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Quantitative Trait Loci Contributing Resistance to Aflatoxin Accumulation in the Maize Inbred Mp313E Thomas D. Brooks,* W. Paul Williams, Gary L. Windham, Martha C. Willcox, and Hamed K. Abbas 2002). Genotype ⫻ environment interactions tend to be significant, and artificial inoculation has proven necessary to ensure uniform selection pressure (Darrah et al., 1987; Zummo and Scott, 1989; Payne, 1992; Windham et al., 2003). The side-needle inoculation technique developed by Zummo and Scott (1989) has proven to be the most reliable method in Mississippi field trials. Sources of resistance have been identified that exhibit significantly reduced aflatoxin accumulation (Campbell and White, 1995; Scott and Zummo, 1988, 1990, 1992; Williams and Windham, 2001). In a QTL study involving the resistant inbred Tex6 and susceptible B73, Paul et al. (2003) identified loci from both parents that contributed to resistance. The QTL were located on chromosomes 3, 4, 5, and 10, with large environmental effects resulting in most QTL being significant in only one year. Mp313E, an inbred line derived from Tuxpan, is highly resistant to aflatoxin accumulation (Scott and Zummo, 1990). This inbred contributes relatively stable resistance in testcrosses but exhibits undesirable characteristics, such as late maturity and poor combining ability for yield. A previous study involving an F2–derived mapping population from Mp313E ⫻ Va35 was performed to identify QTL affecting aflatoxin accumulation (Davis et al., 2000; M.C. Willcox, 2000, unpublished data). One region on chromosome 4L was associated with aflatoxin resistance in three different environments. To determine if this region was important and stable in other genetic backgrounds and to identify additional QTL, a new study was initiated using a F2:3 mapping population from inbreds Mp313E and B73.

Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

ABSTRACT Aflatoxin is a carcinogenic and toxic compound produced by the fungus Aspergillus flavus (Link:fr) that can be found at detrimentally high concentrations in maize (Zea mays L.) grain. Screening has led to the discovery of sources of resistance to aflatoxin accumulation in maize, but associated poor agronomic characteristics and complex inheritance have limited transfer of resistance to elite inbreds. A set of 210 F2:3 families derived from a cross between inbred lines Mp313E (resistant) and B73 (susceptible) was evaluated in replicated trials in four environments for resistance to aflatoxin accumulation. Families were also genotyped using simple sequence repeat (SSR) markers to develop a genetic map for quantitative trait loci (QTL) analysis. Composite interval mapping (CIM) was used to identify 2, 3, 5, and 3 QTL within the tests at Stoneville (2000) and Mississippi State (2000, 2001, 2002), respectively. The QTL were primarily additive in nature, with Mp313E contributing to reduced aflatoxin concentration in all but one case. Two QTL regions were significant in at least three environments. The afl3 locus, represented by marker bnlg371, was located on chromosome two and accounted for 7 to 18% of variation in aflatoxin levels depending on environment. The afl5 locus, represented by marker bnlg2291, was located on chromosome four, with explained variance ranging from 8 to 18%. This QTL has been noted in earlier studies whereas afl3 is new. Identified QTL confirm important regions influencing aflatoxin accumulation previously identified and present new ones of equal effect.

A

flatoxins are toxic secondary metabolites produced by the fungal pathogen Aspergillus flavus. These toxins have carcinogenic, immunosuppressive, and hepatotoxic properties in animals (Castegnaro and McGregor, 1998). Aflatoxin B1 is the most potent class of aflatoxin and has been reported to cause liver cancer in humans. Aspergillus flavus is endemic to maize-growing regions, causing ear rot and significant aflatoxin accumulation particularly in southern growing regions (Payne, 1992; Windham and Williams, 1998). As a result, the U.S. Food and Drug Administration prohibits interstate commerce of feed grain containing more than a maximum limit of 20 ng g⫺1 aflatoxin (Park and Liang, 1993). Efforts have been underway to identify factors contributing to reduced aflatoxin accumulation in maize. To date, commercially available hybrids continue to lack appreciable levels of resistance to aflatoxin accumulation (Windham and Williams, 1999; Abbas et al.,

MATERIALS AND METHODS Population Construction and Evaluation An F2 population was derived from a cross between maize inbred lines Mp313E (resistant) and B73 (susceptible). B73 represents the Iowa Stiff Stalk Synthetic heterotic group, has a different genetic background than Va35, and is an ancestor to more current hybrid parents than Va35. F2 plants from a single, selfed F1 ear were also selfed to create 210 ear-to-row F2:3 families. Each F2:3 family was sib-mated to generate sufficient seed for replicated trials while maintaining variation within the family. The parents, F1, and 210 F2:3 families were grown in a randomized complete block design with four replications. Planting sites included the Delta Research and Extension Center, Stoneville, MS, in 2000 (Stone2000), and the R.R. Foil Plant Science Research Center, Mississippi State University, in 2000, 2001, and 2002 (MSU2000, MSU2001, MSU2002, respectively). Plots were 5.1 m in length and thinned to 20 plants per plot at approximately the V5 stage (Ritchie et al., 1986). Standard

T.D. Brooks, W.P. Williams, G.L. Windham, and M.C. Willcox, USDAARS Corn Host Plant Resistance Research Unit, Mississippi State, MS 39762; and H.K. Abbas, USDA-ARS Crop Genetics and Production Research Unit, Stoneville, MS 38776. This paper is a joint contribution of USDA-ARS and the Mississippi Agricultural and Forestry Experiment Station and is published as journal no. J10454 of the Miss. Agric. and Forestry Exp. Stn. Received 21 May 2004. *Corresponding author ([email protected]). Published in Crop Sci. 45:171–174 (2005). © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA

Abbreviations: CIM, composite interval mapping; LOD, log10-likelihood ratio; QTL, quantitative trait locus or loci; SSR, simple sequence repeat.

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cultural practices were followed at each location to maximize yield. Midsilk (50% plants in a plot had emerged silks) dates were recorded. Developing ears were inoculated with A. flavus using the side-needle technique (Zummo and Scott, 1989). This inoculation technique utilizes an Idico tree-marking gun (Idico Products Co., New York) fitted with a 14-guage hypodermic needle. Aspergillus flavus isolate NRRL 3357, known to produce high levels of aflatoxin in maize grain (Scott and Zummo, 1988), was increased on sterile maize cob grits, and conidia were collected as described by Windham and Williams (1999). The top ear of each plant was inoculated 14 d after midsilk with a 3.4-mL suspension containing 3 ⫻ 108 A. flavus conidia. All inoculated ears in each plot were harvested by hand 65 to 75 d after midsilk and dried at 38⬚C for 7 d. Ears were then machine shelled, and grain samples from each plot were poured into a sample splitter twice to mix the grain. Samples were ground using a Romer mill (Romer Industries, Inc., Union, MO). Aflatoxin concentration in a 50-g subsample from each plot was determined using the VICAM AflaTest (VICAM, Watertown, MA). This procedure can detect aflatoxins (B1, B2, G1, G2) at concentrations as low as 1 ng g⫺1. Aflatoxin concentration data were transformed using ln (y ⫹ 1) before analysis to normalize distributions. Analyses of variance were performed across environments using the general linear model procedure in SAS (SAS Institute, Cary, NC) to accommodate missing data, with environments and genotypes considered random. Broad-sense heritability estimates within each environment were determined using variance components derived from expected mean squares in the ANOVA as described by Hallauer and Miranda (1981).

Genotyping and Linkage Analysis Leaf tissue from 20 to 25 plants of each F2:3 family was bulked, treated in liquid nitrogen, and freeze dried. Lypholized samples were ground and DNA was extracted by the CTAB method (Saghai Maroof et al., 1984). Approximately 225 oligonucleotide primer pairs amplifying genomic regions containing SSRs or microsatellites that span the maize genome were obtained from the Maize Genetic and Genomics Database (http://www.maizegdb.org/). Primer oligonucleotides were synthesized by Research Genetics, part of Invitrogen (Carlsbad, CA), and Integrated DNA Technologies (Coralville, IA). Polymerase chain reaction was performed using JumpStart REDAccuTaq and its recommended protocol (Sigma, St. Louis, MO). Thermocycling was performed in a 96-well, thin-walled plate using a count-down profile with the following steps: 95⬚C for 1 min, 65⬚C for 1 min, 72⬚C for 1.5 min; repeat steps, decreasing second step 1⬚C each cycle until 55⬚C is reached; repeat final cycle 30 times. Amplified products were visualized on 4% gels of Amresco’s SFR agarose (Solon, OH). Where resolution was not sufficient to differentiate between the inbred parents, a high resolution, nondenaturing acrylamide gel system from C.B.S. Scientific (Del Mar, CA) was used to obtain resolution of polymorphisms as small as 2 bp (Wang et al., 2003). Of these, 85 SSRs that proved to be polymorphic between the

parents were genotyped on the F2:3 families and included in linkage analysis. Marker groups and order were determined using Carthagene mapping software with a minimum LOD score of 3.0 and a maximum recombination fraction of 0.4 (Schiex and Gaspin, 1997). Carthagene produces results similar to MAPMAKER (Lincoln et al., 1992) while also handling larger data sets more easily and allowing consensus maps to be later created from multiple mapping studies (Schiex and Gaspin, 1997). Mapping results followed closely with marker orders listed in the MaizeGDB IBM consensus map (Polacco et al., 2002). Additional markers were added to linkage groups when recombination frequency was ⬎0.4 between two markers identified as being linked on the consensus map. Eighty-five markers fell into groups corresponding to all 10 chromosomes. The map spanned 1553 cM with a mean interval of 18.1 cM.

Quantitative Trait Analysis Composite interval mapping was performed by QTLCartographer version 2.0 (Zeng, 1993, 1994; Basten et al., 1999). To estimate the 0.05 significance threshold for QTL, 1000 permutations were performed with each data set and across all data (Doerge and Churchill, 1996; Doerge and Rebai, 1996). The standard control model was selected using five markers and the forward regression method was selected to perform CIM. The QTL analysis was performed within each environment as well as on family means across all environments.

RESULTS Phenotypic Description Mean levels of aflatoxin accumulation across F2:3 families ranged from 212 ng g⫺1 at Stone2000 to 1639 ng g⫺1 at MSU2002 (Table 1). The Stone2000 test mean was 75% lower than the next lowest test mean and it was the only test where mean aflatoxin levels did not differ significantly between B73 and Mp313E (P ⫽ 0.05). The susceptible inbred B73 accumulated extremely high levels of aflatoxin in the other environments differing from Mp313E by a factor of 10 overall. In addition, B73 had a much higher standard error than Mp313E (338.3 vs. 50.5 across all tests), suggesting differences between the inbreds for stability. Analysis of variance indicated that significant differences occurred in aflatoxin levels among environments and families (Table 2). Replications within environments also were significant. Estimates of broadsense heritability within environments ranged from 0.27 to 0.42. Table 2. Analysis of variance of aflatoxin concentration in F2:3 families from the cross Mp313E ⫻ B73 grown in four Mississippi environments. Source†

Table 1. Mean (⫾ SE) of aflatoxin accumulation of the parents and 210 F2:3 families grown in Stoneville and Mississippi State, MS, in 2000, 2001, and 2002. MSU2000

Stone2000

MSU2001

MSU2002

ng g⫺1 B73 2215 (⫾ 318) 378 (⫾ 134) 6352 (⫾ 69) 5036 (⫾ 719) Mp313E 722 (⫾ 318) 313 (⫾ 155) 150 (⫾ 692) 385 (⫾ 719) F2:3 families 877 (⫾ 27) 212 (⫾ 12) 1522 (⫾ 62) 1639 (⫾ 58)

Environments Families Environments ⫻ Families Error R2 CV, %

df

MS

3 209 622 2405

900.77** 4.38** 1.08** 0.74 0.71 13.74

** Significant at P ⫽ 0.01. † Test contained 3360 observations, including 120 missing values. Plot means were transformed [ln(y ⫹ 1)] before analysis.

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BROOKS ET AL.: QTL AND RESISTANCE TO AFLATOXIN ACCUMULATION

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QTL Analysis Analysis of the Mp313E ⫻ B73 F2:3 mapping population identified 3, 2, 5, and 3 significant QTL within the MSU2000, Stone2000, MSU2001, and MSU2002 environments, respectively (Table 3). Permutation test estimates of significance levels within environments were consistently around LOD ⫽ 3.5. The Stone2000 test, having the least phenotypic variation, revealed the fewest QTL (two). Two QTL were significant in at least three of the four environments. A 35-cM region on chromosome 2, designated afl3 and represented by left flanking SSR marker bnlg371, accounted for 7 to 18% of phenotypic variance within environments and 23% across all environments. This QTL displayed additive gene action with the Mp313E allele responsible for reduced aflatoxin levels. It was significant in three environments with a recognizable, though not significant, LOD ⫽ 2.89 in the MSU2001 environment. The QTL were found on chromosome 4 in each test that displayed a more complex segregation. A 22-cM region represented by left flanking SSR marker bnlg2291 (afl5) had probability scores greater than the minimum threshold within all tests. Maximum estimated phenotypic variance per test explained by this QTL ranged from 8 to 18%, and 20% overall. Broad chromosomal regions, however, both to the left and right of bnlg2291 were also found to be higher than the significance threshold in some environments, suggesting that multiple, unresolved QTL are present on this chromosome. Except for afl1, which was significant in two environments, additional QTL identified were not significant across multiple environments and contributed little to explained phenotypic variance. The afl4 locus is noteworthy in that it was only significant in the MSU2001 test. Clearly defined, though not significant, LOD peaks (1.45, 2.19, 1.76) were generated in the other environments such that overall this QTL was significant, accounting for 8% of phenotypic variation. Most of the QTL identified, including all the major

loci leading to reduced levels of aflatoxin, were contributed by Mp313E.

DISCUSSION Previous QTL analysis with Mp313E and Va35 as parents identified loci on chromosome 4 (bins 7–9) that significantly contributed to reduced aflatoxin levels across three different environments (Davis et al., 2000; M.C. Willcox, 2000, unpublished data). Other QTL regions varied in significance depending on the environment, and tended to have relatively minor effects. Using a different source of resistance (Tex6) crossed to B73 in their mapping study, Paul et al. (2003) found that important QTL regions were rarely the same from year to year, with a significant number having resistance contributed by B73. Resistance to aflatoxin accumulation was contributed by Mp313E in all but one QTL in the present study, including the two major QTL that were significant across environments. Negligible contributions from B73 could be partially attributed to its lack of adaptation to the Southeastern environment, possibly enhancing its susceptibility to A. flavus infection/aflatoxin accumulation. This study further confirms that a QTL from Mp313E, located on chromosome 4, and designated afl5 in this study, is consistently important in determining aflatoxin accumulation levels even in different genetic backgrounds. afl5 generally accounts for approximately 18% of the phenotypic variance for aflatoxin levels and exhibits an additive gene action. Likelihood estimates also suggest that this QTL may contain multiple linked loci that could not be resolved. Analysis combining these data with the Mp313E ⫻ Va35 mapping data is being performed in an attempt to separate QTL in chromosome 4. In addition, a new QTL on chromosome 2, afl3, has similar effects and has stable expression across environments. This QTL may represent important differences between Va35 and B73 with respect to genomic regions influencing susceptibility to aflatoxin accumulation in

Table 3. Quantitative trait loci (QTL) and their associated markers linked to aflatoxin accumulation as detected by composite interval mapping. Environment MSU2000 (3.53)†† Stone2000 (3.43) MSU2001 (3.54)

MSU2002 (3.58) Overall (3.58)

QTL ID

Marker†

Chromosome

Bin

Add.‡

Dom.§

%Var¶

LOD#

afl3 afl5 afl7 afl3 afl5 afl2 afl3 afl4 afl5 afl6 afl1 afl3 afl5 afl3 afl4 afl5

bnlg371 bnlg2291 bnlg1154 bnlg371 bnlg2291 bnlg439 bnlg371 mmc0022 bnlg2291 mmc0081 bnlg1953 bnlg371 bnlg2291 bnlg371 mmc0022 bnlg2291

2 4 6 2 4 1 2 3 4 5 1 2 4 2 3 4

2.05 4.06 6.05 2.05 4.06 1.03 2.05 3.05 4.06 5.05 1.02 2.05 4.06 2.05 3.05 4.06

⫺0.378 ⫺0.348 0.180 ⫺0.478 ⫺0.324 ⫺2.677 ⫺2.829 ⫺3.376 ⫺4.179 ⫺1.786 ⫺0.224 ⫺0.297 ⫺0.227 ⫺3.557 ⫺1.956 ⫺3.244

0.044 ⫺0.104 ⫺0.221 ⫺0.059 0.059 ⫺0.043 ⫺1.247 ⫺2.012 ⫺1.284 3.777 0.031 ⫺0.174 ⫺0.187 ⫺1.072 ⫺1.532 ⫺1.541

18.4 16.0 7.6 18.3 8.2 5.2 7.1 10.4 14.8 8.7 5.0 12.5 9.3 22.9 8.2 20.1

6.72 7.52 4.15 7.50 3.78 3.29 2.89 5.52 7.77 3.62 3.00 4.30 4.17 10.32 5.46 10.10

† Left most adjacent marker flanking QTL where left corresponds to lower chromosomal bin numbers. ‡ Additive effect. § Dominance effect. ¶ Proportion of phenotypic variance explained by QTL. # LOD scores (log10-likelihood ratio). †† Permutation significance threshold (P ⫽ 0.05) for stated environment.

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crosses with Mp313E. The important contribution of these two QTL across environments lends them to incorporation into elite susceptible inbreds via marker-assisted selection methods, which is being continued from lines of this population.

Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

ACKNOWLEDGMENTS The authors express our appreciation to G.L. Davis, who established a molecular breeding program in Mississippi and identified the first QTL associated with aflatoxin accumulation in maize. This work could not have been accomplished without the technical assistance of J.A. Haynes and L.T. Owens. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.

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