A USA–Africa collaborative strategy for identifying, characterizing, and developing maize germplasm with resistance to aflatoxin contamination

Aflatoxin contamination of maize by Aspergillus flavus poses serious potential economic losses in the US and health hazards to humans, particularly in West Africa. The Southern Regional Research Center of the United States Department of Agriculture, Agricultural Research Service (USDA-ARS-SRRC) and the International Institute of Tropical Agriculture (IITA) initiated a collaborative breeding project to develop maize germplasm with resistance to aflatoxin accumulation. Resistant genotypes from the US and selected inbred lines from IITA were used to generate backcrosses with 75% US germplasm and F₁ crosses with 50% IITA and 50% US germplasm. A total of 65 S₄ lines were developed from the backcross populations and 144 S₄ lines were derived from the F₁ crosses. These lines were separated into groups and screened in SRRC laboratory using a kernel-screening assay. Significant differences in aflatoxin production were detected among the lines within each group. Several promising S₄ lines with aflatoxin values significantly lower than their respective US resistant recurrent parent or their elite tropical inbred parent were selected for resistance-confirmation tests. We found pairs of S₄ lines with 75–94% common genetic backgrounds differing significantly in aflatoxin accumulation. These pairs of lines are currently being used for proteome analysis to identify resistance-associated proteins and the corresponding genes underlying resistance to aflatoxin accumulation. Following confirmation tests in the laboratory, lines with consistently low aflatoxin levels will be inoculated with A. flavus in the field in Nigeria to identify lines resistant to strains specific to both US and West Africa. Maize inbred lines with desirable agronomic traits and low levels of aflatoxin in the field would be released as sources of genes for resistance to aflatoxin production.     

Genetic diversity for restriction fragment length polymorphisms and heterosis for two diallel sets of maize inbreds

Summary Changes that may have occurred over the past 50 years of hybrid breeding in maize (Zea maize L.) with respect to heterosis for yield and heterozygosity at the molecular level are of interest to both maize breeders and quantitative geneticists. The objectives of this study were twofold: The first, to compare two diallels produced from six older maize inbreds released in the 1950's and earlier and six newer inbreds released during the 1970's with respect to (a) genetic variation for restriction fragment length polymorphisms (RFLPs) and (b) the size of heterosis and epistatic effects, and the second, to evaluate the usefulness of RFLP-based genetic distance measures in predicting heterosis and performance of single-cross hybrids. Five generations (parents, F₁; F₂, and backcrosses) from the 15 crosses in each diallel were evaluated for grain yield and yield components in four Iowa environments. Genetic effects were estimated from generation means by ordinary diallel analyses and by the Eberhart-Gardner model. Newer lines showed significantly greater yield for inbred generations than did older lines but smaller heterosis estimates. In most cases, estimates of additive x additive epistatic effects for yield and yield components were significantly positive for both groups of lines. RFLP analyses of inbred lines included two restriction enzymes and 82 genomic DNA clones distributed over the maize genome. Eighty-one clones revealed polymorphisms with at least one enzyme. In each set, about three different RFLP variants were typically found per RFLP locus. Genetic distances between inbred lines were estimated from RFLP data as Rogers' distance (RD), which was subdivided into general (GRD) and specific (SRD) Rogers' distances within each diallel. The mean and range of RDs were similar for the older and newer lines, suggesting that the level of heterozygosity at the molecular level had not changed. GRD explained about 50% of the variation among RD values in both sets. Cluster analyses, based on modified Rogers' distances, revealed associations among lines that were generally consistent with expectations based on known pedigree and on previous research. Correlations of RD and SRD with f₁ performance, specific combining ability, and heterosis for yield and yield components, were generally positive, but too small to be of predictive value. In agreement with previous studies, our results suggest that RFLPs can be used to investigate relationships among maize inbreds, but that they are of limited usefulness for predicting the heterotic performance of single crosses between unrelated lines.Joint contribution from Cereal and Soybean Research Unit, USDA, Agricultural Research Service and Journal Paper no. J-13929 of the Iowa Agric and Home Economics Exp Stn, Ames, IA 50011. Projects no. 2818 and 2778A.E.M. is presently at the Iowa State University on leave from University of Hohenheim, D-7000 Stuttgart 70, Federal Republic of Germany     

Heterosis × nutrition interaction in Drosophila melanogaster

Summary The relationship between heterozygosity and the expression of heterosis at two different nutrition levels was investigated using Drosophila melanogaster. Average daily egg production and egg hatchability were measured in two parental strains and in F₁, F₂ and reciprocal backcross generations. Heterosis was more pronounced in the poor nutritional conditions. Two electrophoretic markers used to estimate the level of heterozygosity in F₂ and backcrosses revealed an excess of heterozygous genotypes. Quantitative genetic effects (an additive line effect and individual and maternal heterosis) were estimated for both traits in the two environments. Although this model gave a reasonable fit in most cases, some epistatic interaction would have to be invoked in order to explain fully the results.     

Epistatic contributions to quantitative traits in Tribolium castaneum

Summary Triple-testcross experiments were used to analyze epistatic contributions to larva weight, pupa weight, pupa width and adult weight in Tribolium castaneum. Seven diverse inbred lines and the F₁. produced by crossing the two tester lines were examined for indications of epistasis. Larva weight was the only trait for which no significant epistasis was detected. There was significant epistasis for pupa weight in three of the inbred lines; for pupa width in four of the inbred lines; for adult weight in five of the inbred lines. Only one inbred line and the F₁ line failed to exhibit significant epistasis for any trait. Each inbred line had a unique pattern of epistasis, suggesting that a number of different loci were contributing to the detected epistasis.This paper (No. 76-5-158) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Assessment of testcross performance and genetic diversity of yellow endosperm maize lines derived from adapted × 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 × 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 × KUSR, with the best 15 testcrosses producing between 289 and 1,056 kg/ha more grain yield than L4001 × KUSR. The best testcrosses were similar to or better than L4001 × 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.     

Inheritance of resistance in maize silks to the corn earworm

The corn earworm,Helicoverpa zea (Boddie), is a perennial economic pest of field crops in the United States. Maize,Zea mays L., is the major host crop promoting the build-up of devastating corn earworm populations that limit full production of cotton, soybean, peanut, and grain sorghum. Resistance to the corn earworm in maize and in particular sweet maize, would provide an environmentally safe, economical method of control for this pest insect. Antibiotic effects of corn silks on this insect are: small larvae, extended developmental period, and reduced fecundity. Silks from individual maize plants of resistant and susceptible lines and progeny in six generations consisting of parents (P₁, P₂), F₁, F₂, and backcrosses BC_1.1 (F₁ × P₁) and BC_1.2 (F₁ × P₂) from each of four crosses were used to determine the genetic basis of the antibiotic resistance of silks to the corn earworm. In the cross of Zapalote Chico × PI340856, genes controlling resistance in the silks to the corn earworm larvae are dominant in PI340856 to those in Zapalote Chico. The cross of Zapalote Chico × GT114 involves parents differing in degree of resistance, and possibly differing for the genetic mechanism by which the resistance is inherited. The inheritance of resistance may involve non-additive (dominance and epistasis) genetic variance. A digenic 6-parameter model indicated (1) the resistance in this cross is controlled by more than one pair of genes and (2) some or all of the genes interact to cause non-allelic interaction. Thus, the resistance in this cross may be controlled by both dominant and recessive genes. The resistance of Zapalote Chico × CI64, an intermediate inbred, is influenced by additive gene effects. The digenic model adequately predicts all generation means of the cross of GT3 × PI340856 except for the F1. Thus, it appears that the additive-dominance model is not satisfactory for this cross involving susceptible and resistant parents. Generation mean analysis indicates that resistance to silk-feeding by corn earworm larvae is under genetic control, but gene action differs from one type of cross to another.     

Genetic control of T-lymphocyte mitogenesis in chickens

The in vitro mitogenic response to PHA and Con A was determined in three inbred lines of chickens. Lymphocytes from one line consistently showed a greater stimulation by PHA than the other two lines. Analysis of F₁ crosses and backcrosses indicated that this quantitative difference was controlled by more than one gene. More substantial differences in Con-A stimulation were also observed between the three lines, and the data indicated that separate genetic systems were controlling the variation in PHA and Con-A stimulation. Analysis of F₁ crosses, backcrosses and assortative matings between backcrosses revealed that the variation in Con-A stimulation was controlled by at least two major genes, one of which may be linked to the major histocompatibility complex. Surprisingly, one line appeared to be segregating for Con-A stimulation in spite of an inbreeding coefficient greater than 0.98.     

Additive, dominant, and epistatic effects for maize grain yield in acid and non-acid soils

Acid soils severely reduce maize (Zea mays L.) yield in the tropics. Breeding for tolerance to soil acidity provides a permanent, environmentally friendly, and inexpensive solution to the problem. This study was carried out to determine the relative importance of additive, dominant, and epistatic effects on maize grain-yields in different tropical genotypes. Divergent selection in three populations (SA4, SA5, and SA7) provided inbred lines tolerant or sensitive to acid soils. The tolerant and sensitive lines from each population were used to obtain the F₁, F₂, F₃, back-crosses, second back-crosses, and selfed back-cross generations. In addition, the tolerant lines from SA4 and SA5 were crossed with a sensitive line from the Tuxpeño Sequía population, from which the same generations were also derived. All generations from each of the five sets of crosses were evaluated in three acid-soil environments and one non-acid-soil environment. A generation-mean analysis was performed on each set for yield. The sequential sum of squares associated with additive, dominance, and digenic epistatic effects were used to estimate the relative importance of each genetic effect. Epistasis was not important in any set in the non-acid-soil environment, with dominance accounting for 80.76% of the total variation among generation means across sets. In acid-soil environments, epistasis was more important. The relative importance of digenic epistasis was greater in those evaluations with large experimental errors. The tolerant line from population SA5 was prone to severe root lodging, suggesting a very poor root system. Apparently, the tolerance to soil acidity in this line is not associated with a large root system.     

Epistatic contributions to quantitative traits in Tribolium castaneum

Summary Triple-testcross experiments were used to analyze epistatic contributions to % hatchability of eggs, age of pupation, number of eggs laid in 24-hour period, and survival from hatching to day 35. Seven diverse inbred lines and the F₁ produced by crossing the two tester lines were examined for the presence of epistasis. There was evidence of epistasis for each of the 4 traits in at least one of the 8 lines tested. Epistasis was a major source of variation in survival in all of the lines tested.     

Effect of selection on the heterozygosity of inbred lines of maize

Although pedigree selection is the most commonly used method for developing inbred lines of maize, there are no studies on its effect on the heterozygosity of the lines. The objective of this work was to study the effect of pedigree selection on their heterozygosity. Thirteen F₅ or F₆ maize inbred lines developed by the pedigree selection method in four breeding programs and their F₁ and F₂ − F₄ ancestors were genotyped with simple sequence repeat markers distributed along the genome. Simulation was also conducted assuming different models of selection to investigate the selective forces needed to explain the data. In the F₂, F₃ and F₄ 40%, 66% and 86% of the markers segregating in the F₁ were fixed; that is, in the F₂ and F₃ fixation was lower than neutral expectation, but higher in the F₄. Due to such opposite apparent directions of selection, the heterozygosity of the lines in the F₅ or F₆ generations did not differ significantly from neutral expectations. The time to fixation differed from that expected with neutrality for most of the chromosomes, indicating that selection is distributed across the genome; but apparent overdominant effects in chromosome 7 were higher than in other chromosomes. In conclusion, the relationship between heterozygosity and vigour may reduce the effectiveness of pedigree selection in its goal of selecting the more vigorous, homozygous individuals. A more effective procedure is proposed using molecular markers for the identification of the more homozygous individuals, the most vigorous of those individuals being selected.     

Efficient design of crossbreeding experiments

Summary The efficiencies of different experimental configurations for estimating additive (A) and heterotic (H) effects in purebred and crossbred populations derived from two parent breeds are examined. Allocation of resources over six groups is considered: the two parental breeds, the F₁, the F₂ and the backcrosses to the two parental breeds. Additive and heterotic effects are best estimated by allocating resources to the two parental groups and F₁ in the proportions 35%, 35%, 30% and for many practical situations, an experiment involving numbers in the region 250–300 is reasonable. If it is not possible to include all three groups, other combinations involving a subset of them and some of the other three groups can be used to give estimates of A and H. However, even the best of these alternatives requires over twice the resources to give the same precision as the optimal design and, further, these estimates may be correlated. Relatively modest reallocation of resources to the F₂ to estimate or test for an epistatic effect (E) leads to a minor reduction in the precision of estimates of A and H while giving reasonable precision for the estimate of E. The inclusion of maternal effects in the model greatly reduces the efficiency of estimation of A and H. Where one of the breeds is introduced through the sire line only, optimal allocation gives roughly equal replication to the pure lines and F₁ but about 63% of allocation is placed in equal amounts on the two backcrosses produced through crossing F₁ dams with pure sires of both breeds. The relevance of these results to the planning of livestock crossbreeding trials, particularly those involving a local and an exotic or imported breed, is discussed.     

Genetic analysis of tolerance to low-phosphorus stress in maize using restriction fragment length polymorphisms

Summary An understanding of the genetic nature underlying tolerance to low-phosphorus (low-P) stress could aid in the efficient development of tolerant plant strains. The objective of this study was to identify the number of loci in a maize (Zea mays L.) population segregating for tolerance to low-P stress, their approximate location, and the magnitude of their effect.Seventy-seven restriction fragment length polymorphisms (RFLPs) were identified and scored in a maize F₂ population derived from a cross between line NY821 and line H99. The F₂ individuals were self-pollinated to produce F₃ families. Ninety F₃ families were grown in a sand-alumina system, which simulated diffusion-limited, low-P soil conditions. The F₃ families were evaluated for vegetative growth in a controlled-environment experiment. To identify quantitative trait loci (QTLs) underlying tolerance to low-P stress, the mean phenotypic performances of the F₃ families were contrasted based on genotypic classification at each of 77 RFLP marker loci.Six RFLP marker loci were significantly associated with performance under low-P stress (P<0.01). One marker locus accounted for 25% of the total phenotypic variation. Additive gene action was predominant for all of the QTLs identified. Significant marker loci were located on four separate chromosomes representing five unlinked genomic regions. Two marker loci were associated with an additive by additive epistatic interaction. A multiple regression model including three marker loci and the significant epistatic interaction accounted for 46% of the total phenotypic variation. Heterozygosity per se was not predictive of phenotypic performance.     

QTL detection for stover yield and quality traits using two connected populations in high-oil maize

Both yield and quality traits for stover portion were important for forage and biofuel production utility in maize. A high-oil maize inbred GY220 was crossed with two normal-oil dent maize inbred lines 8984 and 8622 to generate two connected F_2:3 populations with 284 and 265 F_2:3 families. Seven yield and quality traits were evaluated under two environments. The variance components of genotype (σ_g²), environment (σ_e²) and genotype × environment interactions (σ_ge²) were all significant for most traits in both populations. Different levels of correlations were observed for all traits. QTL mapping was conducted using composite interval mapping (CIM) for data under each environment and in combined analysis in both populations. Totally, 45 and 42 QTL were detected in the two populations. Only five common QTL across the two populations, and one and three common QTL across the two environments in the two populations were detected, reflecting substantial influence of genetic backgrounds and environments on the results of QTL detection for stover traits. Combined analysis across two environments failed to detect most QTL mapped using individual environmental data in both populations. Few of the detected QTL displayed digenic epistatic interactions. Common QTL among all traits were consistent with their correlations. Some QTL herein have been detected in previous researches, and linked with candidate genes for enzymes postulated to have direct and indirect roles in cell wall components biosynthesis.     

Resistance of maize to Meloidogyne arenaria and Meloidogyne javanica

Summary A diallel cross of eight maize, Zea mays L., inbred lines was analyzed for reaction to two species of root-knot nematodes, Meloidogyne arenaria (Neal) Chitwood and M. javanica (Treub) Chitwood. Egg production following inoculation of F₁ hybrid seedlings with nematode eggs was determined in a greenhouse experiment. Data were analyzed using Griffing's Method 4, Model I. General combining ability was a significant source of variation in egg production of both M. arenaria and M. javanica; specific combining ability was not a significant source of variation for either. The correlation between egg production of the two nematode species on the 28 F₁ hybrids was highly significant. Hybrids with Mp313 or SC213 as one parent were the most resistant to both species. This indicates that germ plasm is available for developing inbred lines and hybrids with resistance to both M. arenaria race 2 and M. javanica.This article is a contribution of the Crop Science Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, in cooperation with the Mississippi Agricultural and Forestry Experiment Station, Journal No. J-7481.     

An Analysis of the Mode of Gene Action Affecting Pupa Weight in TRIBOLIUM CASTANEUM

Triple-testcross experiments (Kearsey and Jinks 1968) were employed to investigate the mode of gene action affecting pupa weight in Tribolium castaneum. Their experimental design involves two inbred lines, the F₁ progeny and a segregating population derived from the cross of the inbred lines. In the present experiments, four segregating populations were used. These populations included the F₂ generation, a select line (SEL) and two relaxed select lines (RSI and RSII). In addition, all possible reciprocal crosses were made among the RSI, RSII, and SEL populations. It was observed that: (1) additive, dominant and epistatic gene effects all made significant contributions to the pupa weight of the progeny from all four segregating populations; (2) there was no evidence of either accumulation of epistasis as a result of selection in the SEL population or decline in epistasis as a result of removing selection pressure from the RSI and RSII populations; and (3) significant negative heterosis and maternal effects contributed to the pupa weight of the crossbred progeny of the RSI, RSII and SEL populations.     

Should maize doubled haploids be induced among F<Subscript>1</Subscript> or F<Subscript>2</Subscript> plants?

Maize (Zea mays L.) doubled haploid lines are typically produced from F₁ plants. Studies have suggested that the low frequency of recombinants in doubled haploids may reduce the response to selection. My objective was to determine if, for sustaining long-term response, doubled haploids should be induced in F₁ or F₂ plants during maize inbred development. In simulation experiments, I examined the response to multiple cycles of testcross selection among doubled haploid lines derived from F₁ plants (denoted by DH), doubled haploid lines derived from F₂ plants (DH_F2), and recombinant inbred (RI) lines derived by single-seed descent. For a trait controlled by 100 or more quantitative trait loci (QTL), the cumulative responses to selection were up to 4–6% larger among DH_F2 lines than among DH lines. The cumulative responses were up to 5–8% larger among RI lines than among DH lines. The QTL become unlinked as the number of QTL in a finite genome decreases, and the responses among RI, DH, and DH_F2 lines were equal or nearly equal when only 20 QTL controlled the trait. Metabolic-flux epistasis reduced the differences in the response among RI, DH, and DH_F2 lines. Overall, the results indicated that doubled haploids should be induced from F₂ plants rather than from F₁ plants. If year-round nurseries are used and new F₁ crosses for inbred development are initially created on a speculative basis, the development of doubled haploids from F₂ rather than F₁ plants should not cause a delay in inbred development.     

Epistatic interaction is an important genetic basis of grain yield and its components in maize

A population of 294 recombinant inbred lines (RIL) derived from Yuyu22, an elite maize hybrid extending broadly in China, has been constructed to investigate the genetic basis of grain yield, and associated yield components in maize. The main-effect quantitative trait loci (QTL), digenic epistatic interactions, and their interactions with the environment for grain yield and its three components were identified by using the mixed linear model approach. Thirty-two main-effect QTL and forty-four pairs of digenic epistatic interactions were detected for the four measured traits in four environments. Our results suggest that both additive effects and epistasis (additive × additive) effects are important genetic bases of grain yield and its components in the RIL population. Only 30.4% of main-effect QTL for ear length were involved in epistatic interactions. This implies that many loci in epistatic interactions may not have significant effects for traits alone but may affect trait expression by epistatic interaction with the other loci.     

In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

A Genetic Analysis of Mancozeb Resistance in Typhlodromus Pyri (Acari: Phytoseiidae)

A field population of Typhlodromus pyri (Acari: Phytoseiidae) tolerant to mancozeb was selected in the laboratory. After 10 mancozeb selections the LC₅₀ value for mancozeb was 73 times higher in the selected-10 strain compared to the standard susceptible strain. A genetic analysis using reciprocal crosses and backcrosses of female F₁ progeny found no maternal effect. Resistance in the selected-10 strain was codominant in expression, dominance value was about −0.1. Backcrosses between F₁ females and the susceptible strain indicate that the resistance to mancozeb could be principally conferred by a predominant gene, but additional factors would also be involved.     

Improved tissue culture response of an elite maize inbred through backcross breeding,and identification of chromosomal regions important for regeneration by RFLP analysis

Summary The frequency of initiation of friable, embryogenic callus from immature embryos of the elite maize inbred line B73 was increased dramatically by introgression of chromosomal segments from the inbred line A188 through classical backcross breeding. Less than 0.2% of the immature B73 embryos tested (5 of 3,710) formed embryogenic callus. The breeding scheme consisted of six generations of backcrossing to B73 with selection at each generation for high frequency initiation of embryogenic cultures. BC₆ individuals were selfed for four generations to select homozygous lines. The average embryogenic culture initiation frequency increased to 46% (256/561). Nearly all (91%) of the embryos from one BC₆ S₄ plant formed embryogenic cultures. RFLP analysis was used to determine the locations and effects of the introgressed A188 chromosomal segments. Five segments were retained through at least the fifth backcross generation. The hypothesis that one or more of these five regions contains genes controlling somatic embryogenesis in maize was tested using an F₂ population of the cross A188 X Mo17. A set of five DNA markers (three of them linked) explained 82% of the observed phenotypic variance for percentage of immature embryos forming embryognic callus. Four of the five markers were located in or near introgressed A188 chromosome segments.The region marked by probe c595 on the long arm of chromosome 9 was highly associated with several measures of in vitro culture response (percent embryogenic embryos, plants per embryo, and plants per embryogenic embryo). We propose that there is a major gene (or genes) in this region in A188 that promotes embryogenic callus initiation and plant regeneration in B73, Mo17, and probably many other recalcitrant inbred lines of maize.