The Genetic Basis of Natural Variation in Kernel Size and Related Traits Using a Four-Way Cross Population in Maize

Kernel size is an important component of grain yield in maize breeding programs. To extend the understanding on the genetic basis of kernel size traits (i.e., kernel length, kernel width and kernel thickness), we developed a set of four-way cross mapping population derived from four maize inbred lines with varied kernel sizes. In the present study, we investigated the genetic basis of natural variation in seed size and other components of maize yield (e.g., hundred kernel weight, number of rows per ear, number of kernels per row). In total, ten QTL affecting kernel size were identified, three of which (two for kernel length and one for kernel width) had stable expression in other components of maize yield. The possible genetic mechanism behind the trade-off of kernel size and yield components was discussed.     

Genetic analysis as a tool to investigate the molecular mechanisms underlying seed development in maize

BACKGROUND: In angiosperms the seed is the outcome of double fertilization, a process leading to the formation of the embryo and the endosperm. The development of the two seed compartments goes through three main phases: polarization, differentiation of the main tissues and organs and maturation. SCOPE: This review focuses on the maize kernel as a model system for developmental and genetic studies of seed development in angiosperms. An overview of what is known about the genetic and molecular aspects underlying embryo and endosperm formation and maturation is presented. The role played by embryonic meristems in laying down the plant architecture is discussed. The acquisition of the different endosperm domains are presented together with the use of molecular markers available for the detection of these domains. Finally the role of programmed cell death in embryo and endosperm development is considered. CONCLUSIONS: The sequence of events occurring in the developing maize seed appears to be strictly regulated. Proper seed development requires the co-ordinated expression of embryo and endosperm genes and relies on the interaction between the two seed components and between the seed and the maternal tissues. Mutant analysis is instrumental in unravelling the genetic control underlying the formation of each compartment as well as the molecular signals interplaying between the two compartments.     

Maize seed endophytes

Maize is a vital global crop, and each seed (kernel) hosts an ecosystem of microbes living inside it. However, we know very little about these endophytes and what their role is in plant production and physiology. In this Microreview, I summarize the major questions around maize seed endophytes, including what organisms are present, how they get there, whether and how they transmit across generations, and how they and the plant affect each other. Although several studies touch on each of these areas, ultimately there are far more questions than answers. Future priorities for research on maize seed endophytes should include understanding what adaptations allow microbes to be seed endophytes, how the host genetics and the environment affect these communities, and how maize seed endophytes ultimately contribute to the next generation of plants.     

Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.     

Maize orthologs of rice GS5 and their transregulator are associated with kernel development

Genome information from model species such as rice can assist in the cloning of genes in a complex genome,such as maize.Here,we identified a maize ortholog of rice GS5 that contributes to kernel development in maize.The genomewide association analysis of the expression levels of ZmGS5,and 15 of its 26 paralogs,identified a trans-regulator on chromosome 7,which was a BAKi-like gene.This gene that we named as ZmBAK1-7 could regulate the expression of ZmGS5 and three of the paralogs.Candidate-gene association analyses revealed that these five genes were associated with maize kernel development-related traits.Linkage analyses also detected that ZmGSs and ZmBAK1-7 co-localized with mapped QTLs.A transgenic analysis of ZmGS5 in Arabidopsis thaliana L.showed a significant increase in seed weight and cell number,suggesting that ZmGS5 may have a conserved function among different plant species that affects seed development.     

Seed-specific expression of the wheat puroindoline genes improves maize wet milling yields

The texture of maize ( Zea mays L.) seeds is important to seed processing properties, and soft dent maize is preferred for both wet-milling and livestock feed applications. The puroindoline genes ( Pina and Pinb ) are the functional components of the wheat ( Triticum aestivum L.) Hardness locus and together function to create soft grain texture in wheat. The PINs (PINA and PINB) are believed to act by binding to lipids on the surface of starch granules, preventing tight adhesion between starch granules and the surrounding protein matrix during seed maturation. Here, maize kernel structure and wet milling properties were successfully modified by the endosperm-specific expression of wheat Pins ( Pina and Pinb ). Pins were introduced into maize under the control of a maize γ- Zein promoter. Three Pina/Pinb expression positive transgenic lines were evaluated over two growing seasons. Textural analysis of the maize seeds indicated that the expression of PINs decreased adhesion between starch and protein matrix and reduced maize grain hardness significantly. Reduction in pressure required to fracture kernels ranged from 15.65% to 36.86% compared with control seeds. Further, the PINs transgenic maize seeds had increased levels of extractable starch as characterized by a small scale wet milling method. Starch yield was increased by 4.86% on average without negatively impacting starch purity. The development of softer maize hybrids with higher starch extractability would be of value to maize processors.     

Identification of kernel iron- and zinc-rich maize inbreds and analysis of genetic diversity using microsatellite markers

Development of micronutrient enriched staple foods is an important breeding goal in view of the extensive problem of ‘hidden hunger’ caused by micronutrient malnutrition. In the present study, kernel iron (Fe) and zinc (Zn) concentrations were evaluated in a set of 31 diverse maize inbred lines in three trials at two locations – Delhi (Kharif 2007 & 2008) and Hyderabad (Rabi 2007–08). The ranges of kernel Fe and Zn concentrations were 13.95–39.31 mg/kg and 21.85–40.91 mg/kg, respectively, across the three environments. Pooled analysis revealed significant genotype × environment (G × E) interaction in the expression of both the micronutrient traits, although kernel Fe was found to be more sensitive to G × E as compared to kernel Zn. Seven inbred lines, viz., BAJIM-06-03, DQPM-6, CM212, BAJIM-06-12, DQPM-7, DQPM-2 and CM129, were found to be the most stable and promising inbred lines for kernel Zn concentration, while for kernel Fe concentration, no promising and stable genotypes could be identified. Analysis of molecular diversity in 24 selected inbred lines with phenotypic contrast for the two kernel micronutrient traits, using 50 SSR markers covering the maize genome, revealed high levels of polymorphisms (214 SSR alleles; mean PIC value?=?0.62). The phenotypically contrasting and genetically diverse maize inbred lines identified in this study could be potentially utilized in further studies on QTL analysis of kernel micronutrient traits in maize, and the stable and most promising kernel micronutrient-rich maize genotypes provide a good foundation for developing micronutrient-enriched maize varieties suitable for the Indian context.     

Sugar utilization by developing wild type and shrunken-2 maize kernels

To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose.     

Quantification of Fungal Colonization,Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination^1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (10⁶) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.     

Overexpression of serine acetyltransferase in maize leaves increases seed‐specific methionine‐rich zeins

Maize kernels do not contain enough of the essential sulphur‐amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)‐amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S‐assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S‐assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell‐specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12‐fold higher SAT activity without negative impact on growth. S‐assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10‐kDa δ‐zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40‐fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high‐Met zein accumulation. Moreover, SAT overcomes the shortage of S‐amino acids that limits the expression and accumulation of high‐Met zeins during kernel development.     

Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize

The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms.     

Multiomics approach reveals a role of translational machinery in shaping maize kernel amino acid composition

Maize (Zea mays) seeds are a good source of protein, despite being deficient in several essential amino acids. However, eliminating the highly abundant but poorly balanced seed storage proteins has revealed that the regulation of seed amino acids is complex and does not rely on only a handful of proteins. In this study, we used two complementary omics-based approaches to shed light on the genes and biological processes that underlie the regulation of seed amino acid composition. We first conducted a genome-wide association study to identify candidate genes involved in the natural variation of seed protein-bound amino acids. We then used weighted gene correlation network analysis to associate protein expression with seed amino acid composition dynamics during kernel development and maturation. We found that almost half of the proteome was significantly reduced during kernel development and maturation, including several translational machinery components such as ribosomal proteins, which strongly suggests translational reprogramming. The reduction was significantly associated with a decrease in several amino acids, including lysine and methionine, pointing to their role in shaping the seed amino acid composition. When we compared the candidate gene lists generated from both approaches, we found a nonrandom overlap of 80 genes. A functional analysis of these genes showed a tight interconnected cluster dominated by translational machinery genes, especially ribosomal proteins, further supporting the role of translation dynamics in shaping seed amino acid composition. These findings strongly suggest that seed biofortification strategies that target the translation machinery dynamics should be considered and explored further.

An integrated approach reveals the key role of translational machinery in maize kernel amino acid natural variation and homeostasis, highlighting targets for seed amino acid biofortification.     

Exogenously applied glycinebetaine enhances seed and seed oil quality of maize (Zea mays L.) under water deficit conditions

A field trial was carried out to appraise up to what extent exogenous application of a potential osmoprotectant, glycinebetaine (GB), could ameliorate the inhibitory effects of shortage of water on maize seed and seed oil composition and oil antioxidant potential. Two maize cultivars, Agaiti-2002 (drought tolerant) and EV-1098 (drought sensitive), were exposed to drought treatments at the vegetative growth stage. Both the maize cultivars used in the present study are being widely cultivated in Pakistan and have been an important source of developing different maize hybrids. Two levels of glycinebetaine (0 or 30 mM) were foliar-applied at the vegetative stage. Water stress reduced the kernel sugar, oil, protein, moisture contents and most of the seed micro- and macro-nutrients analyzed of both maize cultivars, but it increased the contents of seed fiber and ash contents. Among different seed oil un-saturated fatty acids, water stress increased the oil oleic acid contents with a decrease in linoleic acid contents, which resulted in increased oil oleic/linoleic ratio of both maize cultivars. However, no variation was observed in oil stearic and palmitic acid contents due to water stress. A considerable increase in seed oil α-, γ-, δ- and total tocopherols and flavonoids was observed in both maize cultivars. However, oil phenolic content and 1,1′-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity decreased. Foliar-applied GB significantly increased the contents of seed sugar, oil, protein, moisture, fiber, ash, GB contents and micro- and macro-nutrients of both maize cultivars under well irrigated and water deficit conditions. Furthermore, exogenous application of GB increased the oil oleic and linoleinic acid contents. All different lipophilic compounds estimated in the seed oil increased due to foliar applied GB. Furthermore, GB also increased seed oil antioxidant activity appraised in terms of oil DPPH free radical scavenging activity. By summarizing the results, it seemed that exogenously applied GB remained in intact form until later stages of growth and counteracted the inhibitory effects of water deficit on seed and seed oil composition similarly of both maize cultivars.     

玉米籽粒性状的遗传效应分析

采用二倍体胚和三倍体胚乳种子遗传模型及其分析方法,以5个玉米自交系及其配制的F1,F2,BC1,BC2世代为材料,研究5个玉米种子性状的胚直接效应、胚乳直接效应、母体效应和细胞质效应。分析结果表明,除粒宽外,各性状的遗传同时由细胞质效应和胚、胚乳、母体基因效应所控制,百粒重主要受胚乳和母体效应的影响,粒长的遗传以母体效应为主,粒宽和粒厚以胚乳效应为主。各部位籽粒百粒重的胚乳直接加性效应与母体加性效应的协方差达到显著或极显著水平,其余性状的胚、胚乳直接效应与母体效应间的协方差均不显著,通过母体植株的遗传表现可以对这些性状进行有效的选择。S22 是改良百粒重的优良亲本。 Abstract：The embryo,endosperm and cytoplasm effects of seven seed traits were studied by genetic model for diploid embryo and triploid endosperm plant seeds using five inbreds and their F1, F2, BC1 and BC2 generations. The estimates of genetic variance components indicated that the inheritance of all other kernel traits was controlled by the four effects except kernel width. The 100?kernel weight was mainly controlled by endosperm and maternal effects , and kernel length was controlled by the maternal effects,while endosperm conrrolled kernel width and kernel thickness. Except the significant or highly significant covariances between the endosperm direct additive and maternal additive effects for 100－kernel weight,all other traits between the embryo or endosperm direct effect and the maternal were not significant. So,maize inbreds could be developed by direct selection based on maternal plants for these traits. S22 was the best inbred of the improvement for kernel weight in this study.     

Sublethal effects of Cry 1F Bt corn and clothianidin on black cutworm (Lepidoptera: Noctuidae) larval development

Black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), is an occasional pest of maize (corn), Zea mays L., that may cause severe stand losses and injury to corn seedlings. The efficacy of the neonicotinoid seed treatment clothianidin at two commercially available rates and their interaction with a transgenic corn hybrid (Bt corn), trait expressing the Bacillus thuringiensis variety aizawai insecticidal toxin Cry 1Fa2, against black cutworm larvae was investigated. Clothianidin at a rate of 25 mg kernel(-1) on Bt corn increased larval mortality and reduced larval weight gains additively. In contrast, weights of larvae fed non-Bt corn seedlings treated with clothianidin at a rate of 25 mg kernel(-1) increased significantly, suggesting either compensatory overconsumption, hormesis, or hormoligosis. Both Bt corn alone and clothianidin at a rate of 125 mg kernel(-1) applied to non-Bt corn seedlings caused increased mortality and reduced larval weight gains. In two field trials, plots planted with Bt corn hybrids consistently had the highest plant populations and yields, regardless of whether they were treated with clothianidin at the lower commercial rate of 25 mg kernel(-1) The use of Bt corn alone or in combination with the low rate of clothianidin (25 mg kernel(-1)) seems suitable as a means of suppressing black cutworm in no-tillage cornfields, although rescue treatments may still be necessary under severe infestations. Clothianidin alone at the low rate of 25 mg kernel(-1) is not recommended for black cutworm control until further studies of its effects on larval physiology and field performance have been completed.     

The Effects of Modifying Sucrose Concentration on the Development of Maize Kernels Grown in vitro

Intact Zea mays L. kernels attached to cob tissue develop tomaturity when grown in vitro. This experiment was designed todetermine if it is possible to prolong kernel growth by refreshingthe culture medium. Blocks of maize kernels were grown in vitroon media containing several concentrations of sucrose. Kernels,at all concentrations of sucrose, developed to maturity at 30–35d post-pollination, indicating that it is not possible to extendthe kernel growth phase by supplying a carbohydrate source.Kernels grown on media containing 80 g 1^–1 or higher sucroseconcentration had a significantly greater percentage of kernelsthat developed to maturity, and had greater weight and starchcontent per seed. Zea mays, kernel culture, seed development, starch     

The retromer protein ZmVPS29 regulates maize kernel morphology likely through an auxin‐dependent process(es)

Kernel size and morphology are two important yield‐determining traits in maize, but their molecular and genetic mechanisms are poorly characterized. Here, we identified a major QTL, qKM4.08, which explains approximately 24.20% of the kernel morphology variance in a recombinant population derived from two elite maize inbred lines, Huangzaosi (HZS, round kernel) and LV28 (slender kernel). Positional cloning and transgenic analysis revealed that qKM4.08 encodes ZmVPS29, a retromer complex component. Compared with the ZmVPS29 HZS allele, the ZmVPS29 LV28 allele showed higher expression in developing kernels. Overexpression of ZmVPS29 conferred a slender kernel morphology and increased the yield per plant in different maize genetic backgrounds. Sequence analysis revealed that ZmVPS29 has been under purifying selection during maize domestication. Association analyses identified two significant kernel morphology‐associated polymorphic sites in the ZmVPS29 promoter region that were significantly enriched in modern maize breeding lines. Further study showed that ZmVPS29 increased auxin accumulation during early kernel development by enhancing auxin biosynthesis and transport and reducing auxin degradation and thereby improved kernel development. Our results suggest that ZmVPS29 regulates kernel morphology, most likely through an auxin‐dependent process(es).     

Oleosin isoforms of high and low molecular weights are present in the oil bodies of diverse seed species

Oleosins are unique and major proteins localized on the surface of oil bodies in diverse seed species. We purified five different oleosins (maize [Zea mays L.] KD 16 and KD 18, soybean [Glycine max L.] KD 18 and KD 24, and rapeseed [Brassica campestris L.] KD 20), and raised chicken antibodies against them. These antibodies were used to test for immunological cross-reactivity among oleosins from diverse seed species. Within the same seed species, antibodies raised against one oleosin isoform did not cross-react with the other oleosin isoform (i.e. between maize oleosins KD 16 and KD 18, and between soybean oleosins KD 18 and KD 24). However, the respective antibodies were able to recognize oleosins from other seed species. Where interspecies cross-reactivity occurred, the results suggest that there are at least two immunologically distinct isoforms of oleosins present in diverse seed species, one of lower M_r, and another one of higher M_r. This suggestion is also supported by the relative similarities between the amino acid sequence of a small portion of rapeseed oleosin KD 20 and those of maize oleosins KD 16 and KD 18. In maize kernel, there was a tissue-specific differential presentation of the three oleosins, KD 16, KD 18, and KD 19, in the oil-storing scutellum, embryonic axis, and aleurone layer. The phylogenetic relationship between the high and low M_r isoforms within the same, and among diverse, seed species is discussed.     

A Foundation for Provitamin A Biofortification of Maize: Genome-Wide Association and Genomic Prediction Models of Carotenoid Levels

Efforts are underway for development of crops with improved levels of provitamin A carotenoids to help combat dietary vitamin A deficiency. As a global staple crop with considerable variation in kernel carotenoid composition, maize (Zea mays L.) could have a widespread impact. We performed a genome-wide association study (GWAS) of quantified seed carotenoids across a panel of maize inbreds ranging from light yellow to dark orange in grain color to identify some of the key genes controlling maize grain carotenoid composition. Significant associations at the genome-wide level were detected within the coding regions of zep1 and lut1, carotenoid biosynthetic genes not previously shown to impact grain carotenoid composition in association studies, as well as within previously associated lcyE and crtRB1 genes. We leveraged existing biochemical and genomic information to identify 58 a priori candidate genes relevant to the biosynthesis and retention of carotenoids in maize to test in a pathway-level analysis. This revealed dxs2 and lut5, genes not previously associated with kernel carotenoids. In genomic prediction models, use of markers that targeted a small set of quantitative trait loci associated with carotenoid levels in prior linkage studies were as effective as genome-wide markers for predicting carotenoid traits. Based on GWAS, pathway-level analysis, and genomic prediction studies, we outline a flexible strategy involving use of a small number of genes that can be selected for rapid conversion of elite white grain germplasm, with minimal amounts of carotenoids, to orange grain versions containing high levels of provitamin A.