Characterization of QTL for oil content in maize kernel

Kernel oil content in maize is a complex quantitative trait. Phenotypic variation in kernel oil content can be dissected into its component traits such as oil metabolism and physical characteristics of the kernel, including embryo size and embryo-to-endosperm weight ratio (EEWR). To characterize quantitative trait loci (QTL) for kernel oil content, a recombinant inbred population derived from a cross between normal line B73 and high-oil line By804 was genotyped using 228 molecular markers and phenotyped for kernel oil content and its component traits [embryo oil content, embryo oil concentration, EEWR, embryo volume, embryo width, embryo length, and embryo width-to-length ratio (EWLR)]. A total of 58 QTL were identified for kernel oil content and its component traits in 26 genomic regions across all chromosomes. Eight main-effect QTL were identified for kernel oil content, embryo oil content, embryo oil concentration, EEWR, embryo weight, and EWLR, each accounting for over 10?% of the phenotypic variation in six genomic regions. Over 90?% of QTL identified for kernel oil content co-localized with QTL for component traits, validating their molecular contribution to kernel oil content. On chromosome 1, the QTL that had the largest effect on kernel oil content (qKO1-1) was associated with embryo width; on chromosome 9, the QTL for kernel oil content (qKO9) was related to EEWR (qEEWR9). Embryo oil concentration and embryo width were identified as the most important component traits controlling the second largest QTL for kernel oil content on chromosome 6 (qKO6) and a minor QTL for kernel oil content on chromosome 5 (qKO5-2), respectively. The dissection of kernel oil QTL will facilitate future cloning and/or functional validation of kernel oil content, and help to elucidate the genetic basis of kernel oil content in maize.     

From QTLs for enzyme activity to candidate genes in maize

In order to facilitate the search for genes underlying QTLs (Quantitative Trait Loci), the activities of key enzymes of the carbohydrate metabolism in maize, and the concentration of their substrates or products were used as quantitative traits. For each of the chosen enzyme, i.e. ADPglucose pyrophosphorylase, sucrose-phosphate-synthase and invertases, the corresponding cDNA was available. Since biochemical traits are more closely related to gene expression than agronomic traits, co-locations could be expected between an enzyme structural gene and a QTL for its enzyme activity, and/or the corresponding product or substrate content. This approach was applied using recombinant inbred lines on leaves at 3- or 4-leaf stage, under control and water stress conditions and on grain, at maturity. Several QTLs were detected for each trait, particularly for two enzyme activities measured in mature leaves. Apparent co-locations between QTL for activity and structural locus were observed for sucrose-phosphate-synthase (chromosome 8) and acid-soluble invertase (chromosome 2 and 5). Leaf acid-soluble (vacuolar) invertase provided an interesting case since QTL, on chromosome 5, explaining 17% of variability was apparently co-located with the Ivr2 gene encoding a vacuolar invertase protein which was strongly water-stress inducible. Similarly, in grain, an amylose QTL co-located with the Sh2 gene of ADPglucose pyrophosphorylase. The reliability of this candidate was further tested through the examination of Sh2 DNA polymorphism in 46 genetically unrelated lines. A correlation was obtained between this polymorphism and kernel starch content, which further validated Sh2 as a candidate. Some improvements or alternatives to this strategy are briefly discussed.      

QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

Background

Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes.

Results

The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue.

Conclusions

A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.     

Molecular mapping of Arabidopsis thaliana lipid-related orthologous genes in Brassica napus

Quantitative Trait Loci (QTL) for oil content has been previously analyzed in a SG-DH population from a cross between a Chinese cultivar and a European cultivar of Brassica napus. Eight QTL with additive and epistatic effects, and with environmental interactions were evaluated. Here we present an integrated linkage map of this population predominantly based on informative markers derived from Brassica sequences, including 249 orthologous A. thaliana genes, where nearly half (112) are acyl lipid metabolism related genes. Comparative genomic analysis between B. napus and A. thaliana revealed 33 colinearity regions. Each of the conserved A. thaliana segments is present two to six?times in the B. napus genome. Approximately half of the mapped lipid-related orthologous gene loci (76/137) were assigned in these conserved colinearity regions. QTL analysis for seed oil content was performed using the new map and phenotypic data from 11 different field trials. Nine significant QTL were identified on linkage groups A1, A5, A7, A9, C2, C3, C6 and C8, together explaining 57.79% of the total phenotypic variation. A total of 14 lipid related candidate gene loci were located in the confidence intervals of six of these QTL, of which ten were assigned in the conserved colinearity regions and felled in the most frequently overlapped QTL intervals. The information obtained from this study demonstrates the potential role of the suggested candidate genes in rapeseed kernel oil accumulation.     

Differential expression profiles of growth-related genes in the elongation zone of maize primary roots

Growth in the apical elongation zone of plant roots is central to the development of functional root systems. Rates of root segmental elongation change from accelerating to decelerating as cell development proceeds from newly formed to fully elongated status. One of the primary variables regulating these changes in elongation rates is the extensibility of the elongating cell walls. To help decipher the complex molecular mechanisms involved in spatially variable root growth, we performed a gene identification study along primary root tips of maize (Zea mays) seedlings using suppression subtractive hybridization (SSH) and candidate gene approaches. Using SSH we isolated 150 non-redundant cDNA clones representing root growth-related genes (RGGs) that were preferentially expressed in the elongation zone. Differential expression patterns were revealed by Northern blot analysis for 41 of the identified genes and several candidate genes. Many of the genes have not been previously reported to be involved in root growth processes in maize. Genes were classified into groups based on the predicted function of the encoded proteins: cell wall metabolism, cytoskeleton, general metabolism, signaling and unknown. In-situ hybridization performed for two selected genes, confirmed the spatial distribution of expression shown by Northern blots and revealed subtle differences in tissue localization. Interestingly, spatial profiles of expression for some cell wall related genes appeared to correlate with the profile of accelerating root elongation and changed appropriately under growth-inhibitory water deficit.     

Natural variations in stearoyl-acp desaturase genes affect the conversion of stearic to oleic acid in maize kernel

Key message

We identified 11 SAD genes, and mined their natural variations associated with the conservation of stearic to oleic acid, especially ZmSAD1 supported by both the QTL and an expression QTL.

Abstract

Maize oil is generally regarded as a healthy vegetable oil owing to its low abundance of saturated fatty acids. Stearoyl-ACP desaturase (SAD) is a key rate-limiting enzyme for the conservation of stearic (C18:0) to oleic (C18:1) acid. Here, 11 maize SAD genes were identified to have more divergent functions than Arabidopsis SAD genes. The genomic regional associations in a maize panel including 508 inbred lines identified 6 SAD genes significantly associated (P < 0.01) with the C18:0/C18:1 ratio or the level of C18:0 or C18:1, one gene of which co-localized with a quantitative trait locus (QTL) and 5 of which co-localized with an expression QTL. ZmSAD1, supported by both the QTL and an expression QTL, had the largest effect on C18:0/C18:1. One nonsynonymous single-nucleotide polymorphism in exon 3 and one 5-bp insertion/deletion in the 3′ untranslated region were further shown to contribute to the natural variation in C18:0/C18:1 according to ZmSAD1-based association mapping. Finally, selection tests of ZmSAD1 in teosinte, regular maize, and high-oil maize indicated that ZmSAD1 was not a selection target during the process of maize domestication and high-oil maize development. These results will guide the manipulation of the ratio between saturated and unsaturated fatty acids in maize.

     

Combined linkage mapping and association analysis reveals genetic control of maize kernel moisture content

The free moisture in crop kernels after being naturally dried is referred to as kernel moisture content (KMC). Maize KMC reflects grain quality and influences transportation and storage of seeds. We used an IBM Syn10 DH maize population consisting of 249 lines and an association panel comprising 310 maize inbred lines to identify the genetic loci affecting maize KMC in three environments. Using the IBM population detected 13 QTL on seven chromosomes, which were clustered into nine common QTL. Genome-wide association analysis (GWAS) identified 16 significant SNPs across the 3 environments, which were linked to 158 genes across the three environments. Combined QTL mapping and GWAS found two SNPs that were located in two of the mapped QTL, respectively. Twenty-three genes were linked with the loci co-localized in both populations. Of these 181 genes, five have previously been reported to be associated with KMC or to regulate seed development. These associations were verified by candidate gene association analysis. Two superior alleles and one favorable haplotype for Zm00001d007774 and Zm00001d047868 were found to influence KMC. These findings provide insights into molecular mechanisms underlying maize KMC and contribute to the use of marker-assisted selection for breeding low-KMC maize.     

Genetic analysis and major QTL detection for maize kernel size and weight in multi-environments

Key Message

Twelve major QTL in five optimal clusters and several epistatic QTL are identified for maize kernel size and weight, some with pleiotropic will be promising for fine-mapping and yield improvement.

Abstract

Kernel size and weight are important target traits in maize (Zea mays L.) breeding programs. Here, we report a set of quantitative trait loci (QTL) scattered through the genome and significantly controlled the performance of four kernel traits including length, width, thickness and weight. From the cross V671 (large kernel) × Mc (small kernel), 270 derived F_2:3 families were used to identify QTL of maize kernel-size traits and kernel weight in five environments, using composite interval mapping (CIM) for single-environment analysis along with mixed linear model-based CIM for joint analysis. These two mapping strategies identified 55 and 28 QTL, respectively. Among them, 6 of 23 coincident were detected as interacting with environment. Single-environment analysis showed that 8 genetic regions on chromosomes 1, 2, 4, 5 and 9 clustered more than 60 % of the identified QTL. Twelve stable major QTLs accounting for over 10 % of phenotypic variation were included in five optimal clusters on the genetic region of bins 1.02–1.03, 1.04–1.06, 2.05–2.07, 4.07–4.08 and 9.03–9.04; the addition and partial dominance effects of significant QTL play an important role in controlling the development of maize kernel. These putative QTL may have great promising for further fine-mapping with more markers, and genetic improvement of maize kernel size and weight through marker-assisted breeding.     

Meta-analysis combined with syntenic metaQTL mining dissects candidate loci for maize yield

Yield potential and stability improvement with the goal of ensuring global food security is an important priority. Yield has a quantitative nature and is controlled by quantitative trait loci (QTL) and environmental factors. An increasingly large number of maize yield QTL have been identified, and how to integrate and re-analyze them is challenging. To this end, we tried to combine QTL meta-analysis with homology-based cloning techniques to dissect candidate loci/genes for maize yield. We first collected maize yield-related QTL from public resources. Then, 351 collected QTL were iteratively projected and meta-analyzed to obtain metaQTL (MQTL). A total of 54 MQTL were identified and tended to cluster in the maize genome. Seven MQTL containing ten maize orthologs of rice yield genes were dissected and temporarily termed syntenic MQTL. Maize orthologs of three functionally-characterized rice yield genes, GIF1, WFP/IPA1, and DEP1, were specially selected to undergo phylogenetic, proliferation, and selective pattern analysis. The results showed that maize orthologs were closely related to rice yield genes and subjected to mixed selective pressures, including positive selection during selective sweeps. The power of the combined techniques reported here was primarily validated not only by the congruency of MQTL and recently reported maize yield QTL but also by mined syntenic MQTL containing the well-characterized Miniature1 (Mn1) gene for maize kernel size and weight determination. Maize MQTL, especially syntenic MQTL regions, could serve not only for QTL fine-mapping and cloning but also for the marker-assisted selection breeding program. The maize yield candidate loci/genes presented here also deserve further investigation and will provide clues to the molecular bases of grain yield. Additionally, the combined technique described here will find its way into further quantitative trait research.     

DNA array profiling of gene expression changes during maize embryo development

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)⁺RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)⁺ RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6. Electronic Publication     

Genetic dissection of tocopherol content and composition in maize grain using quantitative trait loci analysis and the candidate gene approach

Tocopherols are essential micronutrients for humans and animals, with several beneficial effects in plants. Among cereals, only maize grains contain high concentrations of tocopherols. In this investigation we analyzed, during 2004 and 2005, by high-performance liquid chromatography (HPLC), a population of 233 recombinant inbred lines (RIL) which were derived from two diverse parents and had extremely variable tocopherol content and composition. A genetic map was constructed using 208 polymorphic molecular markers including gene-targeted markers based on six candidate genes of the tocopherol biosynthesis pathway (HPPD, VTE1, VTE3, VTE4, P3VTE5, and P4VTE5). Thirty-one quantitative trait loci (QTL) associated with quantitative variation of tocopherol content and composition were identified by composite interval mapping (CIM); these were located on sixteen genomic regions covering all the chromosomes except chromosome 4. Most (65%) QTL were co-located, suggesting that in some cases the same QTL predominantly affected the amounts of more than one tocopherol. Two candidate genes, HPPD and VTE4 showed co-localization with major QTL for tocopherol content and composition whereas only one interval (umc1075–umc1304) on chromosome eight exhibited a QTL for α, δ, γ, and total tocopherols with high LOD and PVE values. The candidate genes associated with tocopherol content and with composition, especially VTE4 and HPPD, could be precisely used for alteration of the tocopherol content and composition of maize grains by development of functional markers. Other identified major QTL especially those on chromosomes 8, 1, and 2 (near candidate gene VTE5) can also be used for improvement of maize grain quality by marker-assisted selection.     

Multiple pyruvate decarboxylase genes in maize are induced by hypoxia

Two cDNA clones corresponding to anaerobically induced maize mRNAs were found to have homology to a previously identified maize pyruvate decarboxylase gene. DNA sequencing and RFLP mapping indicate that these cDNAs represent two additional maize pdc genes. Each of the clones is approximately 85% identical in predicted amino acid sequence to the other two. All three clones are induced by hypoxic stress, but with different levels and kinetics of induction.     

Mapping of quantitative trait loci for kernel row number in maize across seven environments

Genetic factors controlling quantitative inheritance of grain yield and its components have been intensively investigated during recent decades using diverse populations in maize (Zea mays L.). Notwithstanding this, quantitative trait loci (QTL) for kernel row number (KRN) with large and consistent effect have not been identified. In this study, a linkage map of 150 simple sequence repeat (SSR) loci was constructed by using a population of 500 F2 individuals derived from a cross between elite inbreds Ye478 and Dan340. The linkage map spanned a total of 1478 cM with an average interval of 10.0 cM. A total of 397 F2:3 lines were evaluated across seven diverse environments for mapping QTL for KRN. Some QTL for grain yield and its components had previously been confirmed with this population across environments. A total of 13 QTL for KRN were identified, with each QTL explaining from 3.0 to 17.9% of phenotypic variance. The gene action for KRN was mainly additive to partial dominance. A large-effect QTL (qkrn7) with partial dominance effect accounting for 17.9% of the phenotypic variation for KRN was identified on chromosome 7 near marker umc1865 with consistent gene effect across seven diverse environments. This study has laid a foundation for map-based cloning of this major QTL and for developing molecular markers for marker-assisted selection of high KRN.     

基于元分析的抗玉米丝黑穗病QTL比较定位

以玉米遗传连锁图谱IBM2 2005 Neighbors为参考图谱,通过映射整合不同试验中的抗玉米丝黑穗病QTL,构建QTL综合图谱。在国内外种质中,共发现22个抗病QTL,分布在除第7染色体外的9条玉米染色体上。采用元分析技术,获得2个“一致性”抗病QTL,图距分别为8.79 cM和18.92cM。从MaizeGDB网站下载“一致性”QTL区间内基因和标记的原始序列;采用NCBI网站在线软件BLASTx通过同源比对在2个“一致性”QTL区间内初步获得4个抗病位置候选基因。借助比较基因电子定位策略,将69个水稻和玉米抗性基因定位于玉米IBM2图谱上,在2个“一致性”QTL区间内分别发现1个水稻抗性基因,初步推断为抗病位置候选基因。本文结果为抗玉米丝黑穗病QTL精细定位和分子育种提供了基础。     

Quantitative trait loci influencing protein and starch concentration in the Illinois Long Term Selection maize strains

A study was initiated to determine the number, chromosomal location, and magnitude of effect of QTL (quantitative trait loci or locus depending on context) controlling protein and starch concentration in the maize (Zea mays L.) kernel. Restriction fragment length polymorphism (RFLP) analysis was performed on 100 F₃ families derived from a cross of two strains, Illinois High Protein (IHP), X Illinois Low Protein (ILP), which had been divergently selected for protein concentration for 76 generations as part of the Illinois Long Term Selection Experiment. These families were analyzed for kernel protein and starch in replicated field trials during 1990 and 1991. A series of 90 genomic and cDNA clones distributed throughout the maize genome were chosen for their ability to detect RFLP between IHP and ILP. These clones were hybridized with DNA extracted from the 100 F₃ families, revealing 100 polymorphic loci. Single factor analysis of variance revealed significant QTL associations of many loci with both protein and starch concentration (P < 0.05 level). Twenty-two loci distributed on 10 chromosome arms were significantly associated with protein concentration, 19 loci on 9 chromosome arms were significantly associated with starch concentration. Sixteen of these loci were significant for both protein and starch concentration. Clusters of 3 or more significant loci were detected on chromosome arms 3L, 5S, and 7L for protein concentration, suggesting the presence of QTL with large effects at these locations. A QTL with large additive effects on protein and starch concentration was detected on chromosome arm 3L. RFLP alleles at this QTL were found to be linked with RFLP alleles at the Shrunken-2 (Sh2) locus, a structural gene encoding the major subunit of the starch synthetic enzyme ADP-glucose pyrophosphorylase. A multiple linear regression model consisting of 6 significant RFLP loci on different chromosomes explained over 64 % of the total variation for kernel protein concentration. Similar results were detected for starch concentration. Thus, several chromosomal regions with large effects may be responsible for a significant portion of the changes in kernel protein and starch concentration in the Illinois Long Term Selection Experiment.     

The role of regulatory genes during maize domestication: evidence from nucleotide polymorphism and gene expression

We investigated DNA sequence variation in 72 candidate genes in maize landraces and the wild ancestor of maize, teosinte. The candidate genes were chosen because they exhibit very low sequence diversity among maize inbreds and have sequence homology to known regulatory genes. We observed signatures of selection in 17 candidate genes, indicating that they were potential targets of artificial selection during domestication. In addition, 21 candidate genes were identified as potential targets of natural selection in teosinte. A comparison of the proportion of selected genes between our regulatory genes and genes unfiltered for their potential function (but also with very low sequence diversity among maize inbreds) provided some weak evidence that regulatory genes are overrepresented among selected genes. We detected no significant association between the positions of genes identified as potential targets of selection during domestication and quantitative trait loci (QTL) responsible for maize domestication traits. However, a subset of these genes, those identified by sequence homology as kinase/phosphatase genes, significantly cluster with the domestication QTL. We also analyzed expression profiles of genes in distinct maize tissues and observed that domestication genes are expressed on average at a significantly higher level than neutral genes in reproductive organs, including kernels.     

A transposable element insertion within ZmGE2 gene is associated with increase in embryo to endosperm ratio in maize

Most of the maize kernel oil is located in the embryo while the majority of starch is located in the endosperm. Maize kernel composition and value are affected significantly by the ratio of the embryo size to the endosperm size; however, the genetic regulation of embryo to endosperm ratio (EER) in maize is unknown. Here we identified ZmGE2 gene, which encodes a cytochrome p450 protein, as a gene associated with EER variation in maize. We first expressed rice Giant Embryo (GE) gene driven by oleosin promoter in maize and detected a 23.2?% reduction in EER in transgenic seeds, demonstrating the existence of evolutionarily conserved mechanisms for EER determination in rice and maize. We next identified maize GE2, a homolog of rice GE sharing 70?% identity in amino sequence, as a candidate based on the similar expression pattern and co-localization with a previously detected QTL for EER. Followed by linkage and association mapping, a 247-bp transposable element (TE) insertion in 3′-untranslated region of ZmGE2 gene was identified to be associated with increase in EER and kernel oil content. Expression level of the favorable ZmGE2 allele containing the 247-bp TE insertion was strongly reduced. In addition, the 247-bp TE insertion site was a selection target during the artificial long-term selection for the high EER trait in a high oil population. This is the first report that demonstrates an association of ZmGE2 with EER variation in maize and identifies ZmGE2 gene as a promising target for manipulation of EER and grain composition by either transgenic approach or molecular breeding in maize.