Maize Chromosomes Research Articles

Haploid Bio-Induction in Plant through Mock Sexual Reproduction.

SummaryHaploidization is invaluable for basic genetic research and crop breeding. The haploid bio-induction principle is an important topic that remains largely unexplored. In this study, both CenH3 RNAi and in vitro inhibition were used to simulate and induce haploids in allopolyploid crop. Notably, in vitro CenH3 inhibition showed that the results were much the same to that of RNAi in phenotype, chromosome behavior, microspore production, and haploid induction. Cytological analyses of RNAi and inhibitor-treated progenies revealed elimination of chromosomes, defective microspores with empty nuclei, thereby giving rise to pseudo male gametes, and haploid parthenogenesis induction. We found distinct defective empty microspores that were positively correlated with the decrease of CenH3 during RNAi manipulation. Investigation through both in vivo and in vitro studies revealed that haploidization was induced through the pseudo male gamete-mediated mock sexual reproduction. The present results provide insights for the haploid parthenogenesis induction process.

Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.

Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.

A universal chromosome identification system for maize and wild Zea species.

Maize was one of the first eukaryotic species in which individual chromosomes can be identified cytologically, which made maize one of the oldest models for genetics and cytogenetics research. Nevertheless, consistent identification of all 10 chromosomes from different maize lines as well as from wild Zea species remains a challenge. We developed a new technique for maize chromosome identification based on fluorescence in situ hybridization (FISH). We developed two oligonucleotide-based probes that hybridize to 24 chromosomal regions. Individual maize chromosomes show distinct FISH signal patterns, which allow universal identification of all chromosomes from different Zea species. We developed karyotypes from three Zea mays subspecies and two additional wild Zea species based on individually identified chromosomes. A paracentric inversion was discovered on the long arm of chromosome 4 in Z. nicaraguensis and Z. luxurians based on modifications of the FISH signal patterns. Chromosomes from these two species also showed distinct distribution patterns of terminal knobs compared with other Zea species. These results support that Z. nicaraguensis and Z. luxurians are closely related species.

Mapping of QTL for kernel abortion caused by in vivo haploid induction in maize (Zea mays L.).

Kernel abortion is common phenomenon in vivo haploid induction and closely linked with haploid induction rate, but little information of kernel abortion is available and its genetic basis still unclear. We used two mapping populations including 186 and 263 F2.3 family lines to analyze the different degree of kernel abortion and identify quantitative trait loci (QTL) responsible for kernel abortion during haploid induction. In total 62 putative QTL, accounting for 3.27–14.70% of the phenotypic variation in kernel abortion traits, were detected across all 10 chromosomes. Ten QTL with over 10% contribution to phenotypic variation were affecting the fifth level of endosperm abortion (EnA5th), endosperm abortion (EnA) and total abortion (TA). Co-localization among kernel abortion traits QTL was observed in both populations and among different kernel abortion types. Five overlaps were indentified in the QTL for kernel abortion traits and HIR traits. Maize chromosome bins 3.01–3.02, 3.04–3.06, 4.05–4.06, 5.03–5.04, 8.06 were QTL hotspots for three or four traits related to the kernel abortion during haploid induction. Total kernel abortion rate (TAR) and HIR showed highly significant positive correlation. These findings may help to reveal haploid induction mechanisms and improve haploid production efficiency.

Mapping Quantitative Trait Loci Associated With Resistance to Aflatoxin Accumulation in Maize Inbred Mp719.

Aflatoxins are carcinogenic and toxic compounds produced principally by fungal species Aspergillus flavus (Link: Fries) and A. parasiticus (Speare), which are common contaminants of food and feed. Aflatoxins can be found at dangerously high levels and can readily contaminate pre-harvest maize (Zea mays L.) grain. Sources of resistance to aflatoxin accumulation in maize have been identified, however, the highly quantitative nature and complex inheritance of this trait have limited the introgression of aflatoxin accumulation resistance into agronomically desirable lines. Mapping of quantitative trait loci (QTL) was performed on a bi-parental population comprised of 241 F2:3 families derived from the cross of inbred lines Mp705 (susceptible) × Mp719 (resistant). The mapping population was phenotyped in replicated field trials in three environments for resistance to aflatoxin accumulation under artificial inoculation with an A. flavus spore suspension. The genetic linkage map was constructed with 1,276 single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) molecular markers covering a total genetic distance of 1,642 cM across all ten maize chromosomes. Multiple interval mapping revealed that majority of the aflatoxin-reducing alleles and the source for the larger effect QTL identified in this study were contributed from Mp719, the resistant parent. Two QTL identified on chromosome 1 (bin 1.06–1.07) and chromosome 3 (bin 3.09) were the most stable across different environments and when combined, explained 24.6% of the total phenotypic variance across all three environments. Results from the study showed that these chromosomal regions harbor important QTL for influencing aflatoxin accumulation, which is consistent with previous reports with other different mapping populations. These stable QTL were the most promising for controlling aflatoxin accumulation in maize grain. Identifying beneficial alleles derived from Mp719 and closely linked molecular markers through QTL analysis for implementation of MAS could accelerate breeding efforts to reduce aflatoxin accumulation in maize.

Genome-wide identification and expression analysis of HD-ZIP I subfamily genes in maize

Transcription factors (TFs) are indispensable regulators of plant response to abiotic stress and play an important role in the whole growth and development process. HD-ZIP proteins constitute a large family of transcription factors that are found only in plants and are divided into four subfamilies (HD-ZIP I–IV). HD-ZIP I subfamily genes mainly participate in response to extreme environments such as drought and osmotic stress and treatments of ABA and ethylene. Here, we identified 17 HD-ZIP I subfamily genes in the maize genome using the hidden Markov model (HMM), which distributed non-uniformly on six chromosomes of maize and were more closely related to rice than to Arabidopsis . Furthermore, these HD-ZIP I subfamily genes exhibited multiple expression patterns in seven tissues, showing strong tissues-specific expression. Moreover, maize HD-ZIP I subfamily genes showed different response patterns and degrees to different stresses, such as high salinity, waterlogging and cold stress. In addition, maize HD-ZIP I subfamily genes also showed a complex response pattern under treatment of five different hormones. These results provide valuable reference information for dissecting function and molecular mechanism of HD-ZIP I subfamily genes in maize.

The condensin subunits SMC2 and SMC4 interact for correct condensation and segregation of mitotic maize chromosomes.

Structural Maintenance of Chromosomes 2 (SMC2) and Structural Maintenance of Chromosomes 4 (SMC4) are the core components of the condensin complexes, which are required for chromosome assembly and faithful segregation during cell division. Because of the crucial functions of both proteins in cell division, much work has been done in various vertebrates, but little information is known about their roles in plants. Here, we identified ZmSMC2 and ZmSMC4 in maize (Zea mays) and confirmed that ZmSMC2 associates with ZmSMC4 via their hinge domains. Immunostaining revealed that both proteins showed dynamic localization during mitosis. ZmSMC2 and ZmSMC4 are essential for proper chromosome segregation and for H3 phosphorylation at Serine 10 (H3S10ph) at pericentromeres during mitotic division. The loss of function of ZmSMC2 and ZmSMC4 enlarges mitotic chromosome volume and impairs sister chromatid separation to the opposite poles. Taken together, these findings confirm and extend the coordinated role of ZmSMC2 and ZmSMC4 in maintenance of normal chromosome architecture and accurate segregation during mitosis.

Domestication and evolution of maize

Around ten thousand years ago, men mostly relied on hunting and gathering activities before shifting to agriculture and allied practices. By 4000 years ago, ancient people had completed the domestication of all major crop species upon which human survival is dependent, including rice, wheat, and maize. Recent advances have started to point out the genes that were responsible for this change of the cultivated maize crop. The list of genes to date tentatively suggests that diverse plant developmental pathways were the targets of Neolithic “genetic tinkering,” Maize and its closest wild relatives, the teosintes, present a paradox. Even though maize and the teosintes exhibit extreme differences in their adult morphologies their genomes are so similar that they share the same chromosome number, similar or identical chromosome morphologies, and they can be easily cross-hybridized. A very high level of diversity exists among the maize landraces to explain this diversity many workers have proposed that maize landraces were the products of multiple independent domestication from their wild relative. Reports indicate that all maize arose from a single domestication in southern Mexico about 9,000 years ago. This phylogenetic work is consistent with a model based on the archaeological record suggesting that maize was the result of early Holocene domesticates. A few major genes or multiple linked minor genes on the maize chromosome largely govern the drastic change that is seen between teosinte and maize. Genes like teosinte branched1 (tb1) gene, teosinte glume architecture1 (tga1) and Zea Floricaula / Leafy2 (zfl2). There still remain some untold chapters in the origin and early diversification of maize. These questions will require additional archaeological and botanical exploration, more powerful molecular analyses, and perhaps DNA analysis of archaeological materials such that a perfect phylogenetic relationship can be established.

Population-level analysis reveals the widespread occurrence and phenotypic consequence of DNA methylation variation not tagged by genetic variation in maize

BackgroundDNA methylation can provide a source of heritable information that is sometimes entirely uncoupled from genetic variation. However, the extent of this uncoupling and the roles of DNA methylation in shaping diversity of both gene expression and phenotypes are hotly debated. Here, we investigate the genetic basis and biological functions of DNA methylation at a population scale in maize.ResultsWe perform targeted DNA methylation profiling for a diverse panel of 263 maize inbred genotypes. All genotypes show similar levels of DNA methylation globally, highlighting the importance of DNA methylation in maize development. Nevertheless, we identify more than 16,000 differentially methylated regions (DMRs) that are distributed across the 10 maize chromosomes. Genome-wide association analysis with high-density genetic markers reveals that over 60% of the DMRs are not tagged by SNPs, suggesting the presence of unique information in DMRs. Strong associations between DMRs and the expression of many genes are identified in both the leaf and kernel tissues, pointing to the biological significance of methylation variation. Association analysis with 986 metabolic traits suggests that DNA methylation is associated with phenotypic variation of 156 traits. There are some traits that only show significant associations with DMRs and not with SNPs.ConclusionsThese results suggest that DNA methylation can provide unique information to explain phenotypic variation in maize.

Mapping quantitative trait loci for yield-related traits and predicting candidate genes for grain weight in maize

Quantitative trait loci (QTLs) mapped in different genetic populations are of great significance for marker-assisted breeding. In this study, an F2:3 population were developed from the crossing of two maize inbred lines SG-5 and SG-7 and applied to QTL mapping for seven yield-related traits. The seven traits included 100-kernel weight, ear length, ear diameter, cob diameter, kernel row number, ear weight, and grain weight per plant. Based on an ultra-high density linkage map, a total of thirty-three QTLs were detected for the seven studied traits with composite interval mapping (CIM) method, and fifty-four QTLs were indentified with genome-wide composite interval mapping (GCIM) methods. For these QTLs, Fourteen were both detected by CIM and GCIM methods. Besides, eight of the thirty QTLs detected by CIM were identical to those previously mapped using a F2 population (generating from the same cross as the mapping population in this study), and fifteen were identical to the reported QTLs in other recent studies. For the fifty-four QTLs detected by GCIM, five of them were consistent with the QTLs mapped in the F2 population of SG-5 × SG-7, and twenty one had been reported in other recent studies. The stable QTLs associated with grain weight were located on maize chromosomes 2, 5, 7, and 9. In addition, differentially expressed genes (DEGs) between SG-5 and SG-7 were obtained from the transcriptomic profiling of grain at different developmental stages and overlaid onto the stable QTLs intervals to predict candidate genes for grain weight in maize. In the physical intervals of confirmed QTLs qKW-7, qEW-9, qEW-10, qGWP-6, qGWP-8, qGWP-10, qGWP-11 and qGWP-12, there were 213 DEGs in total. Finally, eight genes were predicted as candidate genes for grain size/weight. In summary, the stable QTLs would be reliable and the candidate genes predicted would be benefit for maker assisted breeding or cloning.

Linkage disequilibrium and haplotype block patterns in popcorn populations.

Linkage disequilibrium (LD) analysis provides information on the evolutionary aspects of populations. Recently, haplotype blocks have been used to increase the power of quantitative trait loci detection in genome-wide association studies and the prediction accuracy of genomic selection. Our objectives were as follows: to compare the degree of LD, LD decay, and LD decay extent in popcorn populations; to characterize the number and length of haplotype blocks in the populations; and to determine whether maize chromosomes also have a pattern of interspaced regions of high and low rates of recombination. We used a biparental population, a synthetic, and a breeding population, genotyped for approximately 75,000 single nucleotide polymorphisms (SNPs). The sample size ranged from 190 to 192 plants. For the whole-genome LD and haplotype block analyses, we assumed a window of 500 kb. To characterize the block and step patterns of LD in the populations, we constructed LD maps by chromosome, defining a cold spot as a chromosome segment including SNPs with the same LDU position. The LD and haplotype block analyses were also performed at the intragenic level, selecting 12 genes related to zein, starch, cellulose, and fatty acid biosynthesis. The populations with the higher and lower frequencies of |D'| values greater than 0.75 were the biparental (65–74%) and the breeding population (26–58%), respectively. There were slight differences between the populations regarding the average distance for SNPs with |D'| values greater than 0.75 (in the range of approximately 207 to 229 kb). The level of LD expressed by the r2 values was low in the populations (0.02, 0.04, and 0.04, on average) but comparable to some non-isolated human populations. The frequency of r2 values greater than 0.75 was lower in the biparental population (0.2–0.5%) and higher in the other populations (0.2–1.6%). The average distance for SNPs with r2 values greater than 0.75 was much higher in the biparental population (approximately 80 to 126 kb). In the other populations, the ranges were approximately 6 to 19 and 6 to 35 kb. The heatmaps for the regions covered by the first 100 SNPs in each chromosome, in each population (1 to 3.3 Mb, approximately), provided evidence that the comparatively few high r2 values (close to 1.0) occurred only for SNPs in close proximity, especially in the synthetic and breeding populations. Due to the reduced number of SNPs in the haplotype blocks (2 to 3) in the populations, it is not expected advantage of a haplotype-based association study as well as genomic selection along generations. The results concerning LD decay (rapid decay after 5–10 kb) and LD decay extent (along up to 300 kb) are in the range observed with maize inbred line panels. The LD maps indicate that maize chromosomes had a pattern of regions of extensive LD interspaced with regions of low LD. However, our simulated LD map provides evidence that this pattern can reflect regions with differences in allele frequencies and LD levels (expressed by |D'|) and not regions with high and low rates of recombination.

Genetic analysis of oleic acid content in the oils of maize sugary-2 mutants

Aim. Genetic analysis of oleate content in the carriers of maize mutation su2. Methods. Experiments were performed on a series of unrelated inbreeds – carriers of mutation su2 which were hybridized by the diallel and top – cross crossings. The analysis of the fatty acid composition of oil were carried out using a modified gas-chromatographic Peyskare method. Results. It was found, that su2 mutants were notable as having the increased content of oleate compared to the maize of usual type and this trait has a quantitative nature. The inbreeds – carriers of su2 mutation were significantly differed in the the effects of combining abilities for the oleate content. The inheritance of oleate was carried out by the type of incomplete dominance with the prevailing contribution of additive effects to the variance. Conclusions. In the sixth maize chromosome it is very likely that there is a spatial linkage of the su2 and oleate- encoding locus, the effect of which can be modified by the polygenic complex. Keywords: Zea mays L., mutation su2, oleic acid, genetic analysis.

Genome-wide association analysis of salt tolerance QTLs with SNP markers in maize (Zea mays L.).

Salt-tolerant breeding of maize has great significance to the development and utilization of saline-alkaline soil and the maintenance of grain security. Genome-wide association study (GWAS) has been widely used in maize genetics and breeding. To discover new salt-tolerant genes in maize by association analysis, which can provide technical supports for the innovation and genetic improvement of salt-tolerant germplasm resources in maize. Totally 150 maize inbred lines were genotyped with a high-density chip. GWAS was carried out to identify the significant single nucleotide polymorphisms (SNPs) which were associated with maize salt tolerance. Totally 34,972 SNPs with high quality and diversity were selected from 56,110 SNP markers, which were distributed on 10 chromosomes of maize. The GLM algorithm in TASSEL5.2 was used to analyze the five traits related to salt tolerance. Using a strict LOD threshold of 4.5, totally 7 SNP loci were identified, which were significantly correlated with plant height change rate and fresh weight change rate. The high density fingerprints of 150 inbred lines were clustered by TASSEL5.2 software to construct genetic clustering map to estimate the genetic distance and the subgroups. The 150 maize inbred lines were divided into two groups: SS group and NSS group, and the SNP loci of the salt-tolerant index showed difference in chromosome distribution. Based on previous studies, we screened 8 candidate genes for salt tolerance in maize and four of them were further validated by real-time quantitative PCR. Totally 7 SNP loci and 8 candidate genes related to salt tolerance in maize were identified, which will be of special value in molecular breeding of salt-tolerant maize.

New protocol for colchicine induced efficient doubled haploidy in haploid regenerants of tetraploid and hexaploid wheats at In vitro level

An investigation to standardize the protocol for in vitro application of colchicine for enhancing the doubled haploid production in wheat was done. Two tetraploid (PDW-314 and A-9-30-1); and two hexaploid (DH-40 and C-306) wheat genotypes were used as maternal parents, whereas, the pollen sources involved Zea mays (cv. Bajaura Makka) and Imperata cylindrica. During the rabi seasons of years 2013–14 and 2014–15, wheat × maize and wheat × I. cylindrica hybridization was carried out followed by treatment of their haploids produced as a result of elimination of chromosomes of maize and I. cylindrica respectively, with varied doses of colchicine for different durations The various doses of colchicine were categorized into two groups: lower doses for longer durations (0.01, 0.025, 0.05% each for 5, 7, 9, 11 hrs) and higher doses for shorter durations (0.05, 0.075, 0.10, 0.15, 0.20, 0.25% each for 5, 4, 3, 2 hrs). The response of different concentrations of colchicine applied for varied durations revealed significant differences for various doubled haploidy parameters viz., per cent survived plants, per cent doubled haploid formation and per cent doubled haploid seed formation. In hexaploid and tetraploid wheats, colchicine doses of 0.075% for 4 hrs and 0.15% for 4 hrs, respectively were established as optimum for enhanced doubled haploid production.

Characterization and Molecular Mapping of Two Novel Genes Resistant to Pythium Stalk Rot in Maize.

Pythium stalk rot caused by Pythium inflatum is becoming a more and more serious disease in maize, and it has caused severe yield loss in China in recent years. Deployment of resistant maize varieties is the most effective way to control this disease. Searching for the resistant maize germplasm and identifying the resistance genes are the vital processes in the breeding program. The maize inbred line X178 previously showed high resistance to Pythium stalk rot. Thus, this study aimed to reveal the gene(s) resistance to Pythium stalk rot in X178 by resistance inheritance analysis using the derived F2 and F2:3 genetic populations. The results showed that two independently inherited dominant genes, designated RpiX178-1 and RpiX178-2, carried by X178 are responsible for its resistance relative to the susceptible parent Ye107; they are located on regions of maize chromosome (chr.) 1 bin 1.09 and chr. 4 bin 4.08, respectively, and flanked by markers umc2047 and bnlg1671 as well as bnlg1444 and umc1313, respectively, by linkage analysis. Subsequently, RpiX178-1 was finely mapped between markers SSRZ8 and IDP2347, with genetic distances of 0.6 and 1.1 cM, respectively, and the physical distance of the target region was about 700 kb. RpiX178-2 was also further located between markers bnlg1444 and umc2041, with a genetic distance of 2.4 cM. Moreover, we confirmed that the two genes RpiX178-1 and RpiX178-2 were newly identified and different from those genes known on chrs. 1 and 4 according to results of allelism testing. Herein, we newly identified two genes resistant to P. inflatum, which provided important genetic information for resistance to Pythium stalk rot in maize.

A Portion of the Apomixis Locus of Paspalum Simplex is Microsyntenic with an Unstable Chromosome Segment Highly Conserved Among Poaceae

The introgression of apomixis in major seed crops, would guarantee self-seeding of superior heterotic seeds over generations. In the grass species Paspalum simplex, apomixis is controlled by a single locus in which recombination is blocked. In the perspective of isolating the genetic determinants of apomixis, we report data on sequencing, in silico mapping and expression analysis of some of the genes contained in two cloned genomic regions of the apomixis locus of P. simplex. In silico mapping allowed us to identify a conserved synteny group homoeologous to the apomixis locus, located on a telomeric position of chromosomes 12, 8, 3 and 4 of rice, Sorghum bicolor, Setaria italica and Brachypodium distachyum, respectively, and on a more centromeric position of maize chromosome 1. Selected genes of the apomixis locus expressed sense and antisense transcripts in reproductively committed cells of sexual and apomictic ovules. Some of the genes considered here expressed apomixis-specific allelic variants which showed partial non-overlapping expression patterns with alleles shared by sexual and apomictic reproductive phenotypes. Our findings open new routes for the isolation of the genetic determinants of apomixis and, in perspective, for its introgression in crop grasses.

The analysis of functional genes in maize molecular breeding

Molecular breeding is capable of improving important agricultural crop traits by controlling functional genes, aiming to attain high yield, stability and quality. During this process, the quantity and maneuverability of functional genes are important in determining breeding efficiency. In our research, 186 functional genes relating to maize agronomic traits, which are available for marker-assisted selection or genetic transformation strategies, were collected from the literature and projected onto an integration map. The traits corresponding to these functional genes included disease resistance, stress tolerance, tassel traits, ear traits, kernel-related traits, leaf traits and plant-type characteristics. The integration map demonstrated that these functional genes were unevenly distributed on maize chromosomes. The greatest and fewest numbers of functional genes were found on chromosomes 1 and 8, respectively. Moreover, 36, 25, 27, 23, 14, 15, 11, 6, 9 and 20 genes were found on chromosomes 1 to 10, respectively. Most of disease-resistant genes were found on chromosome 10, while the genes of kernel-related and leaf-related traits were found on chromosomes 4 and 2, respectively. Out of the 186 functional genes, 95 have been characterized using genetic mapping, 19 using map-based cloning, 53 using transposon-tagging cloning strategies and 19 using other methods. Thus, the number of functional genes identified in maize is still limited and further research on functional genes is required.

A maize cytochrome b-c1 complex subunit protein ZmQCR7 controls variation in the hypersensitive response.

The gene GRMZM2G318346 which encodes a cytochrome b-c1 complex subunit 7 is associated with variation in strength of the hypersensitive response in maize. We previously identified a QTL at 3,545,354bp (B73 reference genome V2) on maize chromosome 5 associated with variation in the hypersensitive response (HR) conferred by the autoactive R-gene Rp1-D21 (Olukolu et al. in PLoS Genet 10:e1004562 2014). In this study, we show that a gene at this locus, GRMZM2G318346 which encodes a cytochrome b-c1 complex subunit seven (ZmQCR7), an important part of the mitochondrial electron transport chain, can suppress HR mediated by Rp1-D21 in a transient expression assay. ZmQCR7 alleles from two maize lines, W22 and B73 differ for the encoded proteins at just two sites, amino acid 27 (threonine and alanine in B73 and W22, respectively) and amino acid 109 (asparagine and serine), however, the B73 allele is much more effective at suppressing HR. We show that variation at amino acid 27 controlled this variation in HR-suppressing effects. We furthermore demonstrate that the B73 allele of ZmQCR7 can suppress HR induced by RPM1(D505V), another autoactive R-gene, and that Arabidopsis homologs of ZmQCR7 can also suppress NLR-induced HR. The implications of these findings are discussed.

Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships

Whole-chromosome painting probes were developed for each of the 10 chromosomes of maize by producing amplifiable libraries of unique sequences of oligonucleotides that can generate labeled probes through transcription reactions. These paints allow identification of individual homologous chromosomes for many applications as demonstrated in somatic root tip metaphase cells, in the pachytene stage of meiosis, and in interphase nuclei. Several chromosomal aberrations were examined as proof of concept for study of various rearrangements using probes that cover the entire chromosome and that label diverse varieties. The relationship of the supernumerary B chromosome and the normal chromosomes was examined with the finding that there is no detectable homology between any of the normal A chromosomes and the B chromosome. Combined with other chromosome-labeling techniques, a complete set of whole-chromosome oligonucleotide paints lays the foundation for future studies of the structure, organization, and evolution of genomes.

Using Maize Chromosome Segment Substitution Line Populations for the Identification of Loci Associated with Multiple Disease Resistance.

Southern Leaf Blight (SLB), Northern Leaf Blight (NLB), and Gray Leaf Spot (GLS) caused by Cochliobolus heterostrophus, Setosphaeria turcica, and Cercospora zeae-maydis respectively, are among the most important diseases of corn worldwide. Previously, moderately high and significantly positive genetic correlations between resistance levels to each of these diseases were identified in a panel of 253 diverse maize inbred lines. The goal of this study was to identify loci underlying disease resistance in some of the most multiple disease resistant (MDR) lines by the creation of chromosome segment substitution line (CSSL) populations in multiple disease susceptible (MDS) backgrounds. Four MDR lines (NC304, NC344, Ki3, NC262) were used as donor parents and two MDS lines (Oh7B, H100) were used as recurrent parents to produce eight BC3F4:5 CSSL populations comprising 1,611 lines in total. Each population was genotyped and assessed for each disease in replicated trials in two environments. Moderate to high heritabilities on an entry mean basis were observed (0.32 to 0.83). Several lines in each population were significantly more resistant than the MDS parental lines for each disease. Multiple quantitative trait loci (QTL) for disease resistance were detected for each disease in most of the populations. Seventeen QTL were associated with variation in resistance to more than one disease (SLB/NLB: 2; SLB/GLS: 7; NLB/GLS: 2 and 6 to all three diseases). For most populations and most disease combinations, significant correlations were observed between disease scores and also between marker effects for each disease. The number of lines that were resistant to more than one disease was significantly higher than would be expected by chance. Using the results from individual QTL analyses, a composite statistic based on Mahalanobis distance (Md) was used to identify joint marker associations with multiple diseases. Across all populations and diseases, 246 markers had significant Md values. However further analysis revealed that most of these associations were due to strong QTL effects on a single disease. Together, these findings reinforce our previous conclusions that loci associated with resistance to different diseases are clustered in the genome more often than would be expected by chance. Nevertheless true MDR loci which have significant effects on more than one disease are still much rarer than loci with single disease effects.