Genetic architecture of maize kernel composition in the nested association mapping and inbred association panels

The maize (Zea mays) kernel plays a critical role in feeding humans and livestock around the world and in a wide array of industrial applications. An understanding of the regulation of kernel starch, protein, and oil is needed in order to manipulate composition to meet future needs. We conducted joint-linkage quantitative trait locus mapping and genome-wide association studies (GWAS) for kernel starch, protein, and oil in the maize nested association mapping population, composed of 25 recombinant inbred line families derived from diverse inbred lines. Joint-linkage mapping revealed that the genetic architecture of kernel composition traits is controlled by 21-26 quantitative trait loci. Numerous GWAS associations were detected, including several oil and starch associations in acyl-CoA:diacylglycerol acyltransferase1-2, a gene that regulates oil composition and quantity. Results from nested association mapping were verified in a 282 inbred association panel using both GWAS and candidate gene association approaches. We identified many beneficial alleles that will be useful for improving kernel starch, protein, and oil content.     

QTLs for enzyme activities and soluble carbohydrates involved in starch accumulation during grain filling in maize

ADPglucose, the essential substrate for starch synthesis, is synthesized in maize by a pathway involving at least invertases, sucrose synthase, and ADPglucose pyrophosphorylase, as shown by the starch-deficient mutants, mn1, sh1, and bt2 or sh2, respectively. To improve understanding of the relationship between early grain-filling traits and carbohydrate composition in mature grain, QTLs linked to soluble invertase, sucrose synthase, and ADPglucose pyrophosphorylase activities and to starch, sucrose, fructose, and glucose concentrations were investigated. In order to take into account the specific time-course of each enzyme activity during grain filling, sampling was carried out at three periods (15, 25, and 35 d after pollination) on 100 lines from a recombinant inbred family, grown in the field. The MQTL method associated with QTL interaction analysis revealed numerous QTLs for all traits, but only one QTL was consistently observed at the three sampling periods. Some chromosome zones were heavily labelled, forming clusters of QTLs. Numerous possible candidate genes of the starch synthetic pathway co-located with QTLs. Four QTLs were found close to the locus Sh1 (bin 9.01) coding for the sucrose synthase. In order to confirm the importance of this locus, the CAPS polymorphism of the Sh1 gene was analysed in 45 genetically unrelated maize lines from various geographical origins. The DNA polymorphism was significantly associated with phenotypic traits related to grain filling (starch and amylose content, grain matter, and ADPglucose pyrophosphorylase activity at 35 DAP). Thus, the Sh1 locus could provide a physiologically pertinent marker for maize selection.     

Analysis of genetic mapping in a waxy/dent maize RIL population using SSR and SNP markers

A maize genetic linkage map was generated using SSR and SNP markers in a F_7:8 recombinant inbred line (RIL) population derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 465 markers, including 459 SSR and 6 SNP markers, were assigned to 10 linkage groups which spanned 2,656.5 cM with an average genetic distance between markers of 5.7 cM, and the number of loci per linkage group ranged from 39 to 55. The SSR (85.4%) and SNP (83.3%) markers showed Mendelian segregation ratios in the RIL population at a 5% significance threshold. In linkage analysis of six SNP loci associated with kernel starch synthesis genes (ae1, bt2, sh1, sh2, su1, and wx1), all six loci were successfully mapped and are closely linked with SSR markers in chromosomes 3 (sh2), 4 (su1 and bt2), 5 (ae1), and 9 (sh1 and wx1). The SSR markers linked with genes in starch synthesis may be utilized in marker assisted breeding programs. The resulting genetic map will be useful in dissection of quantitative traits and the identification of superior QTLs from the waxy hybrid corn. Additionally, these data support further genetic analysis and development of maize breeding programs.     

Genetic diversity of maize kernel starch-synthesis genes with SNAPs.

Measuring genetic diversity in populations of a crop species is very important for understanding the genetic structure of and subsequently improving the crop species by genetic manipulation. Single-nucleotide amplified polymorphisms (SNAPs) among and within maize populations of waxy, dent, and sweet corns at 25 single-nucleotide polymorphism (SNP) sites in 6 kernel starch-synthesis genes (sh2, bt2, su1, ae1, wx1, and sh1) were determined. Because of the intensive selection of some favorable alleles in starch-synthesis genes during the breeding process, and the resultant strong linkage disequilibrium (LD), the number of haplotypes in each population was far less than expected. Subsequent phenetic clustering analysis with the SNAPs indicated that the dent, waxy, and sweet corns formed distinct subclusters, except in a few incidences. LD was surveyed among SNAPs of intragenic, intergenic, and intrachromosomal SNPs in whole and subpopulations, which revealed that some SNAPs showed high LD with many other SNAPs, but some SNAPs showed low or no significant LD with others, depending on the subpopulation, indicating that these starch genes have undergone different selection in each subpopulation during the breeding process. Because the starch synthesis genes used in this study are important in maize breeding, the genetic diversity, LD, and accessions having rare SNAP alleles might be valuable in maize improvement programs.     

Scanning Electron Microscopy and Fermentation Studies on Selected Known Maize Starch Mutants Using STARGEN? Enzyme Blends

The conversion of maize (corn) kernels to bio-ethanol is an energy-intensive process involving many stages. One step typically required is the liquefaction of the ground kernel to enable enzyme hydrolysation of the starch to glucose. The enzyme blends STARGEN? (Genencor) are capable of hydrolysing starch granules without liquefaction, reducing energy inputs and increasing efficiency. Studies were conducted on maize starch mutants amylose extender 1 (ae1), dull 1 (du1) and waxy 1 (wx1) in the inbred line Oh43 to determine whether different maize starches affected hydrolysation rates by STARGEN? 001 and STARGEN? 002. All mutants contained similar proportions of starch in the kernel but varied in the amylose to amylopectin ratio. Ground maize kernels were incubated with STARGEN? 001 and viewed using scanning electron microscopy to examine the hydrolysis action of STARGEN? 001 on the starch granules. The ae1 mutant exhibited noticeably less enzymic hydrolysis action, on the granules visualised, than wx1 and background line Oh43. Kernels were batch-fermented with STARGEN? 001 and STARGEN? 002. The ae1 mutant exhibited a 50% lower ethanol yield compared to the wx1 mutant and background line. A final study compared hydrolysation rates of STARGEN? 001 and STARGEN? 002 on purified maize starch, amylopectin and amylose. Though almost twice the amylopectin was hydrolysed using STARGEN? 002 than STARGEN? 001 in this trial, fermentations using STARGEN? 002 resulted in lower ethanol yields than fermentations using STARGEN? 001. Both STARGEN? enzyme blends were more suitable for the fermentation of high amylopectin maize starches than high amylose starches.     

Quantitative trait loci affecting amylose,amylopectin and starch content in maize recombinant inbred lines

The loci explaining the variability of quantitative traits related to starch content and composition (amylose, amylopectin and water soluble fraction) were searched for in maize kernels. Multifactorial genetic methods were used to detect and locate QTLs (quantitative trait loci) on a genetic map consisting mainly of RFLP markers for genes with known function. The genetic material was recombinant inbred lines originating from parents differing in starch structure (dent vs. flint). Kernels were harvested from field grown plants for two successive years and under two pollination systems. Main effect and epistasis QTLs were detected using two methods, composite interval mapping (MQTL) and ANOVA. Despite large year-to-year differences, physiologically meaningful co-locations were observed between trait QTLs. Moreover, the number of expressed sequences on our map allowed the search for co-locations between QTLs and genes involved in carbohydrate metabolism. The main co-location was between an amylose QTL and Shrunken 2 (SH2) locus, on chromosome 3 (SH2 encoding for the large subunit of ADPglucose pyrophosphorylase). The importance of this locus as a candidate gene for a starch QTL is in agreement with previous studies based either on QTL co-locations or on revertant analysis. Other co-locations were observed between amylose and amylopectin QTLs and the two loci of IVR1 invertase genes on chromosomes 2 and 10. Further comparison with previously detected QTLs for carbohydrate metabolism in maize leaves showed consistent co-location in map regions devoid of candidate genes, such as near chromosome 1S telomere. The possible contribution of regulatory genes in this region is discussed.     

Nucleotides and Nucleotide Sugars in Developing Maize Endosperms (Synthesis of ADP-Glucose in brittle-1)

As part of an in vivo study of carbohydrate metabolism during development of Zea mays L. kernels, quantities of nucleotides and nucleotide sugars were measured in endosperm extracts from normal, the single-mutant genotypes shrunken-1 (sh1), shrunken-2 (sh2), and brittle-1 (btl}, and the multiple-mutant genotypes sh1bt1, sh2bt1, and sh1sh2bt1. Results showed that bt1 kernels accumulated more than 13 times as much adenosine 5[prime] diphospho-glucose (ADP-Glc) as normal kernels. Activity of starch synthase in bt1 endosperm was equal to that in endosperm extracts from normal kernels. Thus the ADP-Glc accumulation in bt1 endosperm cells was not due to a deficiency in starch synthase. ADP-Glc content in extracts of sh1bt1 endosperms was similar to that in bt1, but in extracts of the sh2bt1 mutant kernels ADP-Glc content was much reduced compared to bt1 (about 3 times higher than that in normal). Endosperm extracts from sh1sh2bt1, kernels that are deficient in both ADP-Glc pyrophosphorylase (AGPase) and sucrose synthase, had quantities of ADP-Glc much lower than in normal kernels. These results clearly indicate that AGPase is the predominant enzyme responsible for the in vivo synthesis of ADP-Glc in bt1 mutant kernels, but Suc synthase may also contribute to the synthesis of ADP-Glc in kernels deficient in AGPase.     

Mutation of the maize sbe1a and ae genes alters morphology and physical behavior of wx-type endosperm starch granules

In maize, three isoforms of starch-branching enzyme, SBEI, SBEIIa, and SBEIIb, are encoded by the Sbe1a, Sbe2a, and Amylose extender (Ae) genes, respectively. The objective of this research was to explore the effects of null mutations in the Sbe1a and Ae genes alone and in combination in wx background on kernel characteristics and on the morphology and physical behavior of endosperm starch granules. Differences in kernel morphology and weight, starch accumulation, starch granule size and size distribution, starch microstructure, and thermal properties were observed between the ae wx and sbe1a ae wx plants but not between the sbe1a wx mutants when compared to wx. Starch from sbe1a ae wx plants exhibited a larger granule size with a wider gelatinization temperature range and a lower endotherm enthalpy than ae wx. Microscopy shows weaker iodine staining in sbe1a ae wx starch granules. X-ray diffraction revealed A-type crystallinity in wx and sbe1a wx starches and B-type in sbe1a ae wx and ae wx. This study suggests that, while the SBEIIb isoform plays a dominant role in maize endosperm starch synthesis, SBEI also plays a role, which is only observable in the presence of the ae mutation.     

A large-scale screen for artificial selection in maize identifies candidate agronomic loci for domestication and crop improvement

Maize (Zea mays subsp mays) was domesticated from teosinte (Z. mays subsp parviglumis) through a single domestication event in southern Mexico between 6000 and 9000 years ago. This domestication event resulted in the original maize landrace varieties, which were spread throughout the Americas by Native Americans and adapted to a wide range of environmental conditions. Starting with landraces, 20th century plant breeders selected inbred lines of maize for use in hybrid maize production. Both domestication and crop improvement involved selection of specific alleles at genes controlling key morphological and agronomic traits, resulting in reduced genetic diversity relative to unselected genes. Here, we sequenced 1095 maize genes from a sample of 14 inbred lines and chose 35 genes with zero sequence diversity as potential targets of selection. These 35 genes were then sequenced in a sample of diverse maize landraces and teosintes and tested for selection. Using two statistical tests, we identified eight candidate genes. Extended gene sequencing of these eight candidate loci confirmed that six were selected throughout the gene, and the remaining two exhibited evidence of selection in the 3' portion of each gene. The selected genes have functions consistent with agronomic selection for nutritional quality, maturity, and productivity. Our large-scale screen for artificial selection allows identification of genes of potential agronomic importance even when gene function and the phenotype of interest are unknown.     

Genetic mapping of the Isaac-CACTA transposon in maize

We constructed a genetic linkage map with Isaac-TD, SSR, and SNAP markers in a RIL population which had been derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 368 markers, including 241 Isaac-TD, 121 SSR, and 6 SNAP markers, were assigned to 10 linkage groups, encompassing 1687.0 cM, with an average genetic distance of 4.6 cM between markers. SSR markers were utilized as chromosome anchors, in order to assign the Isaac-TD markers to the chromosomes, and the number of markers in each of the linkage groups ranged between 22 and 49. The majority of the Isaac-TD markers were determined to have been distributed throughout the ten maize chromosomes. In linkage analysis of the Isaac-TD markers with genes of agronomic interest, six genes related with maize kernel starch biosynthesis, ae1, bt2, sh1, sh2, su1, and wx1, were analyzed and shown that they were closely linked with either the Isaac-TD or SSR markers on chromosomes of 3, 4, 5, and 9. We observed and mapped segregation-distorted markers on chromosomes 1, 5, 6, 7, 8, and 10, where these markers were clustered. The Isaac-TD or SSR markers which were closely linked with starch synthesis genes may prove useful in marker-assisted breeding programs.     

Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels

Fusarium verticillioides, a fungal pathogen of maize, produces fumonisin mycotoxins that adversely affect human and animal health. Basic questions remain unanswered regarding the interactions between the host plant and the fungus that lead to the accumulation of fumonisins in maize kernels. In this study, we evaluated the role of kernel endosperm composition in regulating fumonisin B1 (FB1) biosynthesis. We found that kernels lacking starch due to physiological immaturity did not accumulate FB1. Quantitative polymerase chain reaction analysis indicated that kernel development also affected the expression of fungal genes involved in FB1 biosynthesis, starch metabolism, and nitrogen regulation. A mutant strain of F. verticillioides with a disrupted a-amylase gene was impaired in its ability to produce FB1 on starchy kernels, and both the wild-type and mutant strains produced significantly less FB1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the sole carbon source, the wild-type strain produced only trace amounts of FB1, but it produced large amounts of FB1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis. We conclude that amylopectin induces FB1 production in F. verticillioides. This study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.     

Genomic screening for artificial selection during domestication and improvement in maize

BACKGROUND: Artificial selection results in phenotypic evolution. Maize (Zea mays L. ssp. mays) was domesticated from its wild progenitor teosinte (Zea mays subspecies parviglumis) through a single domestication event in southern Mexico between 6000 and 9000 years ago. This domestication event resulted in the original maize landrace varieties. The landraces provided the genetic material for modern plant breeders to select improved varieties and inbred lines by enhancing traits controlling agricultural productivity and performance. Artificial selection during domestication and crop improvement involved selection of specific alleles at genes controlling key morphological and agronomic traits, resulting in reduced genetic diversity relative to unselected genes. SCOPE: This review is a summary of research on the identification and characterization by population genetics approaches of genes affected by artificial selection in maize. CONCLUSIONS: Analysis of DNA sequence diversity at a large number of genes in a sample of teosintes and maize inbred lines indicated that approx. 2 % of maize genes exhibit evidence of artificial selection. The remaining genes give evidence of a population bottleneck associated with domestication and crop improvement. In a second study to efficiently identify selected genes, the genes with zero sequence diversity in maize inbreds were chosen as potential targets of selection and sequenced in diverse maize landraces and teosintes, resulting in about half of candidate genes exhibiting evidence for artificial selection. Extended gene sequencing demonstrated a low false-positive rate in the approach. The selected genes have functions consistent with agronomic selection for plant growth, nutritional quality and maturity. Large-scale screening for artificial selection allows identification of genes of potential agronomic importance even when gene function and the phenotype of interest are unknown. These approaches should also be applicable to other domesticated species if specific demographic conditions during domestication exist.     

Quantitative trait loci influencing chemical and sensory characteristics of eating quality in sweet corn.

This study was conducted to ascertain the chromosomal location and magnitude of effect of quantitative trait loci (QTL) associated with the chemical and sensory properties of sweet corn (Zea mays L.) eating quality. Eighty-eight RFLPs, 3 cloned genes (sh1, sh2, and dhn1), and 2 morphological markers (a2 and se1) distributed throughout the sweet corn genome were scored in 214 F2:3 families derived from a cross between the inbreds W6786su1Se1 and IL731Asu1se1. Kernel properties associated with eating quality (kernel tenderness and starch, phytoglycogen, sucrose, and dimethyl sulfide concentrations) were quantified on F2:3 sib-pollinated ears harvested at 20 days after pollination. Sensory evaluation was conducted on a subset of 103 F2:3 families to determine intensity of attributes associated with sweet corn eating quality (corn aroma, grassy aroma, sweetness, starchiness, grassy flavor, crispness, tenderness, and juiciness) and overall liking. Single factor analysis of variance revealed significant QTL for all these traits, which accounted for from 3 to 42% of the total phenotypic variation. A proportion of the RFLP markers associated with human sensory response were also found to be associated with kernel characteristics. To our knowledge this is the first report of the identification of QTL associated with human flavor preferences in any food crop. Key words : sweet corn, RFLP, quantitative trait loci, eating quality, sensory evaluation.     

Can genetic variability for nitrogen metabolism in the developing ear of maize be exploited to improve yield?

Quantitative trait loci (QTLs) for the main steps of nitrogen (N) metabolism in the developing ear of maize (Zea mays L.) and their co-localization with QTLs for kernel yield and putative candidate genes were searched in order to identify chromosomal regions putatively involved in the determination of yield. During the grain-filling period, the changes in physiological traits were monitored in the cob and in the developing kernels, representative of carbon and N metabolism in the developing ear. The correlations between these physiological traits and traits related to yield were examined and localized with the corresponding QTLs on a genetic map. Glycine and serine metabolism in developing kernels and the cognate genes appeared to be of major importance for kernel production. The importance of kernel glutamine synthesis in the determination of yield was also confirmed. The genetic and physiological bases of N metabolism in the developing ear can be studied in an integrated manner by means of a quantitative genetic approach using molecular markers and genomics, and combining agronomic, physiological and correlation studies. Such an approach leads to the identification of possible new regulatory metabolic and developmental networks specific to the ear that may be of major importance for maize productivity.     

Change of granular and molecular structures of waxy maize and potato starches after treated in alcohols with or without hydrochloric acid

Starches from waxy maize and potato were treated in methanol and 2-propanol either with or without 0.36% hydrochloric acid at 65 °C for 1 h. The granule morphology, molecular structure and pasting properties of the starches were determined and the effects of treatments on the granule and molecular structures of starch were investigated. Starch treated in alcohols without acid showed loss of native order through the hilum of granules, and no obvious molecular degradation was found. However, acid–alcohol treated starch showed many cracks inside granules, and both waxy maize and potato starches showed obvious molecular degradation after treated. Furthermore, the amylose chains and long chains of amylopectin of starch were more easily degraded with acid–alcohol treatment. The pasting viscosity of acid–alcohol treated starches were also obviously less than that of their counterpart native starch and starch after alcohol treatment. The extent of degradation of molecules and the decrease of pasting viscosity on potato starch after acid–alcohol treated were more obvious than that of waxy maize starch. The result indicates that the degradation preferentially occur in the amorphous region when starch treated by acid–alcohol, and the degradation of starch molecules enhances the amorphous excretion and the occurrence of cracks inside the granules.     

QTLs for grain dry milling properties, composition and vitreousness in maize recombinant inbred lines

Vitreousness and kernel hardness are important properties for maize processing and end-product quality. In order to examine the genetic basis of these traits, a recombinant inbred line population resulting from a cross between a flint line (F-2) and a semident line (Io) was used to search for vitreousness and kernel composition QTLs. Vitreousness was measured by image processing from a kernel section, while NIR spectroscopy was used to estimate starch, protein, cellulose, lipid and semolina yield. In addition, thousand-grain weight and grain weight per ear were measured. The MQTL method was used to map the QTLs for the different traits. An additional program allowed for the detection of interaction QTLs between markers. The total number of main-effect and interaction QTLs was similar. The QTLs were not evenly distributed but tended to cluster. Such clusters, mixing main-effect and interaction QTLs, were observed at six positions : on chromosomes 1, 2, 3, 6, 8 and 9. Two of them, on chromosomes 6 and 9, concerned both QTLs for kernel-weight traits and QTLs for kernel-composition traits (protein and cellulose). Technological-trait QTLs (vitreousness or semolina yield) were located less than 16 cM from a protein-content QTL on chromosome 2, and were co-located with lipid- and starch-content QTLs on chromosome 8. The co-location of a vitreousness and a semolina-yield QTL at the telomeric end of the chromosome 2 (Bin 2.02) is likely to be meaningful since measurement of these related traits, made by completely different methods (NIRS vs image processing), yielded very close QTLs. A similar location was previously reported independently for a kernel-friability QTL. Comparing the map location of the numerous loci for known-function genes it was shown that three zein loci were closely linked to QTLs for vitreousness on chromosome 3, for semolina yield and starch on chromosome 4, and for protein, cellulose and grain weight on chromosome 9. Some other candidate genes linked to starch precursor metabolism were also suggested on chromosomes 6 and 8. Received: 27 April 2000 / Accepted: 3 July 2000     

Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions

Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.