Temporal profiling of essential amino acids in developing maize kernel of normal,opaque - 2 and QPM germplasm

Maize, an important cereal crop, has a poor quality of endosperm protein due to the deficiency of essential amino acids, especially lysine and tryptophan. Discovery of mutants such as opaque-2 led to the development of nutritionally improved maize with a higher concentration of lysine and tryptophan. However, the pleiotropic effects associated with opaque-2 mutants necessitated the development of nutritionally improved hard kernel genotype, the present-day quality protein maize (QPM). The aim of present study was to analyze and compare the temporal profile of lysine and tryptophan in the developing maize kernel of normal, opaque-2 and QPM lines. A declining trend in protein along with tryptophan and lysine content was observed with increasing kernel maturity in the experimental genotypes. However, opaque-2 retained the maximum concentration of lysine (3.43) and tryptophan (1.09) at maturity as compared to QPM (lysine-3.05, tryptophan-0.99) and normal (lysine-1.99, tryptophan-0.45) lines. Opaque-2 mutation affects protein quality but has no effect on protein quantity. All maize types are nutritionally rich at early stages of kernel development indicating that early harvest for cattle feed would ensure a higher intake of lysine and tryptophan. Two promising lines (CML44 and HKI 1105) can be used for breeding high value corn for cattle feed or human food in order to fill the protein inadequacy gap. Variation in lysine and tryptophan content within QPM lines revealed that differential expression of endosperm modifiers with varying genetic background significantly affects nutritional quality, indicating that identification of alleles affecting amino acid composition can further facilitate QPM breeding program.     

Endosperm Protein Quality and Kernel Modification in the Quality Protein Maize Inbred Lines

Twenty-two selected quality protein maize (QPM) lines, including 13 lines developed in India (DMRQPM series) and nine lines released by CIMMYT, Mexico (CML series), were evaluated for their endosperm protein content and quality, besides kernel modification in terms of vitreousness. Endosperm protein contents in 13QPMlines were on par or better than that of the normal maize ‘checks’ (Trishulata and Parkash). The QPM endosperm proteins showed significantly higher % tryptophan as well as EF-1α (a multifunctional protein with a positive and highly significant correlation with lysine content in the endosperm) contents, in comparison with the normal maize genotypes. Evaluation of kernel modification revealed considerable scope for accumulation of endosperm modifiers in some of the QPM lines. Positive and highly significant correlation was revealed between tryptophan and EF-1α contents in the endosperm proteins, whereas the correlations between the quality parameters with kernel modification in the QPM genotypes were found to be non-significant. The study led to the identification of some promising QPM lines, such as DMRQPM-37, DMRQPM-44, CML176, CML142 and CML149, which could be effectively deployed in the QPM breeding programmes.     

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.     

Two-generation marker-aided backcrossing for rapid conversion of normal maize lines to quality protein maize (QPM)

The low nutritive value of maize endosperm protein is genetically corrected in quality protein maize (QPM), which contains the opaque2 gene along with numerous modifiers for kernel hardness. We report here a two generation marker-based backcross breeding program for incorporation of the opaque2 gene along with phenotypic selection for kernel modification in the background of an early maturing normal maize inbred line, V25. Using the flanking marker distances from opaque2 gene in the cross V25×CML176, optimum population size for the BC₂ generation was computed in such a way that at least one double recombinant could be obtained. Whole genome background selection in the BC₂ generation identified three plants with 93 to 96% recurrent parent genome content. The three BC₂F₂ families derived from marker identified BC₂ individuals were subjected to foreground selection and phenotypic selection for kernel modification. The tryptophan concentration in endosperm protein was significantly enhanced in all the three classes of kernel modification viz., less than 25%, 25–50% and more than 50% opaqueness. BC₂F₃ lines developed from the hard endosperm kernels were evaluated for desirable agronomic and biochemical traits in replicated trials and the best line was chosen to represent the QPM version of V25, with tryptophan concentration of 0.85% in protein. The integrated breeding strategy reported here can be applied to reduce genetic drag as well as the time involved in a conventional line conversion program, and would prove valuable in rapid development of specialty corn germplasm.     

RNA interference can rebalance the nitrogen sink of maize seeds without losing hard endosperm

Background

One of the goals of plant breeding is to create crops to provide better nutrition for humans and livestock. Insufficient intake of protein is one of the most severe factors affecting the growth and development of children in developing countries. More than a century ago, in 1896, Hopkins initiated the well-known Illinois long-term selection for maize seed protein concentration, yielding four protein strains. By continuously accumulating QTLs, Illinois High Protein (IHP) reached a protein level 2.5-fold higher than normal maize, with the most increased fraction being the zein protein, which was shown to contain no lysine soon after the long-term selection program initiated. Therefore, IHP is of little value for feeding humans and monogastric animals. Although high-lysine lines of non-vitreous mutants were based on reduced zeins, the kernel soft texture precluded their practical use. Kernel hardness in opaque 2 (o2) could be restored in quality protein maize (QPM) with quantitative trait loci called o2 modifiers (Mo2s), but those did not increase total protein levels.

Methods

The most predominant zeins are the 22- and 19-kDa α-zeins. To achieve a combination of desired traits, we used RNA interference (RNAi) against both α-zeins in IHP and evaluated the silencing effect by SDS-PAGE. Total protein, amino acid composition and kernel texture were analyzed.

Conclusions

The α-zeins were dramatically reduced, but the high total seed protein level remained unchanged by complementary increase of non-zein proteins. Moreover, the residual zein levels still allowed for a vitreous hard seed. Such dramatic rebalancing of the nitrogen sink could have a major impact in world food supply.     

Characterization of <Emphasis Type="Italic">opaque2</Emphasis> modifier QTLs and candidate genes in recombinant inbred lines derived from the K0326Y quality protein maize inbred

Quality protein maize (QPM) is a high lysine-containing corn that is based on genetic modification of the opaque2 (o2) mutant. In QPM, modifier genes convert the starchy endosperm of o2 to the vitreous phenotype of wild type maize. There are multiple, unlinked o2 modifier loci (Opm) in QPM and their nature and mode of action are unknown. We previously identified seven Opm QTLs and characterized 16 genes that are differentially up-regulated at a significant level in K0326Y QPM, compared to the starchy endosperm mutant W64Ao2. In order to further characterize these Opm QTLs and the genes up-regulated in K0326Y QPM, we created a population of 314 recombinant inbred lines (RILs) from a cross between K0326Y QPM and W64Ao2. The RILs were characterized for three traits associated with endosperm texture: vitreousness, density and hardness. Genetic linkage analysis of the RIL population confirmed three of the previously identified QTLs associated with o2 endosperm modification in K0326Y QPM. Many of the genes up-regulated in K0326Y QPM showed substantially higher levels of expression in vitreous compared with opaque RILs. These included genes associated with the upstream regulation of the ethylene response pathway, and a gene encoding a regulatory subunit of pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase, an adaptive enzyme of the glycolytic pathway.     

Influence of genotype and texture on zein content in endosperm of maize grains

The effect of genotypes and texture on the content of proteins in maize grains was examined by assessing absolute amounts of six protein fractions in the whole endosperms of four wild‐type lines with high protein content and four quality protein maize (QPM) varieties and for hand‐dissected hard and soft endosperm regions from eight other lines. As previously reported for six wild‐type lines and their opaque‐2(o2) versions, zeins were predominant for all genetic backgrounds and all types of endosperms. From these data and others the amounts of zeins and true proteins (crude proteins free of non‐protein nitrogen) in developing and mature endosperms of wild‐type lines were correlated. The data points for zeins from hard endosperms lay between the regression line and the upper limit of confidence area. Those for zeins from soft endosperms were located at the lower part of confidence area and on a level with the points corresponding to the most immature endosperms. Furthermore, some data points for zeins from o2 and QPM samples lay near the lower limit while the others were outside the confidence area. This suggested an initial zein accumulation dependent on the genotype at a low relative rate, followed by an accumulation at higher rate. The conditions used for isolating and quantitating zeins are discussed.     

SSB recruitment of Exonuclease I aborts template-switching in Escherichia coli

Misalignment of a nascent strand and the use of an alternative template during DNA replication, a process termed “template-switching”, can give rise to frequent mutations and genetic rearrangements. Mutational hotspots are frequently found associated with imperfect inverted repeats (“quasipalindromes” or “QPs”) in many organisms, including bacteriophage, bacteria, yeast and mammals. Evidence suggests that QPs mutate by a replication template-switch whereby one copy of the inverted repeat templates synthesis of the other. To study quasipalindrome-associated mutagenesis (“QPM”) more systematically, we have engineered mutational reporters in the lacZ gene of Escherichia coli, that revert to Lac⁺ specifically by QPM. We and others have shown that QPM is more efficient during replication of the leading strand than it is on the lagging strand. We have previously shown that QPM is elevated and that the leading-strand bias is lost in mutants lacking the major 3′ ssDNA exonucleases, ExoI and ExoVII. This suggests that one or both of these exonucleases more efficiently abort template-switches on the lagging strand. Here, we show that ExoI is primarily responsible for this bias and that its ability to be recruited by single-strand DNA binding protein plays a critical role in QPM avoidance and strand bias. In addition to these stand-alone exonucleases, loss of the 3′ proofreading exonuclease activity of the replicative DNA polymerase III also greatly elevates QPM. This may be because template-switching is initiated by base misincorporation, leading to polymerase dissociation and subsequent nascent strand misalignment; alternatively or additionally, the proofreading exonuclease may scavenge displaced 3′ DNA that would otherwise be free to misalign.     

Characterization of the desiccation of wheat kernels by multivariate imaging

Variations in the quality of wheat kernels can be an important problem in the cereal industry. In particular, desiccation conditions play an essential role in both the technological characteristics of the kernel and its ability to sprout. In planta desiccation constitutes a key stage in the determinism of the functional properties of seeds. The impact of desiccation on the endosperm texture of seed is presented in this work. A simple imaging system had previously been developed to acquire multivariate images to characterize the heterogeneity of food materials. A special algorithm for the use under principal component analysis (PCA) was developed to process the acquired multivariate images. Wheat grains were collected at physiological maturity, and were subjected to two types of drying conditions that induced different kinetics of water loss. A data set containing 24 images (dimensioned 702 × 524 pixels) corresponding to the different desiccation stages of wheat kernels was acquired at different wavelengths and then analyzed. A comparison of the images of kernel sections highlighted changes in kernel texture as a function of their drying conditions. Slow drying led to a floury texture, whereas fast drying caused a glassy texture. The automated imaging system thus developed is sufficiently rapid and economical to enable the characterization in large collections of grain texture as a function of time and water content.     

优质蛋白玉米02基因控制赖氨酸超量积累的分析

以３８个ＱＰＭ（或０２）和对照普通玉米为实验材料,进行０２基因控制赖氨酸超量积累的生化和遗传分析。主要实验结果如下：（１）ＱＰＭ玉米０２基因为隐性的单基因遗传,它控制着胚乳、雄穗和幼苗期叶片中赖氨酸的超量积累,一些修饰因子和遗传背景对胚乳物理性状产生影响;（２）ＱＰＭ玉米、普通玉米的胚较之胚乳,或者ＱＰＭ玉米胚乳较之普通玉米胚乳都含有较多的天门冬氮酸、甘氨酸、赖氨酸和精氨酸,含有较少的脯氨酸、谷氨酸、亮氨酸和苯丙氨酸;（３）两种玉米之间,在胚乳蛋白质含量及胚乳可溶性蛋白、醇溶蛋白、谷蛋白的赖氨酸含量方面没有什么不同;（４）已经育成一批ＱＰＭ或０２玉米自交系,并配制出几个强优势杂交组合。     

Relationship between structural and biochemical characteristics and texture of corn grains

The texture of corn grains is a fundamental characteristic for the industry as well as for grain producers and processors. To further understand the mechanisms involved in grain hardness, contrasting corn cultivars for grain hardness and protein quality were evaluated. The cultivars were Cateto L237/67 (hard endosperm and low protein value), QPM BR 451 (semi-hard endosperm and high protein value); Bolivia-2 (floury endosperm and low protein value), and Opaque-2 (floury endosperm and high protein value). Evaluations were carried out at 10, 20, 30, 40, 50, and 60 days after pollination in developing corn grains and in the mature grain. In developing grains, evaluation consisted in the determination of the area, percentage of starch granules, distribution of starch granules, and protein bodies in the endosperm. In mature corn grains, the parameters evaluated were: density, percentage of total proteins, levels of lysine and tryptophan, and banding pattern of zeins. The results indicate that the higher physical resistance of corn grains from the cultivars analyzed is influenced by the high percentage of total proteins, high synthesis of 27-kDa zeins, presence of protein bodies, and perfect organization of starch granules in the endosperm, independent of their sizes.     

Puroindolines: the molecular genetic basis of wheat grain hardness

The variation in grain hardness is the single most important trait that determines end-use quality of wheat. Grain texture classification is based primarily on either the resistance of kernels to crushing or the particle size distribution of ground grain or flour. Recently, the molecular genetic basis of grain hardness has become known, and it is the focus of this review. The puroindoline proteins a and b form the molecular basis of wheat grain hardness or texture. When both puroindolines are in their `functional' wild state, grain texture is soft. When either one of the puroindolines is absent or altered by mutation, then the result is hard texture. In the case of durum wheat which lacks puroindolines, the texture is very hard. Puroindolines represent the molecular-genetic basis of the Hardness locus on chromosome 5DS and the soft (Ha) and hard (ha) alleles present in hexaploid bread wheat varieties. To date, seven discrete hardness alleles have been described for wheat. All involve puroindoline a or b and have been designated Pina-D1b and Pinb-D1b through Pinb-D1g. A direct role of a related protein, grain softness protein (as currently defined), in wheat grain texture has yet to be demonstrated.     

花生镉污染研究进展

花生既是世界主要的油料作物,又是重要的植物蛋白来源和食品加工原料.随着花生直接食用和食品加工的不断增加,国际上对花生籽粒Cd含量问题越来越关注.我国是世界上重要的花生生产国和出口国.近年来,花生Cd含量偏高已经成为制约我国出口贸易的重要因素.本文从花生籽粒Cd富集能力、花生Cd含量的种内差异、籽粒中Cd的分布规律、影响花生籽粒Cd积累的机制和降低花生籽粒Cd含量技术等方面,对花生Cd污染研究的现状与问题进行了论述.指出在花生Cd污染控制方面有2种策略可以考虑,一是降低花生对土壤Cd的吸收;二是控制Cd向籽粒的迁移富集.为此需要从3个方面加强对花生籽粒Cd积累机制的研究,即花生根系活性特征参数及其与籽粒Cd积累的关系;花生果荚Cd吸收机制及其对籽粒Cd含量的贡献;花生植株体内Cd迁移机制及其与籽粒Cd含量的关系.     

Molecular genetics of puroindolines and related genes: regulation of expression,membrane binding properties and applications

Kernel texture of wheat is a primary determinant of its technological properties. Soft kernel texture phenotype results when the Puroindoline a and Puroindoline b genes are present and encode the wild-type puroindolines PINA and PINB, respectively, and various mutations in either or both gene(s) result in hard phenotypes. A wealth of information is now available that furthers our understanding regarding the spatial and temporal regulation of expression of Puroindoline genes. Through the use of model membranes and synthetic peptides we also have a clearer understanding of the significance of the cysteine backbone, the tryptophan-rich domain (TRD) and the helicoid tertiary structures of PIN proteins in relation to their membrane-active properties. Many studies suggest individual yet co-operative modes of action of the PIN proteins in determining kernel texture, and significant evidence is accumulating that the proteins have in vivo and in vitro antimicrobial activities, shedding light on the biological roles of this unique ensemble of proteins. The puroindolines are now being explored for grain kernel texture modifications as well as antimicrobial activities.     

Genome mapping of kernel characteristics in hard red spring wheat breeding lines

Kernel characteristics, particularly kernel weight, kernel size, and grain protein content, are important components of grain yield and quality in wheat. Development of high performing wheat cultivars, with high grain yield and quality, is a major focus in wheat breeding programs worldwide. Here, we report chromosome regions harboring genes that influence kernel weight, kernel diameter, kernel size distribution, grain protein content, and grain yield in hard red spring wheat breeding lines adapted to the Upper Midwest region of the United States. A genetic linkage map composed of 531 SSR and DArT marker loci spanned a distance of 2,505 cM, covering all 21 chromosomes of wheat. Stable QTL clusters influencing kernel weight, kernel diameter, and kernel size distribution were identified on chromosomes 2A, 5B, and 7A. Phenotypic variation explained by individual QTL at these clusters varied from 5 to 20% depending on the trait. A QTL region on chromosome 2B confers an undesirable pleiotropic effect or a repulsion linkage between grain yield (LOD = 6.7; R ² = 18%) and grain protein content (LOD = 6.2; R ² = 13.3%). However, several grain protein and grain yield QTL independent of each other were also identified. Because some of the QTL identified in this study were consistent across environments, DNA markers will provide an opportunity for increasing the frequency of desirable alleles through marker-assisted selection.     

Predicting complex traits using a diffusion kernel on genetic markers with an application to dairy cattle and wheat data

Background

Arguably, genotypes and phenotypes may be linked in functional forms that are not well addressed by the linear additive models that are standard in quantitative genetics. Therefore, developing statistical learning models for predicting phenotypic values from all available molecular information that are capable of capturing complex genetic network architectures is of great importance. Bayesian kernel ridge regression is a non-parametric prediction model proposed for this purpose. Its essence is to create a spatial distance-based relationship matrix called a kernel. Although the set of all single nucleotide polymorphism genotype configurations on which a model is built is finite, past research has mainly used a Gaussian kernel.

Results

We sought to investigate the performance of a diffusion kernel, which was specifically developed to model discrete marker inputs, using Holstein cattle and wheat data. This kernel can be viewed as a discretization of the Gaussian kernel. The predictive ability of the diffusion kernel was similar to that of non-spatial distance-based additive genomic relationship kernels in the Holstein data, but outperformed the latter in the wheat data. However, the difference in performance between the diffusion and Gaussian kernels was negligible.

Conclusions

It is concluded that the ability of a diffusion kernel to capture the total genetic variance is not better than that of a Gaussian kernel, at least for these data. Although the diffusion kernel as a choice of basis function may have potential for use in whole-genome prediction, our results imply that embedding genetic markers into a non-Euclidean metric space has very small impact on prediction. Our results suggest that use of the black box Gaussian kernel is justified, given its connection to the diffusion kernel and its similar predictive performance.     

玉米籽粒淀粉含量遗传基础与调控机制*

玉米是世界上种植面积最大、总产量最高的粮食作物,其籽粒重量的70%来自于淀粉。淀粉不仅是人类及其他动物的主要能量来源,同时也是化工等行业的重要原料。利用拟南芥、水稻等模式植物,淀粉合成相关基因克隆与功能研究已取得较多进展。近年来,随着玉米淀粉含量相关遗传学研究的深入开展,通过数量性状位点(quantitative trait locus mapping,QTL)定位、全基因组关联分析(genome-wide association study, GWAS)及各种组学分析方法,发现了较多新的与淀粉含量相关的遗传位点及候选基因,但是尚缺乏归纳总结。综述了玉米籽粒淀粉合成与调控研究进展,对玉米籽粒淀粉含量相关的QTL和基因进行汇总和分析,通过构建一致性物理图谱,提炼玉米籽粒淀粉含量遗传定位热点区间,这为进一步解析玉米籽粒淀粉合成与代谢相关基因的功能提供参考,并为分子标记辅助育种提供遗传资源。     

Wide variability in kernel composition, seed characteristics, and zein profiles among diverse maize inbreds, landraces, and teosinte

All crop species have been domesticated from their wild relatives, and geneticists are just now beginning to understand the consequences of artificial (human) selection on agronomic traits that are relevant today. The primary consequence is a basal loss of diversity across the genome, and an additional reduction in diversity for genes underlying traits targeted by selection. An understanding of attributes of the wild relatives may provide insight into target traits and valuable allelic variants for modern agriculture. This is especially true for maize (Zea mays ssp. mays), where its wild ancestor, teosinte (Z. mays ssp. parviglumis), is so strikingly different than modern maize. One obvious target of selection is the size and composition of the kernel. We evaluated kernel characteristics, kernel composition, and zein profiles for a diverse set of modern inbred lines, teosinte accessions, and landraces, the intermediate between inbreds and teosinte. We found that teosinte has very small seeds, but twice the protein content of landraces and inbred lines. Teosinte has a higher average alpha zein content (nearly 89% of total zeins as compared to 72% for inbred lines and 76% for landraces), and there are many novel alcohol-soluble proteins in teosinte relative to the other two germplasm groups. Nearly every zein protein varied in abundance among the germplasm groups, especially the methionine-rich delta zein protein, and the gamma zeins. Teosinte and landraces harbor phenotypic variation that will facilitate genetic dissection of kernel traits and grain quality, ultimately leading to improvement via traditional plant breeding and/or genetic engineering.     

Genetic analysis of opaque2 modifier loci in quality protein maize

Quality protein maize (QPM) was created by selecting genetic modifiers that convert the starchy endosperm of an opaque2 (o2) mutant to a hard, vitreous phenotype. Genetic analysis has shown that there are multiple, unlinked o2 modifiers (Opm), but their identity and mode of action are unknown. Using two independently developed QPM lines, we mapped several major Opm QTLs to chromosomes 1, 7 and 9. A microarray hybridization performed with RNA obtained from true breeding o2 progeny with vitreous and opaque kernel phenotypes identified a small group of differentially expressed genes, some of which map at or near the Opm QTLs. Several of the genes are associated with ethylene and ABA signaling and suggest a potential linkage of o2 endosperm modification with programmed cell death.