Mapping quantitative trait loci associated with southern leaf blight resistance in maize (Zea mays L.)

Southern leaf blight (SLB) caused by the fungus Cochliobolus heterostrophus (Drechs.) Drechs. is a major foliar disease of maize worldwide. Our objectives were to identify quantitative trait loci (QTL) for resistance to SLB and flowering traits in recombinant inbred line (RIL) population derived from the cross of inbred lines LM5 (resistant) and CM140 (susceptible). A set of 207 RILs were phenotyped for resistance to SLB at three time intervals for two consecutive years. Four putative QTL for SLB resistance were detected on chromosomes 3, 8 and 9 that accounted for 54% of the total phenotypic variation. Days to silking and anthesis–silking interval (ASI) QTL were located on chromosomes 6, 7 and 9. A comparison of the obtained results with the published SLB resistance QTL studies suggested that the detected bins 9.03/02 and 8.03/8.02 are the hot spots for SLB resistance whereas novel QTL were identified in bins 3.08 and 8.01/8.04. The linked markers are being utilized for marker‐assisted mobilization of QTL conferring resistance to SLB in elite maize backgrounds. Fine mapping of identified QTL will facilitate identification of candidate genes underlying SLB resistance.     

Genetic variation at bx1 controls DIMBOA content in maize

The main hydroxamic acid in maize (Zea mays L.) is 2-4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to leaf-feeding by several corn borers. Most genes involved in the DIMBOA metabolic pathway are located on the short arm of chromosome 4, and quantitative trait loci (QTLs) involved in maize resistance to leaf-feeding by corn borers have been localized to that region. However, the low resolution of QTL linkage mapping does not allow convincing proof that genetic variation at bx loci was responsible for the variability for resistance. This study addressed the following objectives: to determine the QTLs involved in DIMBOA synthesis across genetically divergent maize inbreds using eight RIL families from the nested association mapping population, to check the stability of QTLs for DIMBOA content across years by evaluating two of those RIL families in 2 years, and to test the involvement of bx1 by performing association mapping with a panel of 281 diverse inbred lines. QTLs were stable across different environments. A genetic model including eight markers explained approximately 34% of phenotypic variability across eight RIL families and the position of the largest QTL co-localizes with the majority of structural genes of the DIMBOA pathway. Candidate association analysis determined that sequence polymorphisms at bx1 greatly affects variation of DIMBOA content in a diverse panel of maize inbreds, but the specific causal polymorphism or polymorphisms responsible for the QTL detected in the region 4.01 were not identified. This result may be because the causal polymorphism(s) were not sequenced, identity is masked by linkage disequilibrium, adjustments for population structure reduce significance of causal polymorphisms or multiple causal polymorphisms affecting bx1 segregate among inbred lines.     

Targeted discovery of quantitative trait loci for resistance to northern leaf blight and other diseases of maize

To capture diverse alleles at a set of loci associated with disease resistance in maize, heterogeneous inbred family (HIF) analysis was applied for targeted QTL mapping and near-isogenic line (NIL) development. Tropical maize lines CML52 and DK888 were chosen as donors of alleles based on their known resistance to multiple diseases. Chromosomal regions (“bins”; n = 39) associated with multiple disease resistance (MDR) were targeted based on a consensus map of disease QTLs in maize. We generated HIFs segregating for the targeted loci but isogenic at ~97% of the genome. To test the hypothesis that CML52 and DK888 alleles at MDR hotspots condition broad-spectrum resistance, HIFs and derived NILs were tested for resistance to northern leaf blight (NLB), southern leaf blight (SLB), gray leaf spot (GLS), anthracnose leaf blight (ALB), anthracnose stalk rot (ASR), common rust, common smut, and Stewart’s wilt. Four NLB QTLs, two ASR QTLs, and one Stewart’s wilt QTL were identified. In parallel, a population of 196 recombinant inbred lines (RILs) derived from B73 × CML52 was evaluated for resistance to NLB, GLS, SLB, and ASR. The QTLs mapped (four for NLB, five for SLB, two for GLS, and two for ASR) mostly corresponded to those found using the NILs. Combining HIF- and RIL-based analyses, we discovered two disease QTLs at which CML52 alleles were favorable for more than one disease. A QTL in bin 1.06–1.07 conferred resistance to NLB and Stewart’s wilt, and a QTL in 6.05 conferred resistance to NLB and ASR.     

Precise mapping of quantitative trait loci for resistance to southern leaf blight, caused by Cochliobolus heterostrophus race O, and flowering time using advanced intercross maize lines

The intermated B73 × Mo17 (IBM) population, an advanced intercross recombinant inbred line population derived from a cross between the maize lines B73 (susceptible) and Mo17 (resistant), was evaluated in four environments for resistance to southern leaf blight (SLB) disease caused by Cochliobolus heterostrophus race O. Two environments were artificially inoculated, while two were not inoculated and consequently had substantially lower disease pressure. Four common SLB resistance quantitative trait loci (QTL) were identified in all environments, two in bin 3.04 and one each in bins 1.10 and 8.02/3. There was no significant correlation between disease resistance and days to anthesis. A direct comparison was made between SLB QTL detected in two populations, independently derived from the same parental cross: the IBM advanced intercross population and a conventional recombinant inbred line population. Several QTL for SLB resistance were detected in both populations, with the IBM providing between 5 and, in one case, 50 times greater mapping resolution.     

Beyond pathways: genetic dissection of tocopherol content in maize kernels by combining linkage and association analyses

Although tocopherols play an important role in plants and animals, the genetic architecture of tocopherol content in maize kernels has remained largely unknown. In this study, linkage and association analyses were conducted to examine the genetic architecture of tocopherol content in maize kernels. Forty‐one unique quantitative trait loci (QTLs) were identified by linkage mapping in six populations of recombinant inbred lines (RILs). In addition, 32 significant loci were detected via genome‐wide association study (GWAS), 18 of which colocalized with the QTLs identified by linkage mapping. Fine mapping of a major QTL validated the accuracy of GWAS and QTL mapping results and suggested a role for nontocopherol pathway genes in the modulation of natural tocopherol variation. We provided genome‐wide evidence that genes involved in fatty acid metabolism, chlorophyll metabolism and chloroplast function may affect natural variation in tocopherols. These findings were confirmed through mutant analysis of a particular gene from the fatty acid pathway. In addition, the favourable alleles for many of the significant SNPs/QTLs represented rare alleles in natural populations. Together, our results revealed many novel genes that are potentially involved in the variation of tocopherol content in maize kernels. Pyramiding of the favourable alleles of the newly elucidated genes and the well‐known tocopherol pathway genes would greatly improve tocopherol content in maize.     

Mapping of QTL for downy mildew resistance in maize

Quantitative trait loci (QTLs) of maize involved in mediating resistance to Peronosclerospora sorghi, the causative agent of sorghum downy mildew (SDM), were detected in a population of recombinant inbred lines (RILs) derived from the Zea mays L. cross between resistant (G62) and susceptible (G58) inbred lines. Field tests of 94 RILs were conducted over two growing seasons using artificial inoculation. Heritability of the disease reaction was high (around 70%). The mapping population of the RILs was also scored for restriction fragment length polymorphic (RFLP) markers. One hundred and six polymorphic RFLP markers were assigned to ten chromosomes covering 1648 cM. Three QTLs were detected that significantly affected resistance to SDM combined across seasons. Two of these mapped quite close together on chromosome 1, while the third one was on chromosome 9. The percentage of phenotypic variance explained by each QTL ranged from 12.4% to 23.8%. Collectively, the three QTLs identified in this study explained 53.6% of the phenotypic variation in susceptibility to the infection. The three resistant QTLs appeared to have additive effects. Increased susceptibility was contributed by the alleles of the susceptible parent. The detection of more than one QTL supports the hypothesis that several qualitative and quantitative genes control resistance to P. sorghi.     

QTLs for resistance to Setosphaeria turcica in an early maturing Dent×Flint maize population

Quantitative trait loci (QTLs) for resistance to the fungal pathogen Setosphaeria turcica, the cause of northern corn leaf blight (NCLB), were mapped in a population of 220 F₃ families derived from a cross between two moderately resistant European inbred lines, D32 (dent) and D145 (flint). The population was genotyped with 87 RFLP and 7 SSR markers. Trials were conducted in the field in Switzerland, and in the greenhouse with selected F₃ families in Germany. The F₃ population segregated widely for resistance with transgression of the parents. By composite interval mapping, a total of 13 QTLs were detected with two disease ratings (0 and 3 weeks after flowering). Together these QTLs explained 48% and 62% of the phenotypic variation. Gene action at most QTLs was partially dominant. Eight out of the 13 QTL alleles for resistance were contributed by the more-resistant parent, D145. On chromosomes 3, 5 and 8, QTLs were located in the same chromosomal regions as QTLs in tropical and U.S. Corn Belt germplasm. Some QTLs affected NCLB, head smut and common rust at the same time, with alleles at these loci acting isodirectionally. Received: 25 January 1999 / Accepted: 20 Februar 1999     

Identification of a locus in maize controlling response to a host-selective toxin derived from <Emphasis Type="Italic">Cochliobolus heterostrophus</Emphasis>, causal agent of southern leaf blight

Key Message

A host-selective, proteinaceous maize toxin was identified from the culture filtrate of the maize pathogen Cochliobolus heterostrophus. A dominant gene for toxin susceptibility was identified on maize chromosome 4.

Abstract

A toxic activity was identified from the culture filtrate (CF) of the fungus Cochliobolus heterostrophus, causal agent of the maize disease southern leaf blight (SLB) with differential toxicity on maize lines. Two independent mapping populations; a 113-line recombinant inbred line population and a 258-line association population, were used to map loci associated with sensitivity to the CF at the seedling stage. A major QTL on chromosome 4 was identified at the same locus using both populations. Mapping in the association population defined a 400 kb region that contained the sensitivity locus. By comparing CF-sensitivity of the parents of the RIL population with that of the F₁ progeny, we determined that the sensitivity allele was dominant. No relationship was observed between CF-sensitivity in seedlings and SLB susceptibility in mature plants; however, a significant correlation (??0.58) was observed between SLB susceptibility and CF-sensitivity in seedlings. The activity of the CF was light-dependent and was sensitive to pronase, indicating that the toxin was proteinaceous.

     

Allelic analysis of sheath blight resistance with association mapping in rice

Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Global attention has focused on examining individual mapping populations for quantitative trait loci (QTLs) for ShB resistance, but to date no study has taken advantage of association mapping to examine hundreds of lines for potentially novel QTLs. Our objective was to identify ShB QTLs via association mapping in rice using 217 sub-core entries from the USDA rice core collection, which were phenotyped with a micro-chamber screening method and genotyped with 155 genome-wide markers. Structure analysis divided the mapping panel into five groups, and model comparison revealed that PCA5 with genomic control was the best model for association mapping of ShB. Ten marker loci on seven chromosomes were significantly associated with response to the ShB pathogen. Among multiple alleles in each identified loci, the allele contributing the greatest effect to ShB resistance was named the putative resistant allele. Among 217 entries, entry GSOR 310389 contained the most putative resistant alleles, eight out of ten. The number of putative resistant alleles presented in an entry was highly and significantly correlated with the decrease of ShB rating (r = −0.535) or the increase of ShB resistance. Majority of the resistant entries that contained a large number of the putative resistant alleles belonged to indica, which is consistent with a general observation that most ShB resistant accessions are of indica origin. These findings demonstrate the potential to improve breeding efficiency by using marker-assisted selection to pyramid putative resistant alleles from various loci in a cultivar for enhanced ShB resistance in rice.     

Construction of high-density linkage map and identification of QTLs for resistance to sorghum downy mildew in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Sorghum downy mildew (SDM), caused by obligate biotrophic fungi Peronosclerospora sorghi, is an economically important disease of maize. The genetics of resistance was reported to be polygenic thereby necessitating identification of QTLs for resistance to SDM to initiate effective marker-assisted selection programs. During post-rainy and winter season of 2012, 645 F_2:3 progeny families from the cross CML153 (susceptible) × CML226 (resistant) were screened for their reaction to SDM. Characterization of QTLs affecting resistance to SDM was undertaken using the genetic linkage map with 319 polymorphic SSR and SNP marker loci and the phenotypic data of F_2:3 families. Three QTLs conferring resistance to SDM were consistently identified on chromosomes 2, 3 and 6 in both seasons. The resistant parent CML226 contributed all the QTL alleles conferring resistance to SDM. The major QTL located on chromosome 2 explained 38.68% of total phenotypic variation in the combined analysis with a LOD score of 9.12. All the three QTL showed partially dominant gene effects in combined analysis. The detection of more than one QTL supports the hypothesis that quantitative genes control resistance to P. sorghi. The generation was advanced to F₆ using markers linked to major QTLs on chromosomes 2 and 3 to derive 33 SDM resistant maize inbred lines.     

RETRACTED ARTICLE: An analysis of population structure and linkage disequilibrium using multilocus data in 187 maize inbred lines

This study analyzes population structure and linkage disequilibrium (LD) among 187 commonly used Chinese maize inbred lines, representing the genetic diversity among public, commercial and historically important lines for corn breeding. Seventy SSR loci, evenly distributed over 10 chromosomes, were assayed for polymorphism. The identified 290 alleles served to estimate population structure and analyze the genome-wide LD. The population of lines was highly structured, showing 6 subpopulations: BSSS (American BSSS including Reid), PA (group A germplasm derived from modern U.S. hybrids in China), PB (group B germplasm derived from modern U.S. hybrid in China), Lan (Lancaster Surecrop), LRC (derivative lines from Lvda Reb Cob, a Chinese landrace) and SPT (derivative lines from Si-ping-tou, a Chinese landrace). Forty lines, which formerly had an unknown and/or miscellaneous origin and pedigree record, were assigned to the appropriate group. Relationship estimates based on SSR marker data were quantified in a Q matrix, and this information will inform breeder’s decisions regarding crosses. Extensive inter- and intra-chromosomal LD was detected between 70 microsatellite loci for the investigated maize lines (2109 loci pairs in LD with D′ > 0.1 and 93 out of them at P < 0.01).This suggests that rapidly evolving microsatellites may track recent population structure. Interlocus LD decay among the diverse maize germplasm indicated that association studies in QTLs and/or candidate genes might avoid nonfunctional and spurious associations since most of the LD blocks were broken between diverse germplasm. The defined population structure and the LD analysis present the basis for future association mapping. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of quantitative trait loci controlling resistance to maize chlorotic dwarf virus

Ineffective screening methods and low levels of disease resistance have hampered genetic analysis of maize (Zea mays L.) resistance to disease caused by maize chlorotic dwarf virus (MCDV). Progeny from a cross between the highly resistant maize inbred line Oh1VI and the susceptible inbred line Va35 were evaluated for MCDV symptoms after multiple virus inoculations, using the viral vector Graminella nigrifrons. Symptom severity scores from three rating dates were used to calculate area under the disease progress curve (AUDPC) scores for vein banding, leaf twist and tear, and whorl chlorosis. AUDPC scores for the F₂ population indicated that MCDV resistance was quantitatively inherited. Genotypic and phenotypic analyses of 314 F₂ individuals were compared using composite interval mapping (CIM) and analysis of variance. CIM identified two major quantitative trait loci (QTL) on chromosomes 3 and 10 and two minor QTL on chromosomes 4 and 6. Resistance was additive, with alleles from Oh1VI at the loci on chromosomes 3 and 10 contributing equally to resistance.     

Strain-specific and recessive QTLs involved in the control of partial resistance to Fusarium oxysporum f. sp. melonis race 1.2 in a recombinant inbred line population of melon

Fusarium oxysporum f. sp. melonis (FOM) causes serious economic losses in melon (Cucumis melo L.). Two dominant resistance genes have been identified, Fom-1 and Fom-2, which provide resistance to races 0 and 2 and races 0 and 1, respectively, however FOM race 1.2 overcomes these resistance genes. A partial resistance to FOM race 1.2 that has been found in some Far East accessions is under polygenic control. A genetic map of melon was constructed to tag FOM race 1.2 resistance with DNA markers on a recombinant inbred line population derived from a cross between resistant (Isabelle) and susceptible (cv. Védrantais) lines. Artificial root inoculations on plantlets of this population using two strains, one that causes wilting (FOM 1.2w) and one that causes yellowing (FOM 1.2y), resulted in phenotypic and genotypic data that enabled the identification of nine quantitative trait loci (QTLs). These QTLs were detected on five linkage groups by composite interval mapping and explained between 41.9% and 66.4% of the total variation. Four digenic epistatic interactions involving seven loci were detected and increased the total phenotypic variation that was explained. Co-localizations between QTLs and resistance gene homologs or resistance genes, such as Fom-2 and Vat, were observed. A strain-specific QTL was detected, and some QTLs appeared to be recessive.     

Detection of putative quantitative trait loci in line crosses under infinitesimal genetic models

Quantitative trait locus (QTL) mapping studies often employ segregating generations derived from a cross between genetically divergent inbred lines. In the analysis of such data it is customary to fit a single QTL and use a null hypothesis which assumes that the genomic region under study contributes no genetic variance. To explore the situation in which multiple linked genes contribute to the genetic variance, we simulated an F₂-mapping experiment in which the genetic difference between the two original inbred strains was caused by a large number of loci, each having equal effect on the quantitative trait. QTLs were either in coupling, dispersion or repulsion phase in the base population of inbred lines, with the expected F₂ genetic variance explained by the QTLs being equivalent in the three models. Where QTLs were in coupling phase, one inbred line was fixed for all plus alleles, and the other line was fixed for minus alleles. Where QTLs were in dispersion phase, they were assumed to be randomly fixed for one or other allele (as if the inbred lines had evolved from a common ancestor by random drift). Where QTLs were in repulsion phase alleles within an inbred line were alternating plus and minus at adjacent loci, and alternative alleles were fixed in the two inbred lines. In all these genetic models a standard interval mapping test statistic used to determine whether there is a QTL of large effect segregating in the population was inflated on average. Furthermore, the use of a threshold for QTL detection derived under the assumption that no QTLs were segregating would often lead to spurious conclusions regards the presence of genes of large effects (i.e. type I errors). The employment of an alternative model for the analysis, including linked markers as cofactors in the analysis of a single interval, reduced the problem of type I error rate, although test statistics were still inflated relative to the case of no QTLs. It is argued that in practice one should take into account the difference between the strains or the genetic variance in the F₂ population when setting significance thresholds. In addition, tests designed to probe the adequacy of a single-QTL model or of an alternative infinitesimal coupling model are described. Such tests should be applied in QTL mapping studies to help dissect the true nature of genetic variation.     

The Wheat Puroindoline Genes Confer Fungal Resistance in Transgenic Corn

Puroindoline a and b (Pina and Pinb), together make up the functional components of the wheat grain hardness locus (Ha) and have antimicrobial properties. The antifungal activity of puroindoline proteins, PINA and PINB, has been demonstrated in vitro and in vivo. In this study, Pina and Pinb were introduced into corn under the control of a corn Ubiquitin promoter. Two Pina/Pinb expression–positive transgenic events were evaluated for resistance to Cochliobolus heterostrophus, the corn southern leaf blight (SLB) pathogen. Transgenic corn expressing Pins showed significantly increased tolerance to C. heterostrophus, averaging 42.1% reduction in symptoms. Pins are effective in vivo as antifungal proteins and could be valuable tools in corn SLB control.     

A first step toward the development of a barley NAM population and its utilization to detect QTLs conferring leaf rust seedling resistance

Key message

We suggest multi-parental nested association mapping as a valuable innovation in barley genetics, which increases the power to map quantitative trait loci and assists in extending genetic diversity of the elite barley gene pool.

Abstract

Plant genetic resources are a key asset to further improve crop species. The nested association mapping (NAM) approach was introduced to identify favorable genes in multi-parental populations. Here, we report toward the development of the first explorative barley NAM population and demonstrate its usefulness in a study on mapping quantitative trait loci (QTLs) for leaf rust resistance. The NAM population HEB-5 was developed from crossing and backcrossing five exotic barley donors with the elite barley cultivar ‘Barke,’ resulting in 295 NAM lines in generation BC₁S₁. HEB-5 was genetically characterized with 1,536 barley SNPs. Across HEB-5 and within the NAM families, no deviation from the expected genotype and allele frequencies was detected. Genetic similarity between ‘Barke’ and the NAM families ranged from 78.6 to 83.1 %, confirming the backcrossing step during population development. To explore its usefulness, a screen for leaf rust (Puccinia hordei) seedling resistance was conducted. Resistance QTLs were mapped to six barley chromosomes, applying a mixed model genome-wide association study. In total, four leaf rust QTLs were detected across HEB-5 and four QTLs within family HEB-F23. Favorable exotic QTL alleles reduced leaf rust symptoms on two chromosomes by 33.3 and 36.2 %, respectively. The located QTLs may represent new resistance loci or correspond to new alleles of known resistance genes. We conclude that the exploratory population HEB-5 can be applied to mapping and utilizing exotic QTL alleles of agronomic importance. The NAM concept will foster the evaluation of the genetic diversity, which is present in our primary barley gene pool.     

Molecular mapping of soybean aphid resistance genes in PI 567541B

The soybean aphid (Aphis glycines Matsumura) is an important pest of soybean [Glycine max (L.) Merr.] in North America since it was first reported in 2000. PI 567541B is a newly discovered aphid resistance germplasm with early maturity characteristics. The objectives of this study were to map and validate the aphid resistance genes in PI 567541B using molecular markers. A mapping population of 228 F₃ derived lines was investigated for the aphid resistance in both field and greenhouse trials. Two quantitative trait loci (QTLs) controlling the aphid resistance were found using the composite interval mapping method. These two QTLs were localized on linkage groups (LGs) F and M. PI 567541B conferred resistant alleles at both loci. An additive × additive interaction between these two QTLs was identified using the multiple interval mapping method. These two QTLs combined with their interaction explained most of the phenotypic variation in both field and greenhouse trials. In general, the QTL on LG F had less effect than the one on LG M, especially in the greenhouse trial. These two QTLs were further validated using an independent population. The effects of these two QTLs were also confirmed using 50 advanced breeding lines, which were all derived from PI 567541B and had various genetic backgrounds. Hence, these two QTLs identified and validated in this study could be useful in improving soybean aphid resistance by marker-assisted selection.     

Loci and candidate gene identification for resistance to Sclerotinia sclerotiorum in soybean (Glycine max L. Merr.) via association and linkage maps

Soybean white mold (SWM), caused by Sclerotinia sclerotiorum ((Lib.) W. Phillips), is currently considered to be the second most important cause of soybean yield loss due to disease. Research is needed to identify SWM‐resistant germplasm and gain a better understanding of the genetic and molecular basis of SWM resistance in soybean. Stem pigmentation after treatment with oxaloacetic acid is an effective indicator of resistance to SWM. A total of 128 recombinant inbred lines (RILs) derived from a cross of ‘Maple Arrow’ (partial resistant to SWM) and ‘Hefeng 25’ (susceptible) and 330 diverse soybean cultivars were screened for the soluble pigment concentration of their stems, which were treated with oxalic acid. Four quantitative trait loci (QTLs) underlying soluble pigment concentration were detected by linkage mapping of the RILs. Three hundred and thirty soybean cultivars were sequenced using the whole‐genome encompassing approach and 25 179 single‐nucleotide polymorphisms (SNPs) were detected for the fine mapping of SWM resistance genes by genome‐wide association studies. Three out of five SNP markers representing a linkage disequilibrium (LD) block and a single locus on chromosome 13 (Gm13) were significantly associated with the soluble pigment content of stems. Three more SNPs that represented three minor QTLs for the soluble pigment content of stems were identified on another three chromosomes by association mapping. A major locus with the largest effect on Gm13 was found both by linkage and association mapping. Four potential candidate genes involved in disease response or the anthocyanin biosynthesis pathway were identified at the locus near the significant SNPs (<60 kbp). The beneficial allele and candidate genes should be useful in soybean breeding for improving resistance to SWM.     

Combined linkage mapping and association analysis reveals genetic control of maize kernel moisture content

The free moisture in crop kernels after being naturally dried is referred to as kernel moisture content (KMC). Maize KMC reflects grain quality and influences transportation and storage of seeds. We used an IBM Syn10 DH maize population consisting of 249 lines and an association panel comprising 310 maize inbred lines to identify the genetic loci affecting maize KMC in three environments. Using the IBM population detected 13 QTL on seven chromosomes, which were clustered into nine common QTL. Genome-wide association analysis (GWAS) identified 16 significant SNPs across the 3 environments, which were linked to 158 genes across the three environments. Combined QTL mapping and GWAS found two SNPs that were located in two of the mapped QTL, respectively. Twenty-three genes were linked with the loci co-localized in both populations. Of these 181 genes, five have previously been reported to be associated with KMC or to regulate seed development. These associations were verified by candidate gene association analysis. Two superior alleles and one favorable haplotype for Zm00001d007774 and Zm00001d047868 were found to influence KMC. These findings provide insights into molecular mechanisms underlying maize KMC and contribute to the use of marker-assisted selection for breeding low-KMC maize.