 (HapMap 3), built from whole-genome sequencing data from 1218 maize lines, covering predomestication and domesticated Zea mays varieties across the world.ResultsA new computational pipeline was set up to process more than 12 trillion bp of sequencing data, and a set of population genetics filters was applied to identify more than 83 million variant sites.ConclusionsWe identified polymorphisms in regions where collinearity is largely preserved in the maize species. However, the fact that the B73 genome used as the reference only represents a fraction of all haplotypes is still an important limiting factor.

Vitamin A deficiency (VAD) compromises immune function and is the leading cause of preventable blindness in children in many developing countries. Biofortification, or breeding staple food crops that are rich in micronutrients, provides a sustainable way to fight VAD and other micronutrient malnutrition problems. Polymorphisms, with associated molecular markers, have recently been identified for two loci, LcyE (lycopene epsilon cyclase) and CrtRB1 (β-carotene hydroxylase 1) that govern critical steps in the carotenoid biosynthetic pathway in maize endosperm, thereby enabling the opportunity to integrate marker-assisted selection (MAS) into carotenoid breeding programs. We validated the effects of 3 polymorphisms (LcyE5′TE, LcyE3′Indel and CrtRB1-3′TE) in 26 diverse tropical genetic backgrounds. CrtRB1-3′TE had a two-ten fold effect on enhancing beta-carotene (BC) and total provitamin A (proA) content. Reduced-function, favorable polymorphisms within LcyE resulted in 0–30 % reduction in the ratio of alpha- to beta-branch carotenoids, and increase in proA content (sometimes statistically significant). CrtRB1-3′TE had large, significant effect on enhancing BC and total ProA content, irrespective of genetic constitution for LcyE5′TE. Genotypes with homozygous favorable CrtRB1-3′TE alleles had much less zeaxanthin and an average of 25 % less total carotenoid than other genotypes, suggesting that feedback inhibition may be reducing the total flux into the carotenoid pathway. Because this feedback inhibition was most pronounced in the homozygous favorable LcyE (reduced-function) genotypes, and because maximum total proA concentrations were achieved in genotypes with homozygous unfavorable or heterozygous LcyE, we recommend not selecting for both reduced-function genes in breeding programs. LcyE exhibited significant segregation distortion (SD) in all the eight, while CrtRB1 in five of eight digenic populations studied, with favorable alleles of both the genes frequently under-represented. MAS using markers reported herein can efficiently increase proA carotenoid concentration in maize.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-012-1987-3) contains supplementary material, which is available to authorized users.

Characterization of genetic diversity is of great value to assist breeders in parental line selection and breeding system design. We screened 770 maize inbred lines with 1,034 single nucleotide polymorphism (SNP) markers and identified 449 high-quality markers with no germplasm-specific biasing effects. Pairwise comparisons across three distinct sets of germplasm, CIMMYT (394), China (282), and Brazil (94), showed that the elite lines from these diverse breeding pools have been developed with only limited utilization of genetic diversity existing in the center of origin. Temperate and tropical/subtropical germplasm clearly clustered into two separate groups. The temperate germplasm could be further divided into six groups consistent with known heterotic patterns. The greatest genetic divergence was observed between temperate and tropical/subtropical lines, followed by the divergence between yellow and white kernel lines, whereas the least divergence was observed between dent and flint lines. Long-term selection for hybrid performance has contributed to significant allele differentiation between heterotic groups at 20% of the SNP loci. There appeared to be substantial levels of genetic variation between different breeding pools as revealed by missing and unique alleles. Two SNPs developed from the same candidate gene were associated with the divergence between two opposite Chinese heterotic groups. Associated allele frequency change at two SNPs and their allele missing in Brazilian germplasm indicated a linkage disequilibrium block of 142 kb. These results confirm the power of SNP markers for diversity analysis and provide a feasible approach to unique allele discovery and use in maize breeding programs.

Tocopherols are a class of four natural compounds that can provide nutrition and function as antioxidant in both plants and animals. Maize kernels have low α-tocopherol content, the compound with the highest vitamin E activity, thus, raising the risk of vitamin E deficiency in human populations relying on maize as their primary vitamin E source. In this study, two insertion/deletions (InDels) within a gene encoding γ-tocopherol methyltransferase, Zea mays VTE4 (ZmVTE4), and a single nucleotide polymorphism (SNP) located ∼85 kb upstream of ZmVTE4 were identified to be significantly associated with α-tocopherol levels in maize kernels by conducting an association study with a panel of ∼500 diverse inbred lines. Linkage analysis in three populations that segregated at either one of these three polymorphisms but not at the other two suggested that the three polymorphisms could affect α-tocopherol content independently. Furthermore, we found that haplotypes of the two InDels could explain ∼33% of α-tocopherol variation in the association panel, suggesting ZmVTE4 is a major gene involved in natural phenotypic variation of α-tocopherol. One of the two InDels is located within the promoter region and associates with ZmVTE4 transcript level. This information can not only help in understanding the underlying mechanism of natural tocopherol variations in maize kernels, but also provide valuable markers for marker-assisted breeding of α-tocopherol content in maize kernels, which will then facilitate the improvement of maize as a better source of daily vitamin E nutrition.

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.

BackgroundMaize breeding germplasm used in Southwest China has high complexity because of the diverse ecological features of this area. In this study, the population structure, genetic diversity, and linkage disequilibrium decay distance of 362 important inbred lines collected from the breeding program of Southwest China were characterized using the MaizeSNP50 BeadChip with 56,110 single nucleotide polymorphisms (SNPs).ResultsWith respect to population structure, two (Tropical and Temperate), three (Tropical, Stiff Stalk and non-Stiff Stalk), four [Tropical, group A germplasm derived from modern U.S. hybrids (PA), group B germplasm derived from modern U.S. hybrids (PB) and Reid] and six (Tropical, PB, Reid, Iowa Stiff Stalk Synthetic, PA and North) subgroups were identified. With increasing K value, the Temperate group showed pronounced hierarchical structure with division into further subgroups. The Genetic Diversity of each group was also estimated, and the Tropical group was more diverse than the Temperate group. Seven low-genetic-diversity and one high-genetic-diversity regions were collectively identified in the Temperate, Tropical groups, and the entire panel. SNPs with significant variation in allele frequency between the Tropical and Temperate groups were also evaluated. Among them, a region located at 130 Mb on Chromosome 2 showed the highest genetic diversity, including both number of SNPs with significant variation and the ratio of significant SNPs to total SNPs. Linkage disequilibrium decay distance in the Temperate group was greater (2.5–3 Mb) than that in the entire panel (0.5–0.75 Mb) and the Tropical group (0.25–0.5 Mb). A large region at 30–120 Mb of Chromosome 7 was concluded to be a region conserved during the breeding process by comparison between S37, which was considered a representative tropical line in Southwest China, and its 30 most similar derived lines.ConclusionsFor the panel covered most of widely used inbred lines in Southwest China, this work representatively not only illustrates the foundation and evolution trend of maize breeding resource as a theoretical reference for the improvement of heterosis, but also provides plenty of information for genetic researches such as genome-wide association study and marker-assisted selection in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3041-3) contains supplementary material, which is available to authorized users.

BackgroundDrought stress is one of the major limiting factors for maize production. With the availability of maize B73 reference genome and whole-genome resequencing of 15 maize inbreds, common variants (CV) and clustering analyses were applied to identify non-synonymous SNPs (nsSNPs) and corresponding candidate genes for drought tolerance.ResultsA total of 524 nsSNPs that were associated with 271 candidate genes involved in plant hormone regulation, carbohydrate and sugar metabolism, signaling molecules regulation, redox reaction and acclimation of photosynthesis to environment were detected by CV and cluster analyses. Most of the nsSNPs identified were clustered in bin 1.07 region that harbored six previously reported QTL with relatively high phenotypic variation explained for drought tolerance. Genes Ontology (GO) analysis of candidate genes revealed that there were 35 GO terms related to biotic stimulus and membrane-bounded organelle, showing significant differences between the candidate genes and the reference B73 background. Changes of expression level in these candidate genes for drought tolerance were detected using RNA sequencing for fertilized ovary, basal leaf meristem tissue and roots collected under drought stressed and well-watered conditions. The results indicated that 70% of candidate genes showed significantly expression changes under two water treatments and our strategies for mining candidate genes are feasible and relatively efficient.ConclusionsOur results successfully revealed candidate nsSNPs and associated genes for drought tolerance by comparative sequence analysis of 16 maize inbred lines. Both methods we applied were proved to be efficient for identifying candidate genes for complex traits through the next-generation sequencing technologies (NGS). These selected genes will not only facilitate understanding of genetic basis of drought stress response, but also accelerate genetic improvement through marker-assisted selection in maize.