Characterization and fine-mapping of a resistance locus for northern leaf blight in maize bin 8.06

Abstract

As part of a larger effort to capture diverse alleles at a set of loci associated with disease resistance in maize, DK888, a hybrid known to possess resistance to multiple diseases, was used as a donor in constructing near-isogenic lines (NILs). A NIL pair contrasting for resistance to northern leaf blight (NLB), caused by Setosphaeria turcica, was identified and associated with bin 8.06. This region of the maize genome had been associated in previous studies with both qualitative and quantitative resistance to NLB. In addition, bins 8.05–8.06 had been associated with quantitative resistance to several other diseases, as well as resistance gene analogs and defense response gene homologs. To test the hypothesis that the DK888 allele at bin 8.06 (designated qNLB8.06 _DK888 ) conditions the broad-spectrum quantitative resistance characteristic of the donor, the NILs were evaluated with a range of maize pathogens and different races of S. turcica. The results revealed that qNLB8.06 _DK888 confers race-specific resistance exclusively to NLB. Allelism analysis suggested that qNLB8.06 _DK888 is identical, allelic, or closely linked and functionally related to Ht2. The resistance conditioned by qNLB8.06 was incompletely dominant and varied in effectiveness depending upon allele and/or genetic background. High-resolution breakpoint analysis, using ~2,800 individuals in F₉/F₁₀ heterogeneous inbred families and 98 F₁₀/F₁₁ fixed lines carrying various recombinant events, delimited qNLB8.06 _DK888 to a region of ~0.46 Mb, spanning 143.92–144.38 Mb on the B73 physical map. Three compelling candidate genes were identified in this region. Isolation of the gene(s) will contribute to better understanding of this complex locus.

Introduction

Plants have evolved diverse mechanisms to combat pathogens. Some defense mechanisms condition complete resistance, while others provide intermediate forms of resistance. Mechanisms of complete resistance include R-genes and non-host resistance. R-gene-mediated resistance has often proven ephemeral, while quantitative resistance has generally been recognized as moderately effective, race non-specific and durable. Quantitative disease resistance (QDR) has, therefore, been more widely utilized in resistance breeding programs. QDR may, however, be conditioned by diverse mechanisms, and may vary in performance (Poland et al. 2009a). When QDR is conditioned by genes involved in the recognition of evolutionarily labile pathogen effectors, it is likely to be both race-specific and non-durable. An understanding of the pathogen- and race-specificity of a locus is more likely to provide predictive power regarding the durability of resistance than its quantitative effect alone.

A range of mechanisms have been associated with QDR, some of which are broader in spectrum and more durable than others. Broad-spectrum resistance has commonly been divided into two classes: (1) resistance effective against all known variants of a given pathogen (“race non-specific resistance”) and (2) resistance effective against more than one pathogen (“multiple disease resistance”). Some major resistance genes have been observed to confer moderate levels of either race-specific [e.g. Rp1 in maize (Smith and Hulbert 2005)] or race-nonspecific resistance [e.g. RB in potato (Song et al. 2003)]. Despite the lower selection pressure caused by genes conditioning incomplete resistance, these genes could be overcome by evolving pathogen races [e.g. R1 in potato (Trognitz and Trognitz 2007)]. Race-nonspecific QDR has also been shown to be associated with mechanisms other than R-genes. For example, the Yr36 gene in wheat contains domains similar to the proteins involved in the signaling of non-R-gene-mediated defenses, including programmed cell death and innate immune response (Fu et al. 2009). In rice, the recessive allele of a susceptibility gene Pi21, encoding a proline-rich protein with putative heavy-metal binding and protein–protein interaction motifs, contributes resistance to blast disease (Fukuoka et al. 2009). The resistance of these non-R-genes has thus far been stable. Disease non-specific QDR have been found to be controlled by genes involved in basal resistance, systemic acquired resistance, and defense signaling pathways [e.g. RPW8.1 and RPW8.2 in Arabidopsis (Wang et al. 2007c); npr1 in Arabidopsis (Cao et al. 1998)]. Agriculturally important genes of this type, including Lr34 in wheat (Krattinger et al. 2009) and mlo in barley (Buschges et al. 1997) have been shown to confer durable resistance to a number of obligate pathogens. Available lines of evidence imply that durability of resistance is associated with non-specificity, as well as non-gene-for-gene recognition in mechanism and incomplete phenotype. Nevertheless, the ambiguity associated with the effectiveness and spectra of defense mechanisms complicates breeding for disease resistance.

A large number of studies have been conducted to map R-genes, resistance gene analogs (RGAs), and loci conditioning QDR (quantitative trait loci for disease, or disease QTL) in plants. Current knowledge in the genetic architecture of disease resistance, as inferred from overview of previous reports, may provide some insights on the types of resistance associated with different genetic loci, which may in turn have implications for the likely performance of genes at these loci. It has been widely noted that R-genes and disease QTL are not randomly distributed across the genome. Apparent clusters of QTL for different diseases have been observed in rice (Wisser et al. 2005), maize (Wisser et al. 2006), barley (Williams 2003) and other plants. The coincidence of defense-related genes and/or QTL for multiple pathogens in certain chromosomal segments has led to the hypothesis that these chromosomal regions are associated with broad-spectrum resistance that could be durable. Likewise, in a range of plant pathosystems, major genes and/or QTL affecting a given disease has been found to overlap. Association of major genes along with QTL [e.g. rhm and QTL for southern leaf blight, and Rp3 and QTL for common rust in maize (Wisser et al. 2006)] may reflect the differential major and minor effects conferred by allelic variants of identical gene(s) (Robertson 1989; Welz and Geiger 2000), or the differential expression of resistance in various genetic backgrounds or environments. At a gene level, complex clustering of homologous or non-homologous R-genes [e.g. Pi5 in rice (Lee et al. 2009) and Rp1-D in maize (Collins et al. 1999)] has been suggested as a genetic hallmark of rapid evolution of R-genes and race specificities (Hulbert et al. 2001; McDowell and Simon 2006).

Chromosomal regions associated with previously reported R-genes, RGAs, and disease QTL can be sources of genes conditioning diverse forms of resistance. However, due to the limitations of QTL analysis, such as low precision of QTL locations and allelic sampling in different studies (Wisser et al. 2006), the implication for resistance specificity should be used with caution. For a given allele at a disease QTL hotspot region, detailed evaluation will be required to clearly determine whether it confers broad spectrum or disease-specific resistance. Race-specificity of disease QTL, particularly for ones that co-localize with known R-genes, needs to be clarified prior to practical application. This is to prevent the deployment of disease QTL under the misleading assumption of QDR conferring non-specific and more durable protection for crops. Expectations for the long-term performance of a disease QTL can be more realistic if its underlying genetic basis is more fully explored. For instance, knowing whether a broad-spectrum phenotype is conditioned by a pleiotropic gene(s), a cluster of defense-related genes, or the linkage of diverse R-genes, is valuable in designing combinations of favorable alleles of resistance genes in crop-breeding programs.

In the maize genome, among the regions that may harbor genes involving diverse defense pathways, the fifth to sixth segment of chromosome 8 (bin 8.05–8.06) can be viewed as one of the most complex, important, and interesting. Bin 8.05–8.06 is known to be associated with QTL for resistance to various diseases, RGAs, and defense response gene homologs (DRHs). Co-localized QTL were mapped in different populations for resistance to northern leaf blight (NLB) (Schechert et al. 1999; Welz et al. 1999a, b), southern leaf blight (SLB) (Bubeck 1992), gray leaf spot (GLS) (Bubeck et al. 1993; Clements et al. 2000; Maroof et al. 1996), common rust (Brown et al. 2001; Kerns et al. 1999), common smut (Luebberstedt et al. 1998), maize streak virus (Pernet et al. 1999), and aflatoxin accumulation in ears (Paul et al. 2003). In silico mapping anchored two RGAs, sharing conserved protein kinase (PK) domain with Pto in tomatoes and Pbs1 in Arabidopsis, to bins 8.05 and 8.06 (Xiao et al. 2006, 2007). Several DRHs, including five members of the S-adenosyl methionine synthetase family involved in amino acid metabolism and an oxalate oxidase-like protein gene associated with hypersensitive responses, were mapped to the same region using genetic and in silico analysis (Wang et al. 2007a).

Bin 8.05–8.06 is also a locus accounting for a significant proportion of NLB resistance in maize germplasm. NLB, caused by Setosphaeria turcica (anamorph Exserohilum turcicum, syn. Helminthosporium turcicum), is a foliar disease of maize that causes periodic epidemics associated with significant yield losses (Perkins and Pedersen 1987; Raymundo and Hooker 1981; Ullstrup and Miles 1957) in most maize-growing regions of the world. In diverse biparental populations, a number of NLB QTL (Schechert et al. 1999; Welz et al. 1999a, b) as well as two major gene loci, Ht2 (Yin et al. 2003; Zaitlin et al. 1992) and Htn1 (Simcox and Bennetzen 1993), have been mapped to bin 8.05–8.06. Evaluation of a large multi-parental mapping population (known as the nested association mapping population) (McMullen et al. 2009; Yu et al. 2008), consisting of 5,000 recombinant inbred lines developed from 25 diverse maize lines, identified two largest effect NLB QTL in the same region (Poland et al. 2009b). In response to a recurrent selection program for NLB resistance (Ceballos et al. 1991), significant changes in allele frequencies provided evidence of selection acting at several loci in bin 8.05–8.06 (Wisser et al. 2008). To date, in the maize–S. turcica pathosystem, clustering of major genes and QTL has only been observed at bin 8.05–8.06 (Wisser et al. 2006).

As part of a larger attempt to capture diverse alleles at important resistance loci, we selected the maize hybrid DK888 as a source of potentially useful alleles. This genotype has been shown to harbor alleles for resistance to diverse diseases (Kraja et al. 2000) and derived lines have been produced. In the present study, we aimed to fine-map and characterize DK888 allele(s) in bin 8.05–8.06 and to determine their disease- and race-specificity. Identification of the genes underlying the QTL region will be an important basis for detailed mechanistic studies.

The “heterogeneous inbred family” (HIF) approach was utilized to rapidly generate near-isogenic lines (NILs) carrying contrasting alleles at bin 8.05–8.06 (Tuinstra et al. 1997). This approach involves extraction of NILs from nearly fixed lines, such as lines that have been produced by selfing segregating materials for several generations. Being isogenic at most of the genome, but contrasting for specific QTL of interest, HIF-derived NILs have been used to validate the position and effect of QTL (Borevitz and Chory 2004; Kobayashi et al. 2006; Loudet et al. 2005; Pumphrey et al. 2007). When compared with NILs generated by successive backcrossing, NILs derived from HIFs can be put to use in a shorter period of time (particularly if NILs are available, as they were in this case), and can possibly provide recombinant genetic backgrounds in which the QTL effects are well expressed (Tuinstra et al. 1997).

This study was undertaken to genetically dissect a complex genetic region associated with qualitative and quantitative resistance to NLB and a range of other diseases in maize. To identify, validate and characterize QTL, we isolated bin 8.05–8.06 of DK888, a maize hybrid carrying favorable alleles for multiple disease resistance (Kraja et al. 2000; C. Chung, unpublished) in NILs using HIF-based approach. We will hereafter identify this QTL with bin 8.06, as it was initially located to a region spanning the distal end of bin 8.05 to the distal end of bin 8.06 (mostly in bin 8.06), and was ultimately fine-mapped to bin 8.06. NILs differing for the specific region were investigated to gain insights into a series of questions, including the disease- and race-specificity of the QTL, the QTL in relation to the known co-localized major gene loci, and the gene action at the QTL. To further unravel the complex genetic architecture and defense mechanisms, high-resolution mapping was conducted using break-point analysis. Our study has laid the foundation for positional cloning of a S. turcica race-specific resistance gene(s) underlying bin 8.06 of maize. The markers closely linked to the major NLB QTL can also be used for practical resistance breeding.

Materials and methods

Plant materials

The initial plant materials for QTL identification were 17 F₆ heterogeneous inbred families (HIFs) from the cross of S11 × DK888, which were provided by The USDA Germplasm Enhancement of Maize (GEM) Project (Balint-Kurti et al. 2006; Goodman 2005; Lee and Hardin 1997). DK888 is a single-cross hybrid developed by Thailand Charoen Seeds Group in collaboration with US Dekalb Seeds. It was released in Thailand in 1991, and dominated the local Thailand hybrid maize seed market in 1990s (Ekasingh et al. 2001). DK888 is a maize genotype carrying favorable alleles for resistance to NLB, southern leaf blight, gray leaf spot, northern leaf spot and common rust (Kraja et al. 2000). It also exhibited high levels of resistance to common smut and Stewart’s wilt in our repeated field trials (C. Chung, unpublished). The subsequently derived HIFs and NILs were generated by single-seed descent from selected lines in the families segregating for bin 8.06. In this study, “NILs” refers to sets of HIF-derived F₈, F₉, F₁₀ and F₁₁ lines that contrasted for bin 8.06, but were presumably isogenic at >99.2% of the genome.

Two sets of isolines with and without the Ht major genes were obtained from Peter Balint-Kurti of the USDA-ARS unit at North Carolina State University (a total of 6 differential lines: Pa91, Pa91Ht1, Pa91Ht2, Pa91Ht3, B68, and B68Htn1). Ht1, Ht2, and Htn1 were derived from maize lines GE440, NN14B and Peptilla, respectively, while Ht3 was derived from Tripsacum floridanum (Welz and Geiger 2000; M. Carson, pers. comm.). Several F₁ and F₂ populations were developed by crossing the differential lines with the F₉ NILs carrying DK888 or S11 alleles at the QTL region. The differential lines were also used to provide reference phenotypes of major gene resistance to S. turcica.

Disease evaluations

Northern leaf blight

Resistance to NLB was evaluated with S. turcica race 1 (isolate EtNY001) in a greenhouse at Cornell University, and at Cornell’s Robert Musgrave Research Farm in Aurora, NY from 2006 to 2009. The isolate EtNY001, originally collected from an infected leaf collected in Freeville NY in 1983, is compatible on Pa91Ht1, and incompatible on Pa91Ht2, Pa91Ht3, and B68Htn1 (Supplementary Table 1) under the standard greenhouse conditions established for NLB assays (Leonard et al. 1989). Another four S. turcica isolates representing different races, including Et10a (race 0), Et1001A (race 1), Et86A (race 23), and Et28A (race 23N), were obtained from P. Balint-Kurti, and used exclusively for the race-specificity tests in the greenhouse. In the greenhouse, plants at the 5–6-leaf stage were inoculated with 0.5 ml of spore suspension (4 × 10³ conidia per ml in 0.02% Tween 20) in the whorl, and kept in a mist chamber at >85% RH overnight. In the field, plants at the same stage were inoculated with spore suspension along with colonized sorghum grains (1/4 teaspoon, ~1.25 ml) in the whorl. The use of both liquid and solid inoculum was intended to ensure the viability of inoculum under dry weather conditions. S. turcica was cultured on lactose–casein hydrolysate agar (LCA) for 2–3 weeks, under a 12 h/12 h normal light–dark cycle at room temperature. Liquid inoculum was prepared by dislodging the conidia from the plates with sterilized ddH₂O, filtering the suspension through four layers of cheesecloth, and adjusting the concentration with the aid of a haemocytometer. The substrate for solid inoculum consisted of sterilized sorghum grains in 1-gallon milk jugs. For each jug, 900 ml of sorghum grains was soaked overnight in 600 ml of dH₂O and then autoclaved twice. The spore suspension from each heavily colonized LCA plate was distributed between 3–5 jugs of sterilized sorghum grains. The inoculated jugs were shaken every day until use to prevent caking and to accelerate fungal colonization. Incubation period (IP) was rated as the number of days post inoculation (dpi) when observing the appearance of first wilted lesion on a plant. IP was checked every day until 25 dpi. The 50% IP was recorded on a row basis when >50% of the plants in a row started showing the lesions. Primary diseased leaf area (PrimDLA) was rated as the percentage of infected leaf area of the inoculated leaves for individual plants at 2–3 weeks after inoculation. Diseased leaf area (DLA) was rated as the percentage of infected leaf area of the entire plant, disregarding decayed bottom leaves for individual plants or on a row basis for fixed lines. DLA was recorded ~10 days before anthesis (2–3 weeks after the onset of secondary infection).

Southern leaf blight

Resistance to SLB was evaluated with Cochliobolus heterostrophus race O in the greenhouse in September 2007 and in Clayton, North Carolina in 2008. In the greenhouse trial, plants at the 5–6-leaf stage were inoculated with the isolate C5 (ATCC 48332) obtained from G. Turgeon at Cornell University. Inoculum was cultured on complete medium with xylose (CMX) under continuous fluorescent light for 7–10 days, and spore suspension was prepared as described for NLB. About 0.5 ml of spore suspension (5 × 10⁴ conidia per ml, 0.02% Tween 20) was evenly sprayed on the first fully expanded leaf with an airbrush (Badger^® Model 150) at 20 psi. After inoculation, the plants were kept in a mist chamber at >85% RH overnight. Lesion length was measured at 4 dpi from 20 randomly chosen lesions on each plant. Primary DLA was rated at 6 dpi as the percentage of infected leaf area of the inoculated leaf. In the field trial, plants at the 4–6-leaf stage were inoculated as previously described (Carson 1998; Carson et al. 2004). Disease severity was rated based on a 1–9 scale corresponding to the diseased leaf area on primarily the ear leaf. Disease was evaluated for three times with 10–12-day interval from around 2 weeks after anthesis. The disease severity scores were used to calculate area under the disease progress curve ${\text{AUDPC}} = \sum\nolimits_{i = 1}^{n - 1} {{\frac{(y_{i} + y_{i + 1})(t_{i + 1} - t_{i})}{2}}}$, where y _i , disease severity at time i, t _i+1 − t _i , day interval between two ratings; n, number of ratings (Wilcoxson et al. 1974).

Gray leaf spot

Resistance to naturally occurring GLS (caused by Cercospora zeae-maydis and/or Cercospora zeina) was evaluated in Blacksburg, Virginia in 2008. The non-tillage field was located in a valley with regular morning mists and heavy dews, conditions that favor GLS development. Disease severity was scored based on a 1–10 scale with 0.25 increments, according to the disease progress on the ear leaf (Saghai Maroff et al. 1993). The evaluation was conducted four times with a 7–8-day interval from about 2 weeks after anthesis. The AUDPC was calculated as described above.

Anthracnose leaf blight

Resistance to anthracnose leaf blight (ALB) was evaluated in the greenhouse in September 2007 and 2008, with Colletotrichum graminicola (teleomorph: Glomerella graminicola) isolate Cg151 (obtained from G. Bergstrom of Cornell University). Inoculum was cultured on oatmeal agar for 2 weeks under continuous fluorescent light at room temperature (Muimba-Kankolongo and Bergstrom 1990). Each plant at the 5–6-leaf stage was inoculated in the whorl with 0.5 ml of spore suspension (2 × 10⁴ conidia per ml, 0.02% Tween 20, prepared as described above), then kept in a mist chamber at >85% RH overnight. Individual plants were rated for IP, latent period and PrimDLA. LP was rated as the number of dpi when observing the first appearance of black acervuli on the lesions. The ratings of IP and PrimDLA were as described for NLB.

Anthracnose stalk rot

Resistance to anthracnose stalk rot (ASR) was evaluated with C. graminicola isolate Cg151 in the greenhouse in December 2007 and in Aurora NY in 2008. For each plant at tasseling stage (Keller and Bergstrom 1988), the first internode above the brace root was punctured with an ice pick, a 1 ml pipette tip was inserted, and the plant was inoculated with 1 ml of spore suspension (10⁶ conidia per ml, 0.02% Tween 20, prepared as described for ALB) through the tip. At 4 weeks post inoculation, the stalk of each individual plant was split longitudinally, and the percentages of discolored area were rated for 8 (the trial in 2007) or 6 (2008) consecutive internodes (Keller and Bergstrom 1988). Data from all the scored internodes were summed for analysis.

Common rust

Resistance to rust was evaluated in the greenhouse in September 2007 and in Aurora, New York in 2008, with urediniospores of Puccinia sorghi collected from naturally infected leaves at Aurora NY in 2007. In the greenhouse trial, about 200–300 mg of stock urediniospores (preserved at −80°C) were suspended in 100 ml of Sortrol oil (Chevron Phillips Chemical Company, Phillips, TX, USA) (Webb et al. 2002). About 1 ml of suspension was evenly applied on the first two fully expanded leaves of each plant with a spray gun (Preval, Yonkers, NY, USA). Plants were kept in a mist chamber at >85% RH overnight. Individual plants were observed daily and rated for first pustule appearance, which is the number of dpi when the first pustule on a plant is observed. Pustules on the inoculated leaves were counted at 10 dpi. PrimDLA was rated at 14 dpi as described above. For the field trial, inoculum was increased on 3–4-leaf stage seedlings of susceptible sweet corn inoculated in the greenhouse. The urediniospores were collected by agitating infected leaves with matured rust pustules in distilled water, and filtering the spores through four layers of cheesecloth. Field plants at 6–8-leaf stage were inoculated with 1 ml of spore suspension (2 × 10⁵ urediniospores per ml, 0.02% Tween 20) in the whorl (Pataky and Campana 2007). Disease severity was rated on a row basis using a 0–10 scale with 0.5 increments, corresponding to the percentage of infected leaf area of the entire plant (0 = no disease, 1 = 10%, 10 = 100%). The AUDPC was calculated as described above, from three severity scores evaluated with 9-day interval from 4 weeks after inoculation.

Common smut

Resistance to smut was evaluated in the greenhouse in November 2007, and in Aurora, NY in 2008, with six compatible strains of Ustilago maydis (UmNY001, UmNY002, UmNY003, UmNY004, UmNY008 and UmNY009) which were isolated from naturally infected smut galls collected in Aurora, NY in 2007 [isolation procedure: (Thakur et al. 1989b); compatibility test: (Puhalla 1968)]. The first ear of each plant was shoot bagged, and injected with 2 ml of sporidial suspension (10⁶ sporidia per ml in 0.02% Tween 20) through the silk channel, when the silk had emerged 1–5 cm. Inoculum was prepared by culturing the isolates separately in potato-dextrose broth (PDB) on a shaker at 100 rpm at room temperature for 1 day, adjusting the sporidial concentrations with sterilized ddH₂O, and mixing equal amounts of compatible strains right before inoculation (du Toit and Pataky 1999). In the greenhouse trial, the volume (length × width × height) and weight of ear galls were measured. In the field trial, the incidence and severity of ear galls and naturally occurring stalk galls were rated at 4–5 weeks post-anthesis. Severity scores were evaluated for individual plants on a 0–10 scale, corresponding to the number and size of galls, and the disease severity of the entire plant.

Stewart’s wilt

Resistance to Stewart’s wilt was evaluated with Pantoea stewartii (syn. Erwinia stewartii) strain PsNY003 (obtained from H. Dillard of Cornell University) in Aurora, NY in 2008. Plants at the 5–6-leaf stage were inoculated following a modified pinprick method (Blanco et al. 1977; Chang et al. 1977). Whorl leaves of each plant were pierced twice with a specialized inoculator pre-dipped in bacterial suspension [10⁷ colony forming units per ml in sterilized 0.1 M NaCl solution, prepared as described by Suparyono and Pataky (1989)]. Multiple-pin inoculators was made with 30 T-pins (1.5-inch long), pieces of 5.5 × 6.5 cm sponge, and cork board (3/8-inch thick) fastened on two arms of a tong with rubber bands. PrimDLA (as described for NLB) was rated on a row basis at 2 and 3 weeks after inoculation.

Genotyping assays

DNA extraction

Plant genomic DNA was extracted following a modified mini-prep CTAB method (Doyle and Doyle 1987; Qiu et al. 2006). The high-throughput extraction was conducted using 96-well plates (Corning^® Costar 96 Well Polypropylene Cluster Tubes). For each sample, about 0.1 g of leaf tissue was frozen and ground with a stainless steel ball (5/32-inch diameter, OPS Diagnostics, NJ, USA), at 450 strokes per min for 50–120 s using Genogrinder 2000 (SPEX CertiPrep Inc., Metuchen, NJ, USA). Pulverized sample was suspended in 500 µl of CTAB extraction buffer [2% (w/v) hexadecyltrimethylammonium bromide, 1.4 M NaCl, 100 mM Tris–HCl (pH 8.0), 20 mM EDTA (pH 8.0), 0.2% (v/v) of 2-mercaptoethanol; 2-mercaptoethanol was added prior to use], and incubated at 65°C for 30–50 min. The CTAB suspension was mixed thoroughly with 400 µl of chloroform/isoamyl alcohol (24:1, v/v) for 3 min, then centrifuged at 5,200 rpm for 15 min. The supernatant was transferred to a new tube, mixed with 300 µl of isopropanol, and incubated at −20°C overnight. DNA was precipitated by centrifuging the sample at 5,200 rpm at 4°C for 12 min, and recovered by repeatedly discarding the supernatant and rinsing with 70% then 100% ethanol. The air-dried DNA pellet was dissolved in 100–150 µl of Tris–EDTA buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0).

Simple sequence repeat markers

Simple sequence repeat (SSR) primers were chosen from the Maize Genetics and Genomics Database (MaizeGDB) (http://www.maizegdb.org/). To integrate the fluorescent dye in the PCR product, the specific primer pair and a fluorescently labeled universal primer were used in a single-reaction nested PCR (Schuelke 2000). Each PCR reaction was performed as described by Wisser et al. (2008) in a total volume of 13 µl, with the same thermal cycling parameters as described by Schuelke (2000). The resulting amplicons labeled with different dyes were multiplexed (up to four PCR reactions were combined) and analyzed with the Applied BioSystems 3730xl DNA Analyzer at Biotechnology Resource Center at Cornell University. Each sample consisted of 0.7 µl PCR product per primer pair, 0.05–0.1 µl GeneScan-500 LIZ size standard, and 9 µl formamide (Applied Biosystems, Foster City, CA, USA). The sizes of amplicons were scored using GeneMapper v. 3.0 (Applied Biosystems).

Single-nucleotide polymorphism (SNP) and cleaved amplified polymorphic site (CAPS) markers

The B73 genomic sequences were used as a reference map for identifying polymorphisms between DK888 and S11. Various genes across the QTL region were chosen as the templates for marker design. Gene sequences were obtained from the database of the Maize Genome Sequencing Project (the Maize Sequence Database, http://www.maizesequence.org; same annotated genes currently available at http://archive.maizesequence.org), and the specific primers for each gene were designed using Primer 3 (Rozen and Skaletsky 2000). Each PCR reaction was performed in a total volume of 16 µl, containing final concentrations of 1× PCR buffer [10 mM Tris–HCl (pH 8.3), 50 mM KCl, 0.1% (v/v) Triton X-100], 1.5 mM MgCl₂, 1 µM forward-specific primer, 1 µM reverse-specific primer, 1–3 units Taq polymerase, and 20–50 ng template DNA. The thermal cycling parameters for different sets of primers can be found in Supplementary Table 2. PCR products amplified from DK888 and S11 homozygotes were purified with exonuclease I and shrimp alkaline phosphatase (New England Biolabs, Ipswich, MA, USA), and sequenced at Biotechnology Resource Center at Cornell University. The DNA sequencing was performed using BigDye Terminator and AmpliTaq-FS DNA Polymerase, and analyzed on the Applied BioSystems 3730×l DNA Analyzer (Applied Biosystems). The sequencing results were then aligned and analyzed for SNPs, small indels (insertions/deletions), and restriction sites using BioLign version 2.0.9 (developed by T. Hall, http://en.bio-soft.net/dna/BioLign.html). CAPS markers were developed if restriction-site polymorphisms were detected. For CAPS markers, PCR products were completely digested with specific restriction endonucleases (New England Biolabs), and the resulting polymorphic fragments were revealed using standard agarose gel electrophoresis followed by ethidium bromide staining. The SNP and CAPS markers are listed in Supplementary Table 2.

Genetic map

A genetic map of 11 SSR markers spanning the qNLB8.06 _DK888 region was constructed using genotypic data from segregating F₉ families. The map distances between SSR markers were estimated using MapDisto 1.7.0 (Lorieux 2007) based on Kosambi’s mapping function (Kosambi 1944). The relative genetic distances between the 12 and 7 newly developed SNP markers in the intervals of umc2199–umc2210 and umc2210–umc1287, respectively, were calculated by the proportion of identified crossover events between SSR markers. Corresponding physical positions of the markers were obtained from the physical map of the inbred line B73, based on the Maize Sequence Database.

QTL analysis

Single marker analysis and interval mapping (Lander and Botstein 1989) were performed using Windows QTL Cartographer 2.5 (Wang et al. 2007b) to analyze QTL position in segregating heterogeneous inbred families. In interval mapping, QTL were scanned at a walk speed of 0.5 cM. The threshold values were based on the likelihood of odds ratio (LOD) scores from 1,000 permutations of the original at a significance level of P = 0.01 (Churchill and Doerge 1994). The LOD threshold used in the study was averaged from the threshold value calculated for each trait. For the marker locus closest to the QTL peak, the additive effect and the proportion of phenotypic variance explained by the QTL (R ²) were obtained using the Windows QTL Cartographer. The R ² values for single marker analysis were from the analysis of variance (ANOVA) conducted in JMP 7.0 (SAS Institute Inc., Cary, NC, USA). The allele effect was designated as the mean difference between DK888 homozygotes and S11 homozygotes at a locus. The 95% confidence interval for the QTL was estimated according to the “1-LOD support interval”, which includes the QTL peak and its right and left loci with LOD scores dropping within 1 (Lander and Botstein 1989).

Experimental design and statistical analysis

HIFs for the identification and fine-mapping of the QTL

From 2006 to 2008, individual plants in each heterogeneous inbred family were genotyped with segregating markers, and phenotyped for resistance to NLB in a controlled greenhouse at Cornell University, or in Aurora, NY (Table 1). To control environmental variations, plants in a family were grown within a single block. Data were analyzed using Windows QTL Cartographer 2.5 as described in “QTL analysis”. The analysis of variance was also carried out on an individual trait-marker basis using JMP 7.0. The phenotypic differences among different genotypes were determined by pairwise two-tailed Student’s t test at P < 0.05.

Table 1 Summary of QTL analysis for qNLB8.06 _DK888 in segregating heterogeneous inbred families (HIFs) derived from S11 × DK888

F₈ and F₉ NILs for the characterization of the QTL

In 2007 and 2008, evaluations for seven different diseases were conducted independently in the field and/or greenhouse using a pair of F₈ NILs, B73 and DK888. Plants were grown in a randomized complete block design (RCBD), with two replications and 10 kernels per genotype (row) per replication in the field, and two replications and 6–8 plants per genotype per replication in the greenhouse. Data were analyzed using JMP 7.0 by fitting linear least squares models with “genotypes”, “environments” and “replications within environments” as independent variables. Differences between the least squares means of the NILs were determined by two-tailed Student’s t test at P < 0.05.

Race-specificity tests were carried out in the greenhouse using a pair of F₈ NILs in September 2007, and a set of six F₉ NILs (4 NILs with qNLB8.06 _DK888 , and 2 NILs with qNLB8.06 _S11 ) in September 2008. This was a split plot design (RCBD on the whole plots) with “S. turcica isolates” as the whole plot treatments and “the alleles at qNLB8.06” as the split plot treatments. In each environment, the trial consisted of two replications, 6–8 plants per NIL per isolate per replication. The maize differential lines Pa91, Pa91Ht1, Pa91Ht2, Pa91Ht3, B68, and B68Htn1 were included as control in 2008. Data were analyzed as described above, with “the alleles at qNLB8.06”, “S. turcica isolates”, “alleles by isolates”, “environments”, and “replications within environments” as variables. Differences among the “alleles by isolates” were determined by Tukey–Kramer HSD (honestly significant difference) test at P < 0.05.

F₁₀ and F₁₁ NILs for high-resolution mapping of the QTL

A total of 13 F₁₀ and 85 F₁₁ NILs were evaluated at Aurora NY in 2008 (for IP and DLA) and 2009 (for IP only), respectively. The NILs were put in rows (10 kernels per row) with two replications per year. In 2008, the 13 F₁₀ NILs were randomized within each replication, with DK888 and B73 rows as control. In 2009, the 85 F₁₁ NILs originated from 11 F₉ families were grown in 11 randomized blocks, according to their parental families (NILs from the same F₉ line were randomized in one block). Two extra control rows, originating from the corresponding F₉ lines, were grown on one side of each block. The resistant and susceptible control rows were two F₁₀ NILs homozygous for DK888 and S11 alleles for the entire QTL region (umc2199–umc1287). In addition to the IP and DLA ratings, the NILs were classified as “resistant” or “susceptible” based on the comparisons with the control lines in the same block. Using JMP 7.0, data from 2008 to 2009 were analyzed separately by fitting a mixed model with each “marker” as a fixed factor, and “replications” and “blocks within replications” as random effects. The analyses were performed on an individual marker–trait basis. Significance levels of marker–QTL associations were represented by the negative logarithm P values (−log P) calculated from the resulting F statistics.

F₁ and F₂ populations for allelic analysis

In 2009, the allelic relationships between qNLB8.06 _DK888 and Ht2, and between qNLB8.06 _DK888 and Htn1, were evaluated in the greenhouse and in the field at Aurora, NY, respectively. The F₁ and F₂ progenies derived from different pairs of Pa91, Pa91Ht2, and B68Htn1 crossed with the NIL carrying DK888 or S11 allele at qNLB8.06, were individually phenotyped for IP, PrimDLA (scored at 18 days after inoculation, greenhouse only), and lesion types. Two F₁₀ NILs contrasting for qNLB8.06, Pa91, Pa91Ht2, B68, and B68Htn1 were used as control. In the greenhouse, different genotypes were arranged following a RCBD with two replications, four blocks per replication. Each block consisted of 5–6 plants per F₁ population and control genotypes, and 10–12 plants per F₂ population. Data from F₁ progenies and control genotypes were analyzed using JMP 7.0 by fitting a linear least squares model with “genotypes”, “replications” and “blocks within replications” as independent variables. Differences among the least squares means of genotypes were determined by Tukey–Kramer HSD test at P < 0.05. In the field, plants were put in rows with 10 kernels per row (average germination rate was 38%). Plants in each population (10 rows per F₁ population, and 24 rows per F₂ population) were grown in one block, with each population bordered by control rows. The F₁ progenies were compared with the control genotypes in the same block. Data were analyzed as described above, with “genotypes” as the variable.

Identification of candidate genes

Putative genes in the B73 genomic sequences have been predicted by the Maize Genome Sequencing Project using the Gramene pipeline (Liang et al. 2009) (data available at http://www.maizesequence.org/ and http://archive.maizesequence.org/). The evidence-based gene prediction was conducted by aligning the sequences of known proteins, full-length cDNAs, and expressed sequence tags (ESTs) from maize as well as cross-species libraries to the bacterial artificial chromosome (BAC) contigs of B73. We surveyed existing predicted genes spanning the fine-mapped QTL interval. The potential identities of the predicted coding sequences were subsequently determined by performing basic local alignment search tool (BLAST) searches at the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Results

Identification of an incompletely dominant NLB QTL (qNLB8.06 _DK888 ) by HIF analysis

Following the HIF methodology described by Tuinstra et al. (1997), the study’s first step was to detect residual heterozygosity at potential disease QTL regions in the HIFs derived from S11 × DK888. Forty-six individuals of 17 F₆ families (1–4 individuals per family) were analyzed with 17 markers covering 12 bins. The marker targeting bin 8.06 was umc1149. An individual heterozygous for umc1149 and another marker at bin 5.06 (umc2216) was identified, and was used to generate the genetic materials for subsequent QTL analysis.

In 2006, a F₇ family consisting of 53 individuals was evaluated for resistance to NLB (Table 1). The F₇ progeny were segregating for umc1149 and umc2216, but isogenic at ~98.4% of the genome. Variation in disease response co-segregated with umc1149 (DK888 allele for resistance; allele effect in IP = 5.9 dpi, LOD = 2.7, R ² = 0.21), not umc2216, indicating the existence of a candidate NLB QTL at bin 8.06. In 2007, the finding was further validated in a F₈ family (96 individuals) segregating for umc1149, but fixed at umc2216. Consistently, the QTL contributed strong effects on reducing disease. As much as 62 and 38% of phenotypic variation in IP and PrimDLA, respectively, were explained by the QTL (Table 1).

To more precisely localize the identified NLB QTL, an additional 15 SSR markers across bins 8.05–8.06 were used to estimate the start and end points of heterozygous loci in the HIFs. Assuming that each end of the QTL segment lies halfway between the last marker for the introgression and the first marker outside it, the QTL was determined to reside in the interval of 386.8–453.7 cM on the IBM 2008 neighbors map, and between 136.2 and 156.0 Mb on the B73 physical map. This is a region spanning bins 8.05 and 8.06, but located mostly in bin 8.06. Among the nine markers analyzed in the F₈ family (umc1287, umc1828, umc2356, umc1149, bnlg240, umc1997, umc1728, umc2361, umc2395), the QTL was closest to umc1287.

The identified QTL locating mostly in bin 8.06, designated as qNLB8.06, showed incompletely dominant resistance (Fig. 1). It was observed that the level of resistance in DK888 homozygotes was much greater than in the heterozygotes or in the S11 homozygotes. The magnitudes and significance levels of the differences among the three genetic classes (PrimDLA: DK888/DK888 − S11/S11 = −22.0%, P < 0.0001; DK888/DK888 − heterozygotes =−14.1%, P < 0.0001; heterozygotes − S11/S11 = −7.9%, P = 0.005) suggested that the resistance performance in heterozygotes is more similar to S11 homozygotes. The same type of gene action was consistently seen in the subsequent mapping populations.

Fig. 1

Gene action at qNLB8.06. DK888 homozygotes showed greater resistance than the heterozygotes and S11 homozygotes, suggesting that resistance conferred by the DK888 allele(s) is incompletely dominant. The phenotypic differences among the three genetic classes were tested in an F₈ family by ANOVA, followed by pairwise Student’s t test at P < 0.05

qNLB8.06 _DK888 is not effective for multiple disease resistance

To understand the resistance spectrum of qNLB8.06 _DK888 , a pair of F₈ NILs was characterized for resistance to GLS, SLB, ALB, ASR, common rust, common smut and Stewart’s wilt (Table 2). The NILs derived from a single F₇ line were contrasting for the QTL region but isogenic at ~99.2% of the genome, according to the theoretical level of heterozygosity in F₈ progeny. Based on the trials conducted in 2007–2008 in the field and/or controlled greenhouse, no significant differences were found between the NIL pairs for response to any of the seven diseases. The result suggested that although DK888 harbors multiple disease resistance, the resistance conferred by qNLB8.06 _DK888 is NLB specific.

Table 2 Resistance spectrum of qNLB8.06 _DK888

qNLB8.06 _DK888 conditions race-specific resistance

Race-specific responses in IP, PrimDLA and lesion type were observed for the NILs (F₈ and F₉) carrying DK888 allele(s) at qNLB8.06. As shown in Fig. 2, races 0 and 1 were avirulent to qNLB8.06 _DK888 , while races 23 and 23N were highly virulent to it. Typical resistance symptoms caused by the incompatible interactions between qNLB8.06 _DK888 and race 0/race 1 were characterized by prolonged IP, decreased DLA and resistant-type lesions. The resistant-type lesions were slightly chlorotic and more restricted, in contrast to the susceptible-type lesions that extended greatly after the first appearance. The chlorosis, likely induced by the hypersensitive response surrounding the infection sites, was more distinct in early stages of lesion development. Once the pathogen grew out from the localized primary-infected region, the resistant or susceptible reactions were differentiable by size rather than the type of mature lesion.

Fig. 2

Race-specificity of qNLB8.06 _DK888 . The F₈ and F₉ NILs carrying DK888 or S11 alleles at bin 8.06 were evaluated for resistance to different races of S. turcica. Photographs were taken 20 days after inoculation. The error bars represent the 95% confidence interval of the least squares means; determined by Tukey–Kramer HSD (honestly significant difference) test at P < 0.05, significant differences for incubation period (IP, bars) and primary diseased leaf area (PrimDLA, dots; scored at 17 days after inoculation) were indicated as different letters below the graph. The results provided evidence that qNLB8.06 _DK888 conditions resistance to race 0 and race 1, but not race 23 and race 23N of S. turcica

The defense mechanism conferred by qNLB8.06 _DK888 was ineffective when inoculated with races 23 and 23N. The observed race-specificity suggested that the QTL could coincide with the major genes Ht2, Ht3, and/or Htn1. While Ht3 locus has not been mapped, Ht2 and Htn1 loci have been mapped to bin 8.05–8.06, which suggests that qNLB8.06 _DK888 may encompass Ht2 and/or Htn1, or some novel modulator(s) conditioning the expression of Ht2 and/or Htn1. Ht2 is a creditable candidate for qNLB8.06 _DK888 based on the compatibility of race 23. The relationship between qNLB8.06 _DK888 and Htn1 is ambiguous, as the compatibility of race 23N may have been caused by Ht2 and Htn1, or Ht2 alone. However, there is no naturally occurring race N isolate available for further resolving the question. It is also worth noting that the resistance reactions of qNLB8.06 _DK888 did not fully resemble those on the maize differential lines Pa91Ht2 or B68Htn1. As illustrated by the control trials (Supplementary Table 1), the lesions on Pa91Ht2 were more chlorotic associated with accumulated reddish pigmentation, and the lesions on B68Htn1 were of the susceptible type, consistent with previously reported lesion types of Ht2 and Htn1 (Welz and Geiger 2000). In contrast to qNLB8.06 _DK888 , Ht2 and Htn1 were effective in delaying lesion formation by 2–3 days and 2–9 days, respectively.

Allelism with known major genes at qNLB8.06

qNLB8.06 in relation to Ht2

To understand the allelism and interactions between qNLB8.06 _DK888 and Ht2, the F₁ and F₂ progenies of qNLB8.06 _DK888  × Ht2 _NN14B , qNLB8.06 _DK888  × Ht2 _Pa91 , qNLB8.06 _S11  × Ht2 _NN14B , and qNLB8.06 _S11  × Ht2 _Pa91 were evaluated in the greenhouse (Fig. 3). Ht2 _NN14B represented the resistance allele (from the donor line NN14B) at the Ht2 locus in the isoline Pa91Ht2, and Ht2 _Pa91 represented the susceptible allele in the recurrent line Pa91. As expected, all the F₁ and F₂ individuals of qNLB8.06 _S11 (S) × Ht2 _Pa91 (S) were susceptible. In contrast, no susceptible plants were found in either F₁ or F₂ individuals of qNLB8.06 _DK888 (R) × Ht2 _NN14B (R) (Fig. 3b). Distinct chlorotic–necrotic lesions were observed in almost all the plants derived from qNLB8.06 _DK888 (R) × Ht2 _NN14B (R). No susceptible individuals were observed, though 3 out of 35 F₁ individuals and 6 out of 72 F₂ individuals showed an intermediate phenotype on lower leaves, which is possibly caused by incomplete expression of resistance under low light intensity (Reuveni et al. 1993; Thakur et al. 1989a). Complementation of the DK888 and NN14B alleles in resistance phenotypes suggests that qNLB8.06 _DK888 is likely to be identical, allelic, or closely linked to the Ht2 locus. A larger F₂ population will be required for differentiating allelism from close linkage.

Fig. 3

Analysis of allelism between qNLB8.06 _DK888 and Ht2. a Crosses were made between the near-isogenic lines (NILs) contrasting for bin 8.06 (alleles designated qNLB8.06 _S11 and qNLB8.06 _DK888 ), Pa91 (allele designated Ht2 _Pa91 ), and Pa91Ht2 (allele designated Ht2 _NN14B ). Plants carrying homozygous qNLB8.06 _DK888 showed chlorotic–necrotic resistance lesions, and plants carrying homozygous Ht2 _NN14B showed chlorotic–necrotic resistance lesions with accumulated reddish pigmentation. The F₁ and F₂ progenies of qNLB8.06 _DK888  × Ht2 _NN14B (b), qNLB8.06 _DK888  × Ht2 _Pa91 (c), qNLB8.06 _S11  × Ht2 _NN14B (d), and qNLB8.06 _S11  × Ht2 _Pa91 (not shown) were evaluated for resistance to race 1 of S. turcica (EtNY001) in the greenhouse. b Complementation between the qNLB8.06 _DK888 and Ht2 _NN14B alleles in resistance phenotypes was observed. c, d Intermediate phenotype (less susceptible-type lesions) was observed in all the F₁ individuals and a considerable proportion of the F₂ individuals, suggesting that the resistance conditioned by either qNLB8.06 _DK888 or Ht2 _NN14B was incompletely dominant. e Significant differences in incubation period (IP, bars) and primary diseased leaf area (PrimDLA, dots; scored at 18 days after inoculation) were observed among the four F₁ progenies. The F₁ individuals were comparable, as they differed at bin 8.06 and Ht2 but isogenic for the rest of the genome. Trait values are least squares means calculated from the linear least squares model with “genotypes”, “replications” and “blocks within replications” as independent variables. Differences were determined by Tukey–Kramer HSD (honestly significant difference) test at P < 0.05, and indicated as different letters below the graph. The result confirmed the incomplete dominance of qNLB8.06 _DK888 and Ht2 _NN14B , and implicated the potential existence of different alleles at bin 8.06. Photographs were taken on the sixth leaves at 19 days after inoculation. Disease phenotypes are denoted as R resistant, M intermediate, and S susceptible

Significantly different levels of NLB resistance were observed in the four F₁ progenies with the same hybrid background: qNLB8.06 _DK888  × Ht2 _NN14B  > qNLB8.06 _DK888  × Ht2 _Pa91  > qNLB8.06 _S11  × Ht2 _NN14B  > qNLB8.06 _S11  × Ht2 _Pa91 (Fig. 3e). Although they showed some levels of resistance, the F₁ progenies of neither qNLB8.06 _DK888  × Ht2 _Pa91 (Fig. 3c) nor qNLB8.06 _S11  × Ht2 _NN14B (Fig. 3d) showed typical resistant chlorotic–necrotic lesions, indicating incomplete dominance of the qNLB8.06 _DK888 and Ht2 _NN14B alleles. The quantitative difference between the F₁ progenies of qNLB8.06 _DK888  × Ht2 _Pa91 and qNLB8.06 _S11  × Ht2 _NN14B also suggested differential allelic effects, though the effectiveness of individual alleles could not be determined.

As expected, marked segregation of resistant, intermediate and susceptible phenotypes was observed in the F₂ populations from the crosses of qNLB8.06 _DK888 (R) × Ht2 _Pa91 (S) and qNLB8.06 _S11 (S) × Ht2 _NN14B (R) (Fig. 3c, d). The intermediate phenotypes in heterozygotes complicated the classification of resistant and susceptible plants. We decided not to pursue Mendelian segregation ratio test because we did not manage to generate F₃ progenies for confirmation, and Mendelian analysis on F₂ individuals would provide meaningful results only if based on complete dominant or recessive genes with high penetrance. Nevertheless, careful observation and ratings were still conducted, from which the incomplete dominance of the qNLB8.06 _DK888 and Ht2 _NN14B alleles, and the likely differential allelic effects were confirmed.

Induced accumulation of reddish pigmentation surrounding chlorotic–necrotic lesions was associated with the Ht2 _NN14B allele and/or Pa91 genetic background. Extensive reddish pigmentation was consistently observed on diseased leaves of Pa91Ht2 (Fig. 3a). In contrast, the pigmentation was never seen on the NILs carrying qNLB8.06 _DK888 or qNLB8.06 _S11 , or their derived lines. All the F₁ and F₂ progenies used in this allelism test, however, showed different degrees of accumulated pigmentation. Relative to the F₂ progeny derived from qNLB8.06 _DK888  × Ht2 _Pa91 , more individuals with higher degrees of reddish pigmentation were seen in the F₂ populations of qNLB8.06 _DK888  × Ht2 _NN14B and qNLB8.06 _S11  × Ht2 _NN14B . Variation in the pigmentation was also seen in the F₂ population of qNLB8.06 _DK888  × Ht2 _NN14B . The variation implies the involvement of the gene(s) controlling the biosynthesis of anthocyanins. However, these results did not clearly differentiate between an influence of the qNLB8.06(Ht2) locus or the genetic background.

qNLB8.06 in relation to Htn1

To understand the allelism and interactions between qNLB8.06 _DK888 and Htn1, the F₁ and F₂ progenies of qNLB8.06 _DK888  × Htn1 _Peptilla and qNLB8.06 _S11  × Htn1 _Peptilla were evaluated in the field (Fig. 4). qNLB8.06 _DK888  × Htn1 _B68 and qNLB8.06 _S11  × Htn1 _B68 were not included due to unavailability of seed. Htn1 _Peptilla represented the resistance allele (from the donor line Peptilla) at the Htn1 locus in the isoline B68Htn1, and Htn1 _B68 represented the susceptible allele in the recurrent line B68. Similar to qNLB8.06 _DK888 and Ht2 _NN14B , Htn1 _Peptilla was much less effective in the heterozygous than homozygous state. Homozygous Htn1 _Peptilla in B68Htn1 (average IP = 23.8 dpi) increased IP by 10.5 days relative to B68 (average IP = 13.3 dpi). Heterozygous Htn1 _Peptilla in the F₁ progeny of qNLB8.06 _S11  × Htn1 _Peptilla (average IP = 15.9 dpi, Fig. 4c), however, only increased IP by 2.6 (P = 0.044) and 2.5 days (P = 0.033) relative to B68 and the NIL carrying qNLB8.06 _S11 , respectively. The incomplete dominance of Htn1 has been described (Raymundo et al. 1981). It was also observed that when Htn1 _Peptilla and qNLB8.06 _DK888 were both heterozygous, the plants displayed an intermediate resistant phenotype that was characterized by slightly chlorotic–necrotic lesions (Fig. 4b) and moderately increased IP [average IP = 18.8 dpi, significantly different from and in between of homozygous Htn1 _Peptilla (average IP = 24.3 dpi) and homozygous qNLB8.06 _DK888 (average IP = 16.4 dpi)]. The intermediate phenotype conforms to previously reported phenotype resulting when heterozygous Htn1 interacts with heterozygous Ht2 (Simcox and Bennetzen 1993). This implies some functional similarity of qNLB8.06 _DK888 and Ht2.

Fig. 4

Analysis of allelism between qNLB8.06 _DK888 and Htn1. a Crosses were made between the near-isogenic lines (NILs) contrasting for bin 8.06 (alleles designated qNLB8.06 _S11 and qNLB8.06 _DK888 ), and B68Htn1 (allele designated Htn1 _Peptilla ). The line B68 (allele designated Htn1 _B68 ) was used as control. Plants carrying homozygous qNLB8.06 _DK888 showed chlorotic–necrotic resistance lesions, and plants carrying homozygous Htn1 _Peptilla showed extraordinarily delayed formation of lesions (until 25 days after inoculation, only a few lesions were observed on B68Htn1). The F₁ and F₂ progenies of qNLB8.06 _DK888  × Htn1 _Peptilla (b) and qNLB8.06 _S11  × Htn1 _Peptilla (c) were evaluated for resistance to race 1 of S. turcica (EtNY001) in the field. b qNLB8.06 _DK888 and Htn1 _Peptilla appeared to be non-allelic, based on the segregation of plants exhibiting chlorotic–necrotic lesions, delayed lesion formation, intermediate phenotypes, and susceptible lesions in their F₂ progeny. c The resistance conditioned by Htn1 _Peptilla was incompletely dominant. Photographs were taken on the seventh leaves at 25 days after inoculation. Disease phenotypes are denoted as R resistant, M intermediate, and S susceptible

Unlike the complementation of qNLB8.06 _DK888 and Ht2 _NN14B , resistance phenotypes of homozygous qNLB8.06 _DK888 (chlorotic–necrotic), homozygous Htn1 _Peptilla (extremely prolonged IP), and heterozygous qNLB8.06 _DK888 in combination with heterozygous Htn1 _Peptilla (intermediate) segregated in the F₂ progeny of qNLB8.06 _DK888  × Htn1 _Peptilla (Fig. 4b). The Mendelian segregation ratio test was not employed because of the ambiguity in phenotypic classification. Nonetheless, it was clearly observed that 4 out of 82 F₂ individuals showed a susceptible phenotype (shorter IP with extended long lesions), which is presumably associated with recombination events between qNLB8.06 and Htn1. The result indicates that qNLB8.06 and Htn1 are non-allelic. Considering the results of the allelism analysis, the locus designation was modified to qNLB8.06(Ht2).

Fine-mapping of qNLB8.06(Ht2)

Breakpoint analysis was conducted to refine qNLB8.06(Ht2) _DK888 . Around 2,800 individuals (from 26 F₉ families and 13 F₁₀ families) segregating for bin 8.06 were used for QTL analysis. Disease evaluations were carried out in three environmental conditions: Oct–Dec in the greenhouse, Apr–Jun in the greenhouse, and May–Aug in the field (Table 1). In the space-limited greenhouse, plants were initially all genotyped for flanking markers of the target QTL interval. Subsequently, only the identified recombinant individuals were kept for disease evaluations. The mapping results from single marker analysis and interval mapping are summarized in Table 1, and displayed in the QTL likelihood map in Fig. 5.

Fig. 5

Likelihood map of qNLB8.06(Ht2) _DK888 . a The likelihood that the loci in bin 8.06 are associated with NLB resistance was analyzed by single marker analysis (circle, triangle, and square dots) and interval mapping (solid and dashed lines). The likelihood of odds ratio (LOD) lines and dots for incubation period (IP) are shown in black, and the LOD lines and dots for primary diseased leaf area (PrimDLA) and DLA are shown in gray. The average LOD threshold for all traits, based on 1,000 permutations at P = 0.01, is 3.3 (not shown in the figure). The resistance of qNLB8.06(Ht2) _DK888 was not as effective in the greenhouse trial conducted in Oct–Dec 2007. The most likely QTL position, based on the data obtained from the evaluations in the greenhouse in Apr–Jun 2008 and in the field in May–Aug 2008, was located between ctg358-18 to ctg358-44. This interval corresponds to 9.86–11.20 cM on the S11 × DK888 genetic map, and 143.88–144.38 Mb on B73 physical map. b The genetic and physical positions of the markers are shown

In a population consisting of 571 F₉ individuals, qNLB8.06(Ht2) _DK888 was found to be likely located between umc2199–umc1287. The LOD scores at this interval were >10 for IP and >5 for PrimDLA, whereas the LOD values dropped to <3 from around 27–58 cM (between umc1287 and umc1828). A high-resolution map was constructed with 19 newly developed SNP and CAPS markers around umc2199–umc1287 (Fig. 5). Initially, two and five SNP/CAPS markers between umc2199–umc2210 and umc2210–umc1287, respectively, were designed to cover the QTL region at low density. By testing the co-segregation of markers and traits, the interval of ctg358-07–ctg358-01 was found to be the most significantly associated with resistance. An additional 12 SNP/CAPS markers were then developed to saturate this region. Marker segregation data showed that the order of the SSR, SNP and CAPS markers used in the study agree with their physical positions in the genome sequence of B73.

To increase efficiency, individuals in mapping populations were selectively genotyped for SNP/CAPS markers, based on their genotypes at the flanking markers of the target interval. Considering the incompletely dominant gene action of qNLB8.06(Ht2) _DK888 , the genotyping strategy aimed to capture the homozygous DK888 segment(s), which provided informative resistance phenotype for QTL analysis. Therefore, all the recombinant individuals that were homozygous for the DK888 allele at either one of the flanking markers were genotyped for intermediate SNP/CAPS markers. Individuals that were homozygous for identical alleles at the two flanking markers were assumed homozygous for the entire interval.

In a population consisting of 1,191 F₁₀ individuals and a population consisting of 1,056 F₉ individuals, qNLB8.06(Ht2) _DK888 was delimited to a region of ~1.34 cM (~0.5 Mb) between ctg358-18–ctg358-44 (Table 1; Fig. 5). In the two experimental environments, the resistance of qNLB8.06(Ht2) _DK888 was well expressed, allowing accurate linkage analysis on the basis of distinct phenotypes. Averaged from the effects estimated from single marker analysis and interval mapping, the DK888 allele increased IP by ~5 days and decreased DLA by ~17%. About 35–47% and 60% of the variance in IP and DLA, respectively, were explained by qNLB8.06(Ht2) _DK888 . Significant evidence of QTL (LOD > 3.3, the average threshold for all traits) was consistently found between umc2199–umc1287 (0–22.4 cM). In this interval, QTL peaks were detected at approximately the same map position (~10.2 cM) for IP and DLA (highest LOD scores: ~97 for IP, and ~210 for DLA). If adopting the 1-LOD drop method, the most likely QTL position can be predicted to a tight region between ctg358-18–ctg358-44 (Fig. 5; 9.86–11.20 cM on the S11 × DK888 genetic map, and 143.88–144.38 Mb on B73 physical map).

To further confirm the location of qNLB8.06(Ht2) _DK888 , a total of 13 F₁₀ and 85 F₁₁ NILs were evaluated for IP, DLA (only in the 2008 trial) and lesion types at Aurora NY in 2008 and 2009, respectively. The NILs were derived from selected lines covering different breakpoints around umc2199–umc1287. The result of single marker–trait analysis and the genotypic compositions of nine representative fixed NILs are shown in Fig. 6. Evaluations conducted in 2008 and 2009 led to the same results. Because F₁₁ NILs captured more recombination events in more homogeneous backgrounds, the data from the 2009 trial was shown to represent the overall result. Markers ctg358-20, ctg358-05, and ctg358-37 were found to be the most significantly associated with disease traits (−log of P > 200, Fig. 6). Among the three markers, ctg358-20 is likely to reside outside of the QTL region, based on the “resistant” phenotype of NIL7 (S11/S11 at ctg358-20, DK888/DK888 at ctg358-05 and ctg358-37). Evidence of the QTL tightly linked to ctg358-05 and ctg358-37 was also found in the rows of NIL9 (Fig. 6; heterozygous at ctg358-05 and ctg358-37), where individual plants segregated for resistance. The number of resistant: intermediate/susceptible plants were 4:21, which does not deviate from the expected 1:3 segregation ratio (χ² = 1.2, P = 0.3) of a single incompletely dominant gene. qNLB8.06(Ht2) _DK888 was thus validated to locate between (but not overlapping with) ctg358-20 and ctg358-44 in bin 8.06 (10.20–11.20 cM on the S11 × DK888 genetic map, and 143.92–144.38 Mb on B73 physical map). There is some ambiguity regarding the precise boundary between bins 8.05 and 8.06. The region 143.92–144.38 Mb was located to bin 8.06 in MaizeGDB, while it was located to a gap between bins 8.05–8.06 in the Maize Sequence Database.

Fig. 6

Validation of qNLB8.06(Ht2) _DK888 position. F₁₀ and F₁₁ near-isogenic lines (NILs) capturing various recombination events at bin 8.06 were evaluated for resistance to northern leaf blight (NLB). The likelihood of each locus being associated with incubation period is represented by negative logarithm P values (−log P) derived from a mixed model analysis. Genotypic compositions and disease phenotypes (R resistant, S susceptible, segregating R and S plants segregating in a row) of nine representative NILs are shown. The solid bars and open bars represent the loci homozygous for DK888 alleles and S11 alleles, respectively. The gray bar represents heterozygous loci. qNLB8.06(Ht2) _DK888 was delimited to a map interval between ctg358-20 and ctg358-44 (10.20–11.20 cM on the S11 × DK888 genetic map, and 143.92–144.38 Mb on B73 physical map)

Candidate genes underlying qNLB8.06 _DK888

On the basis of the annotation of the Maize Genome Sequencing Project (as of August 2009, available at http://archive.maizesequence.org/), the genomic region between ctg358-20 and ctg358-44, spanning 0.46 Mb, harbors a large number of transposable elements (TEs) and 12 putative genes (GRMZM2G135202, GRMZM2G164612, GRMZM2G164640, GRMZM2G091973, GRMZM2G092018, GRMZM2G119720, GRMZM2G018260, GRMZM2G122912, GRMZM2G006188, GRMZM2G042017, GRMZM2G077187 and GRMZM2G065538). The abundance of TEs has been generally observed in the entire maize genome (Wei et al. 2007). Of the 12 non-TE genes, eight genes encode putative proteins with similarities to known protein domains or motifs in the InterPro databases. Putative genes that can be associated with previously reported R-genes or defense-related genes include two protein kinase-like genes (GRMZM2G135202 and GRMZM2G164612 with conserved domains IPR017441, IPR002290, IPR001245, IPR017442, IPR011009, IPR008271) and one serine–threonine-specific protein phosphatase-like gene (GRMZM2G119720 with conserved domain IPR006186). The two protein kinase-like genes are closely linked (2,632 bp apart) and highly homologous to each other (78% genomic sequence identity; 97% putative transcript identity; putative proteins different for 1 out of 290 amino acid residues).

Discussion

Production of NILs for a complex resistance locus using HIFs

Near-isogenic lines carrying contrasting alleles at maize bin 8.06 were successfully generated, and the region was characterized and dissected using HIF analysis. The HIF-based QTL approach was conducted as part of a larger effort to capture diverse alleles at the loci associated with complex types of disease resistance. To increase the probability of finding alleles conditioning broad-spectrum resistance, maize lines possessing multiple disease resistance were used as donors. In the present study, the broadly resistant maize hybrid DK888 was used as a source of alleles. Considering the importance of bins 8.05–8.06 in NLB resistance (2 major genes and many co-localized QTL have been mapped to the region), the effect of DK888 allele(s) at bin 8.06 was first tested for response to NLB.

We detected, validated and localized an NLB QTL at bin 8.06 (designated qNLB8.06) using initially 1 single SSR marker and subsequently 15 additional markers. The F₇ and F₈ families in which qNLB8.06 _DK888 was identified were expected to segregate for <1.6% of the genome. In these HIFs, qNLB8.06 _DK888 appears to be a major QTL explaining a large proportion (14–62%) of phenotypic variations in NLB resistance. qNLB8.06 _DK888 consistently conferred resistance in juvenile and adult plants across greenhouse and field environments. Relative to S11 allele(s), DK888 allele(s) at bin 8.06 was effective for delaying lesion formation by about 2.6–6.8 days, and reducing diseased leaf area by about 12–22% of the primarily inoculated leaves and about 15% of the entire plant. Overall, HIF analysis proved to be an efficient way to extract targeted QTL from the nearly fixed recombinant inbred lines (Tuinstra et al. 1997). Genetic stocks derived during the procedure were readily applicable for subsequent work of characterizing and fine-mapping QTL. Clear expression of the disease phenotypes in the NILs indicated that the QTL was transferred to an appropriate genetic background for QTL examination.

qNLB8.06 conditions race-specific resistance to NLB

The hypothesis that DK888 allele(s) at bin 8.06 conditions disease- and race-nonspecific resistance was tested. The DK888 allele(s) at bin 8.06 conferred resistance only to NLB among the several diseases tested. The resistance was also characterized by its specificity to race 0 and race 1, but not to race 23 and race 23N of S. turcica. The compatibility with race 23 and race 23N led to the question of whether qNLB8.06 _DK888 is the same or different from the known major genes Ht2 and Htn1. We found that qNLB8.06 _DK888 is likely to be identical, allelic, or very closely linked (and functionally related) to Ht2 _NN14B , on the basis of their overlapping map locations, their similarities in race-specificity and resistance phenotypes, and their complementation for resistance in the F₁ and F₂ test progenies. qNLB8.06 _DK888 and Htn1 appear to be linked and functionally dissimilar genes, according to the intermediate resistant phenotype in their F₁ progeny, and the segregation of F₂ individuals showing chlorotic–necrotic lesion type (typical Ht2 phenotype), intermediate lesion type, or delayed formation of lesions (typical Htn1 phenotype). These observations conformed to previously reported non-allelism of Ht2 and Htn1 (Simcox and Bennetzen 1993). Htn1 was mapped to ~10 cM distal to Ht2 in the F₂ progeny of W22Htn1 × A619Ht2. In our group, concurrent work of fine-mapping Htn1 using a population consisting of ~2,600 F₂ individuals derived from B68 × B68Htn1 is underway (J. Kolkman, pers. comm.). The map distance between qNLB8.06(Ht2) and Htn1 will be further clarified.

qNLB8.06(Ht2) shows incomplete dominance

Available evidence on gene action at Ht2 and Htn1 from previous studies is ambiguous. For Ht2, both complete dominance (Yin et al. 2003; Zaitlin et al. 1992) and incomplete dominance (Ceballos and Gracen 1989; Hooker 1977) have been observed in different genetic materials. Reduced resistance in the heterozygotes (incomplete dominance) and the variable expression of resistance in different genetic backgrounds have also been reported for Htn1 (Raymundo et al. 1981). The effects of Ht2 and Htn1 have been found to be highly sensitive to environmental conditions in others’ experiments (Reuveni et al. 1993; Thakur et al. 1989a) and our repeated greenhouse and field trials (data not shown). In the present study, both DK888 and NN14B alleles at qNLB8.06(Ht2) conditioned incomplete dominance and race-specific resistance to S. turcica (NN14B is the resistance donor line used to derive Pa91Ht2 isoline). High levels of resistance and the distinct chlorotic–necrotic lesions were only seen on the plants containing two copies of resistance alleles (DK888/DK888, NN14B/DK888 or DK888/NN14B) at the locus. One copy of the resistance allele along with one copy of a susceptible allele resulted in differential intermediate degrees of disease and susceptible-type lesions.

Incomplete dominance has been widely observed for diverse resistance genes. Examples include the R-genes Cf genes in tomato lines (Hammond-Kosack and Jones 1994), the susceptibility-conferring R-gene LOV1 in Arabidopsis (Lorang et al. 2007), and the detoxification gene Hm2 in maize (Chintamanani et al. 2008). Incomplete dominance is generally associated with a gene dosage effect. Higher expression of resistance gene product in homozygous individuals may lead to more effective perception of pathogen invasion, activation of defensive responses, or elimination of cell damage. The dosage-dependent hypothesis has been tested, to a limited extent, for a few resistance genes. Tomato Cf genes (encoding proteins with extracellular leucine rich repeats and transmembrane domain) against leaf mold caused by Cladosporium fulvum displayed weakened resistance in heterozygous states (Vidhyasekaran 2007). Homozygous Cf lines were capable of responding to a twofold lower concentration of race-specific elicitors than heterozygous lines (Hammond-Kosack and Jones 1994). In the case of the maize Hm2 gene (encoding HC-toxin reductase) against the leaf spot and ear mold caused by Cochliobolus carbonum race 1, intermediate resistance in heterozygotes has been associated with lower abundance of Hm2 transcripts (Chintamanani et al. 2008). Although the underlying genes are currently unknown, the resistance phenotypes conferred by qNLB8.06(Ht2) _DK888 as well as other Ht major genes are expressed in a similar dosage-dependent manner. This is consistent with the observations that triploid (Ht1 Ht1 Ht1) and tetraploid (Ht1 Ht1 Ht1 Ht1) maize seedlings displayed a higher level of resistance to NLB than monoploid (Ht1) and diploid (Ht1 Ht1) seedlings (Dunn and Namm 1970). The dosage-dependent hypothesis and resistance response kinetics can be further characterized by manipulating the isolated resistance gene(s) and its corresponding S. turcica effector(s) in follow-up studies.

Allele- and genetic background-dependent expression of qNLB8.06(Ht2)

The resistance conditioned by qNLB8.06(Ht2) varied depending on allele variants and/or genetic backgrounds. The genetic background effect has been previously reported: Ceballos and Gracen (1989) showed that the expression of Ht2 can be inhibited by a dominant suppressor gene Sht1 in B14-related inbred lines. In this study, the differential performance of DK888/Pa91 and S11/NN14B at qNLB8.06(Ht2) in the same genetic background (F₁ hybrid of the Pa91 and the DK888 × S11 NIL) suggested functional allelic diversity. The existence of allelic series for resistance gene(s) at qNLB8.06(Ht2) can also be inferred from other studies, in which the NN14B allele was more resistant than Oh43 allele, and the Oh43 allele was more resistant than B73 allele at qNLB8.06(Ht2) (Ceballos and Gracen 1989; Dong et al. 2008; Moghaddam and Pataky 1994; Poland et al. 2009b; Zaitlin et al. 1992; Zhang et al. 2007).

While different alleles at bin 8.06 appeared to contribute varying degrees of resistance to NLB, it remained unclear whether the differential expression was conditioned by a single gene or multiple linked genes. Our observation implied the involvement of at least one linked gene in modulating anthocyanin biosynthesis induced in the incompatible reaction of qNLB8.06(Ht2). Anthocyanins are antioxidants that can protect plant cells against the high levels of oxidative stresses in defense reactions (Hammerschmidt 2005). In the maize–Cochliobolus heterostrophus pathosystem, accumulation of anthocyanin has been reported to occur in the uninfected epidermal cells surrounding the lesions (Hipskind et al. 1996). In our allelism analysis, the accumulation of anthocyanins on diseased tissues was associated with the Ht2 _NN14B allele and/or the Pa91 genetic background. Genes controlling anthocyanin biosynthesis in maize have been isolated and mapped to several loci on different chromosomes (Bernhardt et al. 1998), including an a4 locus (dihydroflavonol 4-reductase) residing between umc2210 and umc1287 at bin 8.06 (the map location indicated on MaizeGDB). Since the reddish pigmentation was never observed on the resistant plants carrying qNLB8.06(Ht2) _DK888 in the DK888 × S11 background, the key resistant gene(s) are apparently not anthocyanin-related. Nevertheless, with certain alleles, the anthocyanin-related gene(s) at bin 8.06 and/or other unlinked loci may contribute additive effects to the resistance of qNLB8.06(Ht2).

Map location of qNLB8.06(Ht2)

Several major genes and QTL have been isolated by map-based positional cloning [eg. Pi5-1 and Pi5-2 against rice blast (Lee et al. 2009), Rcg1 against ASR of maize (Broglie et al. 2006), and Yr36 against wheat stripe rust (Fu et al. 2009)]. In the present study, we used ~2,800 individuals in 39 F₉ or F₁₀ heterogeneous inbred families and 98 F₁₀/F₁₁ fixed lines to localize qNLB8.06(Ht2) _DK888 from a region of ~19.8 to 0.46 Mb (1 cM). Within the 1 cM interval delimited by ctg358-20 and ctg358-44, ctg358-20, ctg358-5 and ctg358-37 are closely linked to each other in a 0.08 cM region, whereas ctg358-44 was located at a distance of 0.92 cM. Although there were 34 out of 98 fixed lines capturing recombination events between ctg358-37 and ctg358-44, we have not succeeded in developing polymorphic markers for this region. qNLB8.06(Ht2) can be further delimited by genotyping the 34 lines with more newly developed markers.

The 0.46 Mb region resides within the intervals of Ht2 previously estimated from the F₂ populations of A619Ht2 × W64A (Zaitlin et al. 1992) and W22Htn1 × A619Ht2 (Simcox and Bennetzen 1993). It also resides within the map intervals of the NLB QTL identified in the F_2:3 lines derived from Lo951 × CML202 (Schechert et al. 1999; Welz et al. 1999a), and the NLB QTL identified across the NAM population consisting of RILs derived from 25 diverse maize lines crossed with B73 (Poland et al. 2009b) (J. Poland, pers. comm.). However, some discrepancies were found in previous fine-mapping study using 890 F₂ individuals from the cross of 77Ht2 and Huobai (Yin et al. 2003). The inconsistent Ht2 positions as well as the converse order of linked markers observed in the 77Ht2 × Huobai population suggest that the qNLB8.06(Ht2) locus may be divergent among some maize lines.

It has been recognized that the recombination rate for a given resistance locus can vary depending on the similarity of the haplotypes that are paired [e.g. the maize Rp1 locus (Ramakrishna et al. 2002)]. Lower recombination rate flanking the rice Pi5-1 and Pi5-2 genes was observed in a population derived from the RIL260 and Nipponbare cultivars, for which the resistant and susceptible alleles from the two cultivars are significantly divergent (Lee et al. 2009). Conversely, R-gene clusters have been widely associated with high recombination frequencies (Bakker et al. 2006; Meyers et al. 2005). In the S11 × DK888 mapping population, the ratio of physical to genetic distance in the ~7.4 Mb region between umc2199 and umc1287 at bin 8.06 was ~330 kb/cM. A higher physical to genetic ratio (460 kb/cM) was observed for the 0.46 Mb region of qNLB8.06(Ht2), indicating a lower recombination frequency flanking the resistance gene(s). This implies the possibility of low similarity between the DK888 and S11 alleles at qNLB8.06(Ht2), and the absence of clustering of homologous resistance genes (which facilitates crossovers) in both alleles. More insights on the evolution of qNLB8.06(Ht2) will be gained by detailed investigation of the natural allelic diversity in maize germplasm.

Candidate genes underlying qNLB8.06(Ht2)

Three compelling candidate genes, including two tandem protein kinase (PK)-like genes and one protein phosphatase (PP)-like gene, were identified within the delimited 0.46 Mb interval of qNLB8.06 _DK888 . The two tandem PK-like genes contain the conserved kinase catalytic domain of serine/threonine-specific and tyrosine-specific protein kinases. The PK domain is one of a few conserved domains or motifs shared among R-genes (Xiao et al. 2007). The PP-like gene, on the other hand, has the conserved domain of serine/threonine-specific protein phosphatases, which have been associated with negative regulation of R-gene and non-R-gene-mediated defense signaling in rice, Arabidopsis, and tobacco (He et al. 2004; Park et al. 2008; Schweighofer et al. 2007). Overall, our preliminary analysis suggested that an R-gene(s) equipped with PK domain and/or a serine/threonine-specific PP gene may underlie qNLB8.06(Ht2) _DK888 . Given the high degree of gene non-colinearity among maize lines (Buckler et al. 2006; Fengler et al. 2007; Fu and Dooner 2002), it is possible that the resistance gene(s) or regulatory sequence(s) does not exist in B73 genotype.

Conclusion

Using a HIF-based QTL approach to target a complex genetic region, we identified, characterized and fine-mapped an NLB QTL likely to be identical, allelic, or closely linked to the known major gene Ht2. We provided potentially useful information regarding the resistance spectrum and closely linked markers of the locus. The knowledge will benefit its appropriate deployment in resistance breeding programs. To further delimit qNLB8.06(Ht2) _DK888 and finally isolate the underlying genetic determinant(s), more lines capturing recombination events between flanking markers ctg358-20 and ctg358-44 will be screened, and more polymorphic markers will be developed to saturate the interval. Association analysis based on the three identified candidate genes will be tested in a set of ~300 diverse maize lines, which has been evaluated in our group over three years for resistance to NLB (J. Kolkman, pers. comm.). In light of the potential non-homologies between DK888 and B73 alleles at qNLB8.06, alternatives to candidate gene analysis, such as chromosome walking or construction of a BAC library of qNLB8.06 _DK888 , may be required. Once the genetic determinant(s) underlying qNLB8.06 is elucidated, more intriguing hypotheses about the complex genetic architecture, the evolution of resistance gene(s), gene functions and regulations in response to pathogen attack under different environmental conditions can then be addressed.