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Single-cross hybrids have been critical to the improvement of maize ( Zea mays L.), but the characterization of their genetic architectures remains challenging. Previous studies of hybrid maize have shown the contribution of within-locus complementation effects (dominance) and their differential importance across functional classes of loci. However, they have generally considered panels of limited genetic diversity, and have shown little benefit from genomic prediction based on dominance or functional enrichments. This study investigates the relevance of dominance and functional classes of variants in genomic models for agronomic traits in diverse populations of hybrid maize. We based our analyses on a diverse panel of inbred lines crossed with two testers representative of the major heterotic groups in the U.S. (1106 hybrids), as well as a collection of 24 biparental populations crossed with a single tester (1640 hybrids). We investigated three agronomic traits: days to silking (DTS), plant height (PH), and grain yield (GY). Our results point to the presence of dominance for all traits, but also among-locus complementation (epistasis) for DTS and genotype-by-environment interactions for GY. Consistently, dominance improved genomic prediction for PH only. In addition, we assessed enrichment of genetic effects in classes defined by genic regions (gene annotation), structural features (recombination rate and chromatin openness), and evolutionary features (minor allele frequency and evolutionary constraint). We found support for enrichment in genic regions and subsequent improvement of genomic prediction for all traits. Our results suggest that dominance and gene annotations improve genomic prediction across diverse populations in hybrid maize., (Copyright © 2020 Ramstein et al.)

The emergence of high-throughput, high-density genotyping methods combined with increasingly powerful computing systems has created opportunities to further discover and exploit the genes controlling agronomic performance in elite maize breeding populations. Understanding the genetic basis of population structure in an elite set of materials is an essential step in this genetic discovery process. This paper presents a genotype-based population analysis of all maize inbreds whose Plant Variety Protection certificates had expired as of the end of 2013 (283 inbreds) as well as 66 public founder inbreds. The results provide accurate population structure information and allow for important inferences in context of the historical development of North American elite commercial maize germplasm. Genotypic data was obtained via genotyping-by-sequencing on 349 inbreds. After filtering for missing data, 77,314 high-quality markers remained. The remaining missing data (average per individual was 6.22 percent) was fully imputed at an accuracy of 83 percent. Calculation of linkage disequilibrium revealed that the average r2 of 0.20 occurs at approximately 1.1 Kb. Results of population genetics analyses agree with previously published studies that divide North American maize germplasm into three heterotic groups: Stiff Stalk, Non-Stiff Stalk, and Iodent. Principal component analysis shows that population differentiation is indeed very complex and present at many levels, yet confirms that division into three main sub-groups is optimal for population description. Clustering based on Nei's genetic distance provides an additional empirical representation of the three main heterotic groups. Overall fixation index (FST), indicating the degree of genetic divergence between the three main heterotic groups, was 0.1361. Understanding the genetic relationships and population differentiation of elite germplasm may help breeders to maintain and potentially increase the rate of genetic gain, resulting in higher overall agronomic performance.

Appropriate selection of parents for the development of mapping populations is pivotal to maximizing the power of quantitative trait loci detection. Trait genotypic variation within a family is indicative of the family's informativeness for genetic studies. Accurate prediction of the most useful parental combinations within a species would help guide quantitative genetics studies. We tested the reliability of genotypic and phenotypic distance estimators between pairs of maize inbred lines to predict genotypic variation for quantitative traits within families derived from biparental crosses. We developed 25 families composed of ~200 random recombinant inbred lines each from crosses between a common reference parent inbred, B73, and 25 diverse maize inbreds. Parents and families were evaluated for 19 quantitative traits across up to 11 environments. Genetic distances (GDs) among parents were estimated with 44 simple sequence repeat and 2303 single-nucleotide polymorphism markers. GDs among parents had no predictive value for progeny variation, which is most likely due to the choice of neutral markers. In contrast, we observed for about half of the traits measured a positive correlation between phenotypic parental distances and within-family genetic variance estimates. Consequently, the choice of promising segregating populations can be based on selecting phenotypically diverse parents. These results are congruent with models of genetic architecture that posit numerous genes affecting quantitative traits, each segregating for allelic series, with dispersal of allelic effects across diverse genetic material. This architecture, common to many quantitative traits in maize, limits the predictive value of parental genotypic or phenotypic values on progeny variance.

We compared the genetic architecture of thirteen maize morphological traits in a large population of recombinant inbred lines. Four traits from the male inflorescence (tassel) and three traits from the female inflorescence (ear) were measured and studied using linkage and genome-wide association analyses and compared to three flowering and three leaf traits previously studied in the same population. Inflorescence loci have larger effects than flowering and leaf loci, and ear effects are larger than tassel effects. Ear trait models also have lower predictive ability than tassel, flowering, or leaf trait models. Pleiotropic loci were identified that control elongation of ear and tassel, consistent with their common developmental origin. For these pleiotropic loci, the ear effects are larger than tassel effects even though the same causal polymorphisms are likely involved. This implies that the observed differences in genetic architecture are not due to distinct features of the underlying polymorphisms. Our results support the hypothesis that genetic architecture is a function of trait stability over evolutionary time, since the traits that changed most during the relatively recent domestication of maize have the largest effects., Competing Interests: The authors have declared that no competing interests exist.

US maize yield has increased eight-fold in the past 80 years, with half of the gain attributed to selection by breeders. During this time, changes in maize leaf angle and size have altered plant architecture, allowing more efficient light capture as planting density has increased. Through a genome-wide association study (GWAS) of the maize nested association mapping panel, we determined the genetic basis of important leaf architecture traits and identified some of the key genes. Overall, we demonstrate that the genetic architecture of the leaf traits is dominated by small effects, with little epistasis, environmental interaction or pleiotropy. In particular, GWAS results show that variations at the liguleless genes have contributed to more upright leaves. These results demonstrate that the use of GWAS with specially designed mapping populations is effective in uncovering the basis of key agronomic traits.

Flowering time is a complex trait that controls adaptation of plants to their local environment in the outcrossing species Zea mays (maize). We dissected variation for flowering time with a set of 5000 recombinant inbred lines (maize Nested Association Mapping population, NAM). Nearly a million plants were assayed in eight environments but showed no evidence for any single large-effect quantitative trait loci (QTLs). Instead, we identified evidence for numerous small-effect QTLs shared among families; however, allelic effects differ across founder lines. We identified no individual QTLs at which allelic effects are determined by geographic origin or large effects for epistasis or environmental interactions. Thus, a simple additive model accurately predicts flowering time for maize, in contrast to the genetic architecture observed in the selfing plant species rice and Arabidopsis.

Maize has been targeted for biofortification with provitamin A carotenoids through traditional breeding. Two studies were conducted in gerbils to evaluate factors that may affect provitamin A activity. Maize diets had equal theoretical concentrations of vitamin A (VA) assuming 100% bioefficacy. Study 1 ( n = 57) varied the ratio of beta-cryptoxanthin and beta-carotene but maintained the same theoretical VA. Study 2 ( n = 67) varied lutein and zeaxanthin. Other treatments were oil, VA, or beta-carotene doses. Serum and livers were analyzed for VA and carotenoids. In study 1, total liver VA did not differ among the maize groups. In study 2, total liver VA of the VA and maize groups were higher than controls ( P < 0.05). Conversion factors were 2.1-3.3 mug beta-carotene equivalents to 1 mug retinol. Twice the molar amount of beta-cryptoxanthin was as efficacious as beta-carotene and the proportion of beta-cryptoxanthin or xanthophylls did not appreciably change the VA value of biofortified maize.

Dietary vitamin A deficiency causes eye disease in 40 million children each year and places 140 to 250 million at risk for health disorders. Many children in sub-Saharan Africa subsist on maize-based diets. Maize displays considerable natural variation for carotenoid composition, including vitamin A precursors alpha-carotene, beta-carotene, and beta-cryptoxanthin. Through association analysis, linkage mapping, expression analysis, and mutagenesis, we show that variation at the lycopene epsilon cyclase (lcyE) locus alters flux down alpha-carotene versus beta-carotene branches of the carotenoid pathway. Four natural lcyE polymorphisms explained 58% of the variation in these two branches and a threefold difference in provitamin A compounds. Selection of favorable lcyE alleles with inexpensive molecular markers will now enable developing-country breeders to more effectively produce maize grain with higher provitamin A levels.

Maize is an important source of pro-vitamin A; beta-carotene, alpha-carotene and beta-cryptoxanthin, and the non-pro-vitamin A carotenoids including lutein and zeaxanthin. In the present study, a recombinant inbred (RI) population with 233 RI lines derived from a cross between By804 and B73 was employed to detect QTL for these nutritionally important components in maize grain. High Performance Liquid Chromatography was used to measure amounts of individual carotenoids over 2 years. A genetic linkage map was constructed with 201 molecular markers. In all, 31 putative QTL including 23 for individual and 8 for total carotenoids were detected on chromosome(s) 1, 3, 5, 6, 7, 8 and 10. The notable aspect of this study was that much of the phenotypic variation in contents of carotenoids could be explained by two loci (y1 and y9), and the QTL for carotenoids elucidated the interrelationships among these compounds at the molecular level. A gene targeted marker (Y1ssr) in the candidate gene phytoene synthase 1 (psy1) tightly linked to a major QTL explaining 6.6-27.2% phenotypic variation for levels of carotenoids was identified, which may prove useful to expedite breeding for higher level of carotenoids in maize grain. This functionally characterized gene (psy1) could also be exploited for further development of functional marker for carotenoids in maize. The QTL cluster located at y9 locus may also be used for pyramiding favorable alleles controlling contents of carotenoids from diverse maize germplasm.

Maize tassel inflorescence architecture is relevant to efficient production of F(1) seed and yield performance of F(1) hybrids. The objectives of this study were to identify genetic relationships among seven measured tassel inflorescence architecture traits and six calculated traits in a maize backcross population derived from two lines with differing tassel architectures, and identify Quantitative Trait Loci (QTL) involved in the inheritance of those tassel inflorescence architecture traits. A Principal Component (PC) analysis was performed to examine relationships among correlated traits. Traits with high loadings for PC1 were branch number and branch number density, for PC2 were spikelet density on central spike and primary branch, and for PC3 were lengths of tassel and central spike. We detected 45 QTL for individual architecture traits and eight QTL for the three PCs. For control of inflorescence architecture, important QTL were found in bins 7.02 and 9.02. The interval phi034-ramosa1 (ral) in bin 7.02 was associated with six individual architecture trait QTL and explained the largest amount of phenotypic variation (17.3%) for PC1. Interval bnlg344-phi027 in bin 9.02 explained the largest amount of phenotypic variation (14.6%) for PC2. Inflorescence architecture QTL were detected in regions with candidate genes fasciated ear2, thick tassel dwarf1, and ral. However, the vast majority of QTL mapped to regions without known candidate genes, indicating positional cloning efforts will be necessary to identify these genes.

Maize (Zea mays L.) ear inflorescence architecture is directly relevant to grain yield components, and tassel architecture is relevant to hybrid seed production. The objectives of this study were to (1) determine heritabilities and correlations of a comprehensive set of tassel and ear inflorescence architecture traits in a set of (Illinois Low ProteinxB73) B73 S1 families, (2) identify chromosomal positions of QTL affecting tassel and ear architecture, and (3) identify possible candidate genes associated with these QTL. For tassel traits, the number of detected QTL ranged from one to five, and explained between 6.5 and 35.9% of phenotypic variation. For ear traits, the number of detected QTL ranged from one to nine and phenotypic variation explained by those QTL varied between 7.9 and 53.0%. We detected QTL for tassel architecture traits that required calculation of ratios from measured traits. Some of these calculated traits QTL were detected in regions that did not show QTL for the measured traits, suggesting that calculation of ratios may reveal developmentally relevant patterns of tassel architecture. We detected a QTL on chromosome 7 for tassel branch number near the gene ramosa1 (ra1), which is known to control tassel branch number, making ra1 a candidate gene for tassel branch number. We detected QTL for several traits on chromosomes 6, 8, and 9, where no inflorescence architecture genes have been mapped, thus providing initial information towards new gene discovery for control of inflorescence architecture.

In one of the longest-running experiments in biology, researchers at the University of Illinois have selected for altered composition of the maize kernel since 1896. Here we use an association study to infer the genetic basis of dramatic changes that occurred in response to selection for changes in oil concentration. The study population was produced by a cross between the high- and low-selection lines at generation 70, followed by 10 generations of random mating and the derivation of 500 lines by selfing. These lines were genotyped for 488 genetic markers and the oil concentration was evaluated in replicated field trials. Three methods of analysis were tested in simulations for ability to detect quantitative trait loci (QTL). The most effective method was model selection in multiple regression. This method detected approximately 50 QTL accounting for approximately 50% of the genetic variance, suggesting that >50 QTL are involved. The QTL effect estimates are small and largely additive. About 20% of the QTL have negative effects (i.e., not predicted by the parental difference), which is consistent with hitchhiking and small population size during selection. The large number of QTL detected accounts for the smooth and sustained response to selection throughout the twentieth century.

Cereal starch production forms the basis of subsistence for much of the world's human and domesticated animal populations. Starch concentration and composition in the maize (Zea mays ssp mays) kernel are complex traits controlled by many genes. In this study, an association approach was used to evaluate six maize candidate genes involved in kernel starch biosynthesis: amylose extender1 (ae1), brittle endosperm2 (bt2), shrunken1 (sh1), sh2, sugary1, and waxy1. Major kernel composition traits, such as protein, oil, and starch concentration, were assessed as well as important starch composition quality traits, including pasting properties and amylose levels. Overall, bt2, sh1, and sh2 showed significant associations for kernel composition traits, whereas ae1 and sh2 showed significant associations for starch pasting properties. ae1 and sh1 both associated with amylose levels. Additionally, haplotype analysis of sh2 suggested this gene is involved in starch viscosity properties and amylose content. Despite starch concentration being only moderately heritable for this particular panel of diverse maize inbreds, high resolution was achieved when evaluating these starch candidate genes, and diverse alleles for breeding and further molecular analysis were identified.

The Illinois Long-Term Selection Experiment for grain protein and oil concentration in maize (Zea mays) is the longest continuous genetics experiment in higher plants. A total of 103 cycles of selection have produced nine related populations that exhibit phenotypic extremes for grain composition and a host of correlated traits. The use of functional genomics tools in this unique genetic resource provides exciting opportunities not only to discover the genes that contribute to phenotypic differences but also to investigate issues such as the response of plant genomes to artificial selection, the genetic architecture of quantitative traits and the source of continued genetic variation within domesticated crop genomes.

Carotenoids are a class of fat-soluble antioxidant vitamin compounds present in maize ( Zea mays L.) that may provide health benefits to animals or humans. Four carotenoid compounds are predominant in maize grain: beta-carotene, beta-cryptoxanthin, zeaxanthin, and lutein. Although beta-carotene has the highest pro-vitamin A activity, it is present in a relatively low concentration in maize kernels. We set out to identify quantitative trait loci (QTL) affecting carotenoid accumulation in maize kernels. Two sets of segregating families were evaluated-a set of F2:3 lines derived from a cross of W64a x A632, and their testcross progeny with AE335. Molecular markers were evaluated on the F2:3 lines and a genetic linkage map created. High-performance liquid chromatography was performed to measure beta-carotene, beta-cryptoxanthin, zeaxanthin, and lutein on both sets of materials. Composite interval mapping identified chromosome regions with QTL for one or more individual carotenoids in the per se and testcross progenies. Notably QTL in the per se population map to regions with candidate genes, yellow 1 and viviparous 9, which may be responsible for quantitative variation in carotenoids. The yellow 1 gene maps to chromosome six and is associated with phytoene synthase, the enzyme catalyzing the first dedicated step in the carotenoid biosynthetic pathway. The viviparous 9 gene maps to chromosome seven and is associated with zeta-carotene desaturase, an enzyme catalyzing an early step in the carotenoid biosynthetic pathway. If the QTL identified in this study are confirmed, particularly those associated with candidates genes, they could be used in an efficient marker-assisted selection program to facilitate increasing levels of carotenoids in maize grain.

Increased levels of oleic acid may enhance the nutritional and functional value of corn. Corn oil is primarily composed of palmitic, stearic, oleic, linoleic and linolenic fatty acids. Delta-12 desaturase in plants converts oleic acid (18:1) to linoleic acid (18:2) by inserting a double bond at the delta-12 position. Fatty acid desaturase-2 (fad2) encodes delta-12 desaturase that functions in the endoplasmic reticulum while fatty acid desaturase-6 (fad6) encodes delta-12 desaturase that functions in plastids. Complementary DNA (cDNA) clones from putative maize homologs for fad2 and fad6 were identified and the entire clones DNA sequenced. The maize fad2 cDNAs showed an amino-acid identity of 67-77% to fad2 of Glycine, Arabidopsis and Brassica species. Our cDNA sequence comparisons suggested that more than one fad2 gene is transcribed in maize embryos. Two different fad2 cDNAs from an embryo cDNA library map to separate chromosomal positions, providing evidence consistent with two different isoforms of fad2 expressed in the embryo. The fad2 cDNAs from multiple tissue sources clustered into three groups on a phenogram, and map to different positions on chromosomes 4, 5 and 10, which suggests at least three different isoforms of fad2 may be expressed in the maize plant. The two maize fad6 cDNAs share 81% amino-acid identity with the Arabidopsis fad6 and map to chromosome 1. Northern analysis revealed that fad2 is transcribed in embryos at 14, 21, 28 and 35 days after pollination, with the highest level observed at day 14. None of the fad2 or fad6 clones mapped to maize chromosome bins associated with QTLs for the ratio of oleic/linoleic acid, notably bin 6.04 which contains the linoleic1 locus and the largest reported QTL for the oleic/linoleic ratio. This suggests, but does not prove, that some of the QTLs for the oleic/linoleic acid ratio do not involve allelic variants of fad2 or fad6 but rather involve other genes that may influence flux through the enzymes encoded by fad2 or fad6.

Vitamin E is the common name that describes eight naturally occurring compounds possessing alpha-tocopherol activity. These eight vitamin E compounds are collectively termed tocols, and all have antioxidant activity. There is natural variation among different corn breeding lines for levels of tocols. The two predominant isomers present in corn grain are gamma-tocopherol and alpha-tocopherol. Alpha-tocopherol is considered more desirable for human and animal consumption because it has higher biological activity than gamma-tocopherol. Most corn breeding lines naturally have much more gamma-tocopherol than alpha-tocopherol. Therefore a breeding goal is to increase levels of alpha-tocopherol relative to gamma-tocopherol. However, recent research suggests that gamma-tocopherol and compounds metabolized from it have properties important to human health that are unique from properties of alpha-tocopherol. Therefore it may be desirable to not only increase levels of alpha-tocopherol in corn grain, but also levels of gamma-tocopherol. Determination of levels of tocopherols in corn grain is very laborious, requires HPLC analysis and is too time consuming for use in routine commercial corn breeding programs. Therefore we are performing biotechnology enabled molecular marker mapping of chromosomal regions with genes that control levels and ratios of alpha- and gamma-tocopherol. Breeders can use molecular markers we have identified to expediently select for desirable alleles of genes that will improve levels of alpha- and gamma-tocopherol in corn grain, without having to perform laborious HPLC assays. Another biotechnology strategy we have initiated is genetic transformation of corn with the gamma-tocopherol methyl transferase gene to enhance conversion of gamma-tocopherol to alpha-tocopherol and thus increase levels of alpha-tocopherol. This transgenic strategy has been demonstrated in the model plant Arabidopsis, and we are now applying this approach to corn.

Corn (Zea mays L.) germplasm ownership methods are unwieldy and limit progress. Agronomic performance in hybrids better identifies inbreds for distinctness, i.e., independent varieties (IVs) or for dependency, i.e. essentially derived varieties (EDVs) than do molecular methods alone. Our objectives were to assess hybrid agronomic performance of some popular, closely related inbreds on the basis of molecular methods, and to offer thoughts on developing inbreds and on germplasm ownership. Significant agronomic differences were detected between the following: two hybrids in the same genetic group on the basis of 21 isozymic loci; two closely related inbreds (B73 and LH119) with 88.6% (62/70) RFLP (restriction fragment length polymorphism) similarity; and two closely related inbreds (Mo17 and LH51) with 88.2% (60/68) RFLP similarity. We suggest raising dependency standards for patenting inbreds to 90% or more, allowing the right to use and also paying a small royalty for only 5 yr on EDVs to the owner of the IV, and maintaining a research exemption to provide elite inbreds for developing new inbreds and experimental hybrids to all breeders. This will help maintain genetic gain for corn grain yield and serve the common good.

The F2 generations from two maize crosses were used to compare the ability of RAPD and RFLP marker systems to create a genetic linkage map. Both RFLPs and RAPDs were shown to provide Mendelian-type markers. Most of the RFLPs (80%) could be placed with a good level of certainty (LOD>4) on the genetic linkage map. However, because of their dominant nature, only between 37% and 59% of the RAPDs could be placed with such a LOD score. The use of combined data from RFLPs and RAPDs increases the level of information provided by RAPDs and allows the creation of a combined RFLP/RAPD genetic linkage map. Thus, the RAPD technique was found to be a powerful method to provide improved probes coverage on a previously created RFLP map and to locate markers linked to chromosomal regions of interest.

Four maize (Zea mays L.) populations selected for grain yield (BS10, Iowa Two-ear Synthetic; BS11, formerly Pioneer Two-ear Composite; RBS10, Illinois strain of BS10; and RSSSC, Illinois strain of Iowa Stiff Stalk Synthetic) were assayed for molecular variation in the ribosomal DNA (rDNA) intergenic spacer (IGS) at initial and advanced cycles of selection. RSSSC and RBS10 underwent reciprocal recurrent selection with an inbred tester in a high-yield environment, whereas BS10 and BS11 were subjected to full-sib reciprocal recurrent selection. Maize rDNA, which encodes the ribosomal RNA genes, is highly repetitive and shows IGS length variation within and among individuals. Five different ribosomal spacer-length variants (rslvs) and a polymorphic SstI restriction site in the IGS were detected in the four populations. The five rslvs and the polymorphic restriction fragment were observed in 20 different combinations or hybridization fragment patterns (HP). RSSSC, RBS10, and BS11 showed significant changes in the overall rslv and HP frequencies between cycle 0 and the advanced cycle of selection, whereas BS10 did not. In general, two specific HPs were more frequent in the majority of the advanced cycles of the four populations. The frequency changes between initial and advanced cycles were more dramatic for HPs than rslvs. These results are consistent with earlier findings and further support the hypothesis that certain rDNA HPs and/or linked loci may be responding to selection for grain yield and may be associated with a selective advantage in US Corn Belt environments.

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.

A study was conducted to determine the number and chromosomal location of quantitative trait loci (QTL) influencing the concentration of five fatty acids in 200 F2S1 lines derived from an Illinois High Oil (IHO) by Illinois Low Oil (Early Maturity) (ILO(EM)) cross. Restriction fragment length polymorphism (RFLP) analysis was performed on the 200 S1 lines and concentrations of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids were determined in self-pollinated kernels harvested from plants grown in replicated field trials during 1992 and 1993. A series of 74 cDNA and genomic clones were used and these revealed 80 polymorphic loci spaced, on average, 24 cM apart throughout the maize genome. Analysis of variance detected significant (p < 0.05) associations between several RFLP loci and the concentration of each fatty acid. A total of 15 RFLP loci clustered in 12 chromosomal regions were associated with the concentration of 16:0, 17 loci clustered in 10 regions were associated with the concentration of 18:0, 12 loci clustered in eight regions were associated with the concentration of 18:1 and 18:2, and 17 loci clustered in eight regions were associated with the concentration of 18:3. Multiple linear regression models consisting of four RFLP loci explained 24 and 62% of the total phenotypic and genotypic variation (R2) among the 200 F2S1 lines for 16:0, five loci explained 51 and 71% of the variation for 18:0, three loci explained 67 and 79% of the variation for 18:1, two loci explained 67 and 81% of the variation for 18:2, and seven loci explained 52 and 78% of the variation for 18:3 in these 200 F2S1 lines. The ratio of 18:1 to 18:2 was tightly interrelated as the same QTL were associated with the concentrations of 18:1 and 18:2. A quantitative trait locus that explained 63% of the phenotypic variation in the ratio of 18:1 to 18:2 is tightly linked to umc65 on chromosome 6 in the region of the linoleic acid1 locus.

In order to map the genes conditioning the induction of embryos during our anther culture process, we evaluated F2 plants from three different crosses for their anther culture ability and also performed RFLP analysis on these plants. The results showed that six chromosomal regions appear to be associated with the ability to induce embryo-like structures from maize microspores. These regions are located on chromosomes 1 (two regions), 3, 5, 7, and 8. Some of these chromosomes are identical to those found in previous studies and we have localized the regions more precisely. Notably, all chromosome regions identified, except one, are near viviparous mutant loci. Since the viviparous mutations are known to involve the plant hormone abscisic acid (ABA), these results suggest that ABA or its antagonist, gibberellic acid (GA3), might somehow be related to anther culture ability. We also propose some combinations of probes to screen for anther culture ability in the three genotypes studied.

RFLP marker data from an F2∶3 population derived from a cross between a sugary1 (su1) and a sugary enhancer1 (su1, sel) inbred were used to construct a genetic linkage map of maize. This map includes 93 segregating marker loci distributed throughout the maize genome, providing a saturated linkage map that is suitable for linkage analysis with quantitative trait loci (QTL). This population, which has been immortalized in the form of sibbed F2∶3 families, was derived from each of the 214 F2 plants and along with probe data are available to the scientific community. QTL analysis for kernel sucrose (the primary form of sugar) concentration at 20 days after pollination (DAP) uncovered the segregation of seven major QTL influencing sucrose concentration; a locus linked to umc36a described the greatest proportion of the variation (24.7%). Since maltose concentration has previously been reported to be associated with the se1 phenotype, an analysis of probe associations with maltose concentration at 40 DAP was also conducted. The highly significant association of umc36a with maltose and sucrose concentrations provided evidence that this probe is linked to se1. Phenotypic evaluation for the se1 genotype in each F2∶3 family enabled us to map the gene 12.1 cM distal to umc36a. In contrast to previous work where se1 was reported to be located on chromosome four, our data strongly suggest that the sugary enhancer1 locus maps on the the distal portion of the long arm of chromosome 2 in the maize genome.

The interaction of nuclear and mitochondrial genomes in the alteration of maize (Zea mays L.) mitochondrial orf221 transcript patterns was examined. Northern analyses involving specific maize nuclear genotypes associated with N, C or S cytoplasms revealed considerable orf221 transcript heterogeneity. F1 progenies were developed from maize inbred-cytoplasm combinations that differed for orf221 transcript patterns. Northern analyses revealed that the presence or level of abundance of certain orf221 transcripts was dependent on nuclear genotype. The maize inbred B37(C) exhibits orf221 transcripts of 3500, 3200, 2800, and 1300 nt whereas the F1 of B37(C) x Ky21(N) does not exhibit a 2800-nt transcript but does give transcripts of 2100 and 1250 nt in addition to 3500-, 3200- and 1300-nt transcripts. Northern analyses also suggested that the size or the presence of certain orf221 transcripts was related to the mitochondrial genome configuration. Maize inbred A619 exhibits a 2300-nt orf221 transcript when associated with N cytoplasm and a 2100-nt orf221 transcript when associated with C and S cytoplasms. As a result of deletion of the gene T-urf13, the A188(T7) mitochondrial mutant exhibits only a 3100-nt orf221 transcript and not the very complex T-urf13/orf221 transcript pattern associated with A188(T). The genetic stock A188(T7) x W64A(N)(2) gives a highly abundant 2100-nt orf221 transcript not detected in A188(T7). Deletion of T-urf13 has enabled the nuclear genotype of W64A(N) to alter orf221 transcript patterns in a manner not detected in T cytoplasm. This observation suggests that alteration of the mitochondrial genomic configuration adjacent to orf221 results in a different response to nuclear gene products from that observed when or2f21 is present in the T mitochondrial genome configuration.

Five different ribosomal DNA (rDNA) intergenic spacer-length variants (slvs) were detected among the maize inbreds which were the progenitors of Iowa Stiff Stalk Synthetic (BSSS). One rDNASstI restriction site polymorphism in the 3' region of the 26S gene was detected. Nine different rDNA intergenic spacer (IGS) hybridization fragment patterns (assigned letter designations A-I) were observed among the BSSS progenitors. Following 7 cycles of half-sib recurrent selection in BSSS using the Ia13 double cross as a tester, hybridization fragment pattern E became predominant in the population. In contrast, 11 cycles of reciprocal recurrent selection in BSSS with the Iowa Corn Borer Synthetic No. 1 (BSCB1) population resulted in hybridization pattern D becoming predominant. Hybridization pattern E is present in the elite inbreds B14, B37, B73, and B84, which were derived from different cycles of the BSSS half-sib recurrent selection program with Ia13. Hybridization pattern D is present in the elite inbreds B89 and B94, which were derived from different cycles of the BSSS reciprocal recurrent selection program with BSCB1. Therefore, two different forms of recurrent selection on BSSS resulted in different hybridization patterns becoming predominant in the selected populations and present in elite inbreds derived from the populations. These results also suggest that rDNA IGS hybridization fragment patterns D and E, which both have the longest slv detected, may have a selective or adaptive advantage in BSSS materials grown in the Corn Belt.

Restriction fragment length polymorphism (RFLP) analysis was used to characterize variability in the Illinois Long-Term Selection Experiment oil strains. Considerable polymorphism was detected within each oil strain and among oil strains. Fifty-two individual plants from each of the Illinois High Oil (IHO), Illinois Low Oil (ILO), Reverse High Oil (RHO) and Reverse Low Oil (RLO) strains were sampled to determine RFLP allele/variant frequencies. Generation 90 was sampled for IHO, RHO, and RLO whereas generation 87 was sampled for ILO. Forty-nine RFLP probes distributed throughout the maize genome were used. Chi-square analysis was performed to determine if RFLP genotypes at each of the 49 RFLP loci were significantly different among strains. Oil strains that have been separated for 90 generations showed high levels of significantly-different RFLP genotypic frequencies. The comparison of ILO vs RHO gave only significant chi-square values while the comparisons of IHO vs RLO and RHO vs RLO had 11∶1 ratios of significant to non-significant chi-square values. Strains that have been separated for only 42 generations showed a lower level of significantly-different RFLP genotypic frequencies. The comparisons of IHO vs RHO and ILO vs RLO both had only a 3∶2 ratio of significant to non-significant chi-squares values. Detection of multiple RFLP alleles/variants among the oil strains was common with 59% of the RFLP loci examined exhibiting multiple variants. A number of RFLP loci in RHO (3) and RLO (11) were associated with a trend in RFLP allele/variant frequencies consistent with a response to reverse selection for oil concentration.

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.

RFLP analysis was performed with anther culture-derived callus lines developed from the maize F1 hybrids Pa91 x FR16 (PF), H99 x Pa91 (HP) and H99 x FR16 (HF). Relatively evenly spaced RFLP markers were selected to cover the maize genome with 52, 58 and 35 RFLP markers for the PF, HP and HF callus lines, respectively. The results from populations PF and HP combined with limited information from HF showed that six chromosomal regions on chromosomes 1, 2 (two regions), 3, 6 and 8 appear to be associated with the formation of embryo-like structures (ELSs) from microspores or the subsequent formation of regenerable callus from the ELSs. Regions at the end of the long arm of chromosome 2 and on the long arm of chromosome 8 appear to be associated with ELS formation, and the other regions appear to be associated with either ELS or regenerable callus formation or both. Certain regions that we have identified are the same as those found in other studies to be important for friable, embryogenic callus formation (chromosomes 1 and 3 and near the centromere of 2) and for ESL formation (chromosomes 1 and 3). This study has provided evidence for the genetic basis of the maize anther culture response and callus formation.

The mitochondrial gene T-urf13 in T cytoplasm maize is associated with sensitivity to disease toxins and with cytoplasmic male sterility. T-urf13 is co-transcribed with an open reading frame designated orf221. We have detected alterations in the transcription of the T-urf13/orf221 region that are affected by nuclear genotype. There are multiple mRNA transcripts generated from the T-urf13/orf221 region, one of which is a processed 1538-nucleotide (nt) transcript. This 1538-nt transcript is present in Wf9 (T), but was not found in mitochondrial RNAs (mtRNAs) from maize inbreds B14A (T) and 33-16 (T). For B14A (T) a 1500-nt transcript was detected and for 33-16 (T) a 1400-nt transcript was detected. In F1 progeny of the cross of Wf9 (T) x 33-16 (N), only the 1400-nt transcript was present. Genetic analyses revealed this processing event is nuclear controlled with dominant gene action and is independent of nuclear restorer gene Rf1-associated processing events. T-urf13/orf221 transcriptional patterns were shown to vary in both sterile and fertile states. Segregation analysis of a 1100-nt orf221-specific transcript indicated that the genetic basis of nuclear control for the presence of this transcript was relatively simple. Analysis of the A188 (T4) tissue culture mutant, which has reverted to male fertility but displays the same T-urf13/orf221 transcript pattern as A188 (T), indicated no DNA sequence differences between T4-orf221 and T-orf221. Presence of the nuclear gene Rf2 was not necessary for expression of the T4 cytoplasm-associated malefertile phenotype.

Variation in the intergenic spacer of ribosomal DNA (rDNA) was detected among individual plants of the open-pollinated maize variety 'Hays Golden' and populations derived from this variety. rDNA intergenic spacer-length variants were detected at approximately 200 bp intervals, consistent with the number of 200 bp subrepeats as the basis for this variation. Inheritance data revealed that more than one spacer-length class may be present on an individual chromosome. Fourteen different predominant rDNA intergenic spacer hybridization fragment patterns were detected. C-29, a population developed by 29 cycles of mass-selecting Hay Golden for high grain yield, exhibited a significant change in rDNA intergenic spacer hybridization fragment pattern composition in comparison to Hays Golden. This change included a reduction in frequency of the shortest predominant space-length variant (3.4 kb) and an increase in a 5.2 -kb hybridization fragment. I-31, a population developed through thermal neutron irradiation of Hays Golden and 31 generations of mass selection for high grain yield, did not exhibit a significant change in overall rDNA intergenic spacer composition. I-31 did exhibit an increase in frequency of the 5.2-kb hybridization fragment and a significant change in two specific hybridization fragment patterns that had also changed in C-29. These data, particularly for the C-29 population, suggest that rDNA intergenic spacer-length variants and/or associated loci were influenced by selection.