Performance of testers with contrasting provitamin A content to evaluate provitamin A maize for resistance to Aspergillus flavus infection and aflatoxin production.

In sub-Saharan Africa (SSA), millions of people depend on maize as a primary staple. However, maize consumers in SSA may be exposed to malnutrition due to vitamin A deficiency (VAD) and unsafe aflatoxin levels, which can lead to serious economic and public health problems. Provitamin A (PVA) biofortified maize has been developed to alleviate VAD and may have additional benefits such as reduced aflatoxin contamination. In this study, maize inbred testers with contrasting PVA content in grain were used to identify inbred lines with desirable combining ability for breeding to enhance their level of resistance to aflatoxin. Kernels of 120 PVA hybrids generated by crossing 60 PVA inbreds with varying levels of PVA (5.4 to 51.7 µg/g) and two testers (low and high PVA, 14.4 and 25.0 µg/g, respectively) were inoculated with a highly toxigenic strain of Aspergillus flavus. Aflatoxin had a negative genetic correlation with ß-carotene (r = -0.29, p < 0.0001) and PVA (r = -0.23, p < 0.0001), indicating that hybrids with high PVA content accumulated less aflatoxin than those with low to medium PVA. Both general combining ability (GCA) and specific combining ability (SCA) effects of lines and testers were significant for aflatoxin accumulation, number of spores, PVA, and other carotenoids, with additive gene actions playing a prominent role in regulating the mode of inheritance (GCA/SCA ratio >0.5). Eight inbreds had combined significant negative GCA effects for aflatoxin accumulation and spore count with significant positive GCA effects for PVA. Five testcrosses had combined significant negative SCA effects for aflatoxin with significant positive SCA effects for PVA. The high PVA tester had significant negative GCA effects for aflatoxin, lutein, ß-carotene, and PVA. The study identified lines that can be used as parents to develop superior hybrids with high PVA and reduced aflatoxin accumulation. Overall, the results point out the importance of testers in maize breeding programs to develop materials that can contribute to controlling aflatoxin contamination and reducing VAD.

Maize resistance to witchweed through changes in strigolactone biosynthesis.

Maize (Zea mays) is a major staple crop in Africa, where its yield and the livelihood of millions are compromised by the parasitic witchweed Striga. Germination of Striga is induced by strigolactones exuded from maize roots into the rhizosphere. In a maize germplasm collection, we identified two strigolactones, zealactol and zealactonoic acid, which stimulate less Striga germination than the major maize strigolactone, zealactone. We then showed that a single cytochrome P450, ZmCYP706C37, catalyzes a series of oxidative steps in the maize-strigolactone biosynthetic pathway. Reduction in activity of this enzyme and two others involved in the pathway, ZmMAX1b and ZmCLAMT1, can change strigolactone composition and reduce Striga germination and infection. These results offer prospects for breeding Striga-resistant maize.

Association analysis for resistance to Striga hermonthica in diverse tropical maize inbred lines.

Striga hermonthica is a widespread, destructive parasitic plant that causes substantial yield loss to maize productivity in sub-Saharan Africa. Under severe Striga infestation, yield losses can range from 60 to 100% resulting in abandonment of farmers' lands. Diverse methods have been proposed for Striga management; however, host plant resistance is considered the most effective and affordable to small-scale famers. Thus, conducting a genome-wide association study to identify quantitative trait nucleotides controlling S. hermonthica resistance and mining of relevant candidate genes will expedite the improvement of Striga resistance breeding through marker-assisted breeding. For this study, 150 diverse maize inbred lines were evaluated under Striga infested and non-infested conditions for two years and genotyped using the genotyping-by-sequencing platform. Heritability estimates of Striga damage ratings, emerged Striga plants and grain yield, hereafter referred to as Striga resistance-related traits, were high under Striga infested condition. The mixed linear model (MLM) identified thirty SNPs associated with the three Striga resistance-related traits based on the multi-locus approaches (mrMLM, FASTmrMLM, FASTmrEMMA and pLARmEB). These SNPs explained up to 14% of the total phenotypic variation. Under non-infested condition, four SNPs were associated with grain yield, and these SNPs explained up to 17% of the total phenotypic variation. Gene annotation of significant SNPs identified candidate genes (Leucine-rich repeats, putative disease resistance protein and VQ proteins) with functions related to plant growth, development, and defense mechanisms. The marker-effect prediction was able to identify alleles responsible for predicting high yield and low Striga damage rating in the breeding panel. This study provides valuable insight for marker validation and deployment for Striga resistance breeding in maize.

Novel Sources of Resistance to Fusarium Stalk Rot of Maize in Tropical Africa.

Fusarium stalk rot is one of the most widespread and destructive diseases of maize, and deployment of resistant genotypes is one of the most effective strategies for controlling the disease. Fifty inbred lines and four checks from the breeding program of the International Institute of Tropical Agriculture were evaluated in field trials at Ikenne and Ibadan, Nigeria in 2003 and 2004 to identify new sources of resistance to stalk rot caused by Fusarium verticillioides. Evaluations were conducted under artificial inoculation and natural infection at Ibadan and Ikenne, respectively. Disease severity was recorded using a severity scale (SS) and direct estimation of stalk discoloration (SD). The two methods of disease assessment were compared and combined to classify genotypes into resistance groups using results from rank-sum analysis. In 2003, disease severity ranged from SS = 1 to 5 and SD = 1.3 to 33.8% at both locations. Both SS and SD were significantly (P < 0.01) higher in 2003 than in 2004 at the two locations. In both years, inbred lines significantly differed in SS (P < 0.02) and SD (P < 0.04) at Ibadan. Similarly, inbred lines significantly differed in SS (P < 0.04) and SD (P < 0.04) when genotypes were evaluated at Ikenne. Disease assessments based on SS and SD were significantly correlated (0.68 < r < 0.95, P < 0.01) in both years. Based on the results from rank-sum analysis, inbred lines were separated into highly resistant, resistant, moderately resistant, moderately susceptible, susceptible, and highly susceptible groups. At Ibadan, 6 (11.1%) and 8 (14.8%) were identified as highly resistant and resistant, respectively, whereas 11 (20.4%) were identified as resistant at Ikenne. Inbred lines 02C14609, 02C14643, 02C14654, and 02C14678 were consistently classified as either highly resistant or resistant to stalk rot across locations and years while the check genotypes were classified either as susceptible or moderately susceptible to stalk rot. These four inbred lines identified to have high levels of disease resistance may be used for breeding maize with resistance to Fusarium stalk rot.

Evaluation of Maize Inbred Lines for Resistance to Fusarium Ear Rot and Fumonisin Accumulation in Grain in Tropical Africa.

Fusarium ear rot and fumonisin contamination is a major problem facing maize growers worldwide, and host resistance is the most effective strategy to control the disease, but resistant genotypes have not been identified. In 2003, a total of 103 maize inbred lines were evaluated for Fusarium ear rot caused by Fusarium verticillioides in field trials in Ikenne and Ibadan, Nigeria. Disease was initiated from natural infection in the Ikenne trial and from artificial inoculation in the Ibadan trial. Ear rot severity ranged from 1.0 to 6.0 in both locations in 2003. Fifty-two inbred lines with disease severity ≤3 (i.e., ≤ 10% visible symptoms on ears) were selected and reevaluated in 2004 for ear rot resistance, incidence of discolored kernels, and fumonisin contamination in grain. At both locations, ear rot severity on the selected lines was significantly (P < 0.0020) higher in 2004 than in 2003. The effects of selected inbred lines on disease severity were highly significant at Ikenne (P = 0.0072) and Ibadan (P < 0.0001) in 2004. Inbred lines did not affect incidence of discolored kernels at both locations and across years except at Ikenne (P = 0.0002) in 2004. Similarly, significant effects of inbred lines on fumonisin concentration were observed only at Ikenne (P = 0.0201) in 2004. However, inbred lines 02C14585, 02C14593, 02C14603, 02C14606, 02C14624, and 02C14683 had consistently low disease severity across years and locations. Fumonisin concentration was significantly correlated with ear rot only at Ikenne (R = 0.42, P < 0.0001). Correlation between fumonisin concentration and incidence of discolored kernels was also significant at Ikenne (R = 0.39, P < 0.0001) and Ibadan (R = 0.35, P = 0.0007). At both locations, no significant inbred × year interaction was observed for fumonisin concentration. Five inbred lines, namely 02C14585, 02C14603, 02C14606, 02C14624, and 02C14683, consistently had the lowest fumonisin concentration in both trials. Two of these inbred lines, 02C14624 and 02C14585, had fumonisin levels <5.0 µg/g across years in trials where disease was initiated from both natural infection and artificial inoculation. These lines that had consistently low disease severity are useful for breeding programs to develop fumonisin resistant lines.

Assessment of testcross performance and genetic diversity of yellow endosperm maize lines derived from adapted x exotic backcrosses.

Introduction of exotic maize (Zea mays L.) into adapted tropical germplasm may enhance genetic variability and lead to greater progress from selection. The first objective of this study was to determine if yellow endosperm lines derived from adapted x exotic backcrosses contain exotic alleles that are superior to the recurrent adapted parental line for yield and other agronomic traits in tropical environments. Thirteen exotic yellow maize inbred lines were crossed to an adapted orange line (KUSR) and the F1s were backcrossed to KUSR to generate the first backcrosses. Fifty BC1F4 lines derived from these backcrosses and the recurrent parent were crossed to a common inbred tester (L4001) to form testcrosses, which were evaluated at eight environments in Nigeria. Testcrosses of the BC-derived lines differed significantly for grain yield and other agronomic traits. Only two testcrosses yielded significantly less than L4001 x KUSR, with the best 15 testcrosses producing between 289 and 1,056 kg/ha more grain yield than L4001 x KUSR. The best testcrosses were similar to or better than L4001 x KUSR for other agronomic traits. The second objective of this study was to assess the extent of genetic diversity present among the BC-derived lines. We genotyped 46 BC-derived lines including KUSR and L4001 with 10 AFLP primer pairs and found 491 polymorphic fragments. The average allelic diversity of the lines was 0.30 +/- 0.01. The genetic distance of each BC-derived line from KUSR ranged between 0.49 and 0.91. The average genetic distance for all pairs of the BC-derived lines was 0.68 +/- 0.004, varying from 0.34 to 0.92. The increased grain yield and genetic diversity observed in these studies provide evidence that exotic germplasm can contribute new alleles to expand the genetic base of tropical maize and develop high-yielding hybrids.

Proteomics to identify resistance factors in corn-a review.

The host resistance strategy for eliminating aflatoxins from corn has been advanced by the discovery of natural resistance traits such as proteins. This progress was aided by the development of a rapid laboratory-based kernel screening assay (KSA) used to separate resistant from susceptible seed, and for investigating kernel resistance.A. flavus GUS transformants have also been used, in conjunction with the KSA, to assess the amount of fungal growth in kernels and compare it with aflatoxin accumulation. Several proteins associated with resistance (RAPs) have been identified using 1 D PAGE. However, proteomics is now being used to further the discovery of RAPs. This methodology has led to the identification of stress-related RAPs as well as other antifungals. Characterization studies being conducted, including RNAi gene silencing experiments, may confirm roles for RAPs in host resistance.

Molecular marker-based genetic diversity assessment of Striga-resistant maize inbred lines.

Striga-resistant maize inbred lines are of interest to maize breeding programs in the savannas of Africa where the parasitic weed is endemic and causes severe yield losses in tropical maize. Assessment of the genetic diversity of such inbred lines is useful for their systematic and efficient use in a breeding program. Diversity analysis of 41 Striga-resistant maize inbred lines was conducted using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers to examine the genetic relationships among these lines and to determine the level of genetic diversity that exists within and between their source populations. The two marker systems generated 262 and 101 polymorphic fragments, respectively. Genetic similarity (GS) values among all possible pairs of inbred lines varied from 0.45 to 0.95, with a mean of 0.61+/-0.002 for AFLPs, and from 0.21 to 0.92, with a mean of 0.48+/-0.003, for SSRs. The inbred lines from each source population exhibited a broad range of GS values with the two types of markers. Both AFLPs and SSRs revealed similar levels of within population genetic variation for all source populations. Cluster and principal component analysis of GS estimates with the two markers revealed clear differentiation of the Striga-resistant inbred lines into groups according to their source populations. There was clear separation between early- and late-maturing Striga-resistant inbred lines. Considering the paucity of germplasm with good levels of resistance to Striga in maize, the broad genetic diversity detected within and among source populations demonstrates the genetic potential that exists to improve maize for resistance to Striga.

Grouping of tropical mid-altitude maize inbred lines on the basis of yield data and molecular markers.

The classification of maize inbred lines into heterotic groups is an important undertaking in hybrid breeding. The objectives of our research were to: (1) separate selected tropical mid-altitude maize inbred lines into heterotic groups based on grain yield data; (2) assess the genetic relationships among these inbred lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers; (3) examine the consistency between yield-based and marker-based groupings of the inbred lines. Thirty-eight tropical mid-altitude maize inbred lines were crossed to two inbred line testers representing the flint and dent heterotic pattern, respectively. The resulting testcrosses were evaluated in a trial at three locations for 2 years. Significant general combining ability (GCA) and specific combining ability (SCA) effects for grain yield were detected among the inbred lines. The tester inbred lines classified 23 of the 38 tested inbred lines into two heterotic groups based on SCA effects and testcross mean grain yields. This grouping was not related to endosperm type of the inbred lines. The outstanding performance of testcrosses of the remaining 15 inbred lines indicates the presence of significant genetic diversity that may allow the assignment of the lines into more than two heterotic groups. Diversity analysis of the 40 maize inbred lines using AFLP and SSR markers found high levels of genetic diversity among these lines and subdivided them into two main groups with subdivision into sub-groups consistent with breeding history, origin and parentage of the lines. However, heterotic groups formed using yield-based combining ability were different from the groups established on the basis of molecular markers. Considering the diversity of the genetic backgrounds of the mid-altitude inbred lines, the marker-based grouping may serve as the basis to design and carry out combining ability studies in the field to establish clearly defined heterotic groups with a greater genetic similarity within groups.

Resistance to aflatoxin accumulation in kernels of maize inbreds selected for ear rot resistance in West and Central Africa.

Thirty-six inbred lines selected in West and Central Africa for moderate to high resistance to maize ear rot under conditions of severe natural infection were screened for resistance to aflatoxin contamination using the previously established kernel screening assay. Results showed that more than half the inbreds accumulated aflatoxins at levels as low as or lower than the resistant U.S. lines GT-MAS:gk or MI82. In 10 selected aflatoxin-resistant or aflatoxin-susceptible inbreds, Aspergillus flavus growth, which was quantified using an A. flavus transformant containing a GUS-beta-tubulin reporter gene construct, was, in general, positively related to aflatoxin accumulation. However, one aflatoxin-resistant inbred supported a relatively high level of fungal infection, whereas two susceptibles supported relatively low fungal infection. When kernels of the 10 tested lines were profiled for proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, significant variations from protein profiles of U.S. lines were observed. Confirmation of resistance in promising African lines in field trials may significantly broaden the resistant germplasm base available for managing aflatoxin contamination through breeding approaches. Biochemical resistance markers different from those being identified and characterized in U.S. genotypes, such as ones inhibitory to aflatoxin biosynthesis rather than to fungal infection, may also be identified in African lines. These discoveries could significantly enhance the host resistance strategy of pyramiding different traits into agronomically useful maize germplasm to control aflatoxin contamination.

Comparisons of methods for introgressing exotic germ plasm into adapted sorghum.

The incorporation of exotic germ plasm into breeding populations can broaden and diversify the genetic base of adapted genotypes. To more effectively utilize the genetic resources existing in Sorghum bicolor (L.) Moench, a rapid and efficient method of incorporating exotic genotypes into adapted populations is needed. Therefore, this study was conducted to compare the effectiveness of backcrossing to a broad-based population versus backcrossing to an inbred line for developing improved lines from adapted x exotic crosses. A wild sorghum, a cultivated landrace, and a converted sorghum line were crossed to an inbred line (CK60) and a broad-based population (KP9B). After two generations of backcrossing to the respective adapted parent, 50 F2 lines were derived from each of the backcross generations of every mating and evaluated at three test environments. Backcrossing to an inbred line (CK60) gave fewer high-yielding segregates and generated less genetic variation than backcrossing to a population (KP9B). Also, the number of agronomically acceptable lines derived from each CK60 mating was fewer than that derived from the corresponding mating with KP9B. Overall, the use of a broad-based population as an adapted recurrent parent for introgressing exotic genotypes may provide good opportunities for developing suitable inbred lines from adapted x exotic backcrosses.

Comparison of trait associations in adapted × exotic matings of sorghum developed by two introgression methods.

The association among six traits in the F2 lines derived from adapted × exotic backcrosses of sorghum developed via two introgression methods was studied using principal component analysis. The first principal component defined a hybrid index in matings of the wild accession ('12-26') but not in matings of the cultivated sorghum genotypes ('Segeolane' and 'SC408'), no matter which adapted parent was used. This component accounted for 27-42% of the total variation in each mating. The 'recombination spindle' was wide in all matings of CK60 and KP9B, which indicated that the relationships among traits were not strong enough to restrict recombination among the parental characters. The index scores of both CK60 and KP9B matings showed clear differentiation of the backcross generations only when the exotic parent was the undomesticated wild accession ('12-26'). None of the distributions of the first principal component scores in any backcross population was bimodal. The frequency of recombinant genotypes derived from a mating was determined by the level of domestication and adaptation of the exotic parent and the genetic background of the adapted parent. Backcrossing to a population (KP9B) was found to be superior to backcrossing to an inbred line (CK60) to produce lines with an improved adapted phenotype.