Influence of dent corn genetic backgrounds on QTL detection for plant-height traits and their relationships in high-oil maize

QTL mapping for plant-height traits has not been hitherto reported in high-oil maize. A high-oil maize inbred ‘GY220’ was crossed with two dent maize inbreds (‘8984’ and ‘8622’) to generate two connected F_2:3 populations. Four plant-height traits were evaluated in 284 and 265 F_2:3 families. Single-trait QTL mapping and multiple-trait joint QTL mapping was used to detect QTLs for the traits and the genetic relationship between plant height (PH) and two other plant-height traits. A total of 28 QTLs and 12 pairs of digenic interactions among detected QTLs for four traits were detected in the two F_2:3 families. Only one marker was shared between the two populations. Joint analysis of PH with ear height (EH) and PH with top height (TH) detected 32 additional QTLs. Our results showed that QTL detection for PH was dependent on the genetic background of dent corn inbreds. Multiple-trait joint QTL analysis could increase the number of detected QTLs.     

Parental selection,number of breeding populations,and size of each population in inbred development

Some breeders select inbreds from many F₂ or backcross breeding populations, each with relatively few progenies. Other breeders select inbreds from only a few breeding populations, each with many progenies. My objectives were to: (1) determine the relative importance of parental selection, number of breeding populations, and size of each population, and (2) find optimum combinations between number and size of breeding populations. I assumed that a breeder has resources to test a total of 2,000 recombinant inbreds for a quantitative trait that was controlled by 100 additive loci and had a heritability of 0.20, 0.60, or 1.0. The parental inbreds had an inherent pedigree structure due to advanced cycle breeding. The parental inbreds were ranked according to their mean performance, and breeding populations were made among all parents, the top 25% of parents, and the top 10% of parents. I found that the issue of number versus size of breeding populations was only secondary compared with the ability to identify, prior to making the crosses, the breeding populations with the highest mean performance. For a given level of effectiveness of parental selection, the selection response was largest when the maximum number of breeding populations was used. The effect of the number of breeding populations was minor, however, when selection was practiced among the parents or when heritability was less than 1.0. The results suggested that, in practice, large selection responses could be obtained with a wide range of combinations between number and size of breeding populations.Communicated by H.C. Becker     

Broadening the genetic base of European maize heterotic pools with US Cornbelt germplasm using field and molecular marker data

Maize (Zea mays L.) breeders are concerned about the narrowing of the genetic base of elite germplasm. To reverse this trend, elite germplasm from other geographic regions can be introgressed, but due to lack of adaptation it is difficult to assess their breeding potential in the targeted environment. The objectives of this study were to (1) investigate the relationship between European and US maize germplasm, (2) examine the suitability of different mega-environments and measures of performance to assess the breeding potential of exotics, and (3) study the relationship of genetic distance with mid-parent heterosis (MPH). Eight European inbreds from the Dent and Flint heterotic groups, 11 US inbreds belonging to Stiff Stalk (SS), non-Stiff Stalk (NSS), and CIMMYT Pool 41, and their 88 factorial crosses in F₁ and F₂ generations were evaluated for grain yield and dry matter concentration. The experiments were conducted in three mega-environments: Central Europe (target mega-environment), US Cornbelt (mega-environment where donor lines were developed), and Southeast Europe (an intermediate mega-environment). The inbreds were also fingerprinted with 266 SSR markers. Suitable criteria to identify promising exotic germplasm were F₁ hybrid performance in the targeted mega-environment and F₁ and parental performance in the intermediate mega-environment. Marker-based genetic distances reflected relatedness among the inbreds, but showed no association with MPH. Based on genetic distance, MPH, and F₁ performance, we suggest to introgress SS germplasm into European Dents and NSS into European Flints, in order to exploit the specific adaptation of European flint germplasm and the excellent combining ability of US germplasm in European maize breeding programs.     

Identification of trait-improving quantitative trait loci for grain yield components from a dent corn inbred line in an advanced backcross BC<Subscript>2</Subscript>F<Subscript>2</Subscript> population and comparison with its F<Subscript>2:3</Subscript> population in popcorn

Normal maize germplasm could be used to improve the grain yield of popcorn inbreds. Our first objective was to locate genetic factors associated with trait variation and make first assessment on the efficiency of advanced backcross quantitative trait locus (AB-QTL) analysis for the identification and transfer of favorable QTL alleles for grain yield components from the dent corn inbred. A second objective was to compare the detection of QTL in the BC₂F₂ population with results using F_2:3 lines of the same parents. Two hundred and twenty selected BC₂F₂ families developed from a cross between Dan232 and an elite popcorn inbred N04 were evaluated for six grain yield components under two environments, and genotyped by means of 170 SSR markers. Using composite interval mapping (CIM), a total of 19 significant QTL were detected. Eighteen QTL had favorable alleles contributed by the dent corn parent Dan232. Sixteen of these favorable QTL alleles were not in the same or near marker intervals with QTL for popping characteristics. Six QTL were also detected in the F_2:3 population. Improved N04 could be developed from 210 and 208 families with higher grain weight per plant and/or 100-grain weight, respectively, and 35 families with the same or higher popping expansion volume than N04. In addition, near isogenic lines containing detected QTL (QTL-NILs) for grain weight per plant and/or 100-grain weight could be obtained from 12 families. Our study demonstrated that the AB-QTL method can be applied to identify and manipulate favorable QTL alleles from normal corn inbreds and combine QTL detection and popcorn breeding efficiently.     

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.     

Genetic basis of resistance to sugarcane mosaic virus in European maize germplasm

 Sugarcane mosaic virus (SCMV) causes considerable damage to maize (Zea mays L.) in Europe. The objective of the present study was to determine the genetic basis of resistance to SCMV in European maize germplasm and to compare it with that of U.S. inbred Pa405. Three resistant European inbreds D21, D32, and FAP1360A were crossed with four susceptible inbreds F7, KW1292, D408, and D145 to produce four F₂ populations and three backcrosses to the susceptible parent. Screening for SCMV resistance in parental inbreds and segregating generations was done in two field trials as well as under greenhouse conditions. RFLP markers umc85, bnl6.29, umc10, umc44, and SSR marker phi075 were used in F₂ populations or F₃ lines to locate the resistance gene(s) in the maize genome. Segregation in the F₂ and backcross generations fitted to different gene models depending on the environmental conditions and the genotype of the susceptible parent. In the field tests, resistance in the three resistant European inbreds seems to be controlled by two to three genes. Under greenhouse conditions, susceptibility to SCMV in D32 appears to be governed by one dominant and one recessive gene. Allelism tests indicated the presence of a common dominant gene (denoted as Scm1) in all three resistant European inbreds and Pa405. Marker analyses mapped two dominant genes: Scm1 on chromosome 6S and Scm2 on chromosome 3. Received: 17 November 1997 / Accepted: 25 November 1997     

Connected populations for detecting quantitative trait loci and testing for epistasis: an application in maize

Quantitative trait loci (QTL) detection experiments have often been restricted to large biallelic populations. Use of connected multiparental crosses has been proposed to increase the genetic variability addressed and to test for epistatic interactions between QTL and the genetic background. We present here the results of a QTL detection performed on six connected F₂ populations of 150 F_2:3 families each, derived from four maize inbreds and evaluated for three traits of agronomic interest. The QTL detection was carried out by composite interval mapping on each population separately, then on the global design either by taking into account the connections between populations or not. Epistatic interactions between loci and with the genetic background were tested. Taking into account the connections between populations increased the number of QTL detected and the accuracy of QTL position estimates. We detected many epistatic interactions, particularly for grain yield QTL (R ² increase of 9.6%). Use of connections for the QTL detection also allowed a global ranking of alleles at each QTL. Allelic relationships and epistasis both contribute to the lack of consistency for QTL positions observed among populations, in addition to the limited power of the tests. The potential benefit of assembling favorable alleles by marker-assisted selection are discussed.     

Comparative QTL mapping of resistance to Ustilago maydis across four populations of European flint-maize

 We mapped and characterized quantitative trait loci (QTLs) for resistance to Ustilago maydis and investigated their consistency across different flint-maize populations. Four independent populations, comprising 280 F₃ lines (A×B^I), 120 F₅ lines (A×B^II), 131 F₄ lines (A×C) and 133 F₄ lines (C×D), were produced from four European elite flint inbreds (A, B, C, D) and genotyped at 89, 151, 104, and 122 RFLP marker loci, respectively. All F_n lines were evaluated in field trials with two replications in five German environments. Genotypic variances were highly significant for the percentage of U. maydis infected plants (UST) in all populations, and heritabilities exceeded 0.69. Between five and ten QTLs were detected in individual populations by composite interval mapping, explaining between 39% and 58% of the phenotypic variance. These 19 different QTLs were distributed over all ten chromosomes without any clustering on certain chromosomes. In most cases, gene action was dominant or overdominant. Fourteen pairs of the detected QTLs for UST displayed significant digenic epistatic interactions, but only two of them did so after arcsin √UST/100 transformation. Significant QTL× environment interactions occurred frequently. Between two to four QTLs were common between pairs of populations. Population C×D was also grown in Chartres, a location with a high U. maydis incidence. Two out of six QTLs identified for Chartres were in common with QTLs detected across five German environments for C×D. Consequently, marker-assisted or phenotypic selection based on results from natural infection seem to be suitable breeding strategies for improving the resistance of maize to U. maydis. Received: 3 July 1998 / Accepted: 24 July 1998     

Parental contribution and coefficient of coancestry among maize inbreds: pedigree, RFLP, and SSR data

The genetic relationship between inbreds i and j can be estimated from pedigree or from molecular marker data. The objectives of this study were to: (1) determine whether pedigree, restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) data give similar estimates of parental contribution and coefficient of coancestry (f _ij ) among a set of maize (Zea mays L.) inbreds, and (2) compare the usefulness of RFLP and SSR markers for estimating genetic relationship. We studied 13 maize inbreds with known pedigrees. The inbreds were genotyped using 124 RFLP and 195 SSR markers. For each type of marker, parental contributions were estimated from marker similarity among an inbred and both of its parents, and were subsequently used to estimate f _ij . Estimates of parental contribution differed significantly (α<0.05) between pedigree data and either type of marker, but not between the marker systems. The RFLP estimates of parental contribution failed to sum to 1.0, reflecting a higher frequency of non-parental bands with RFLP than with SSR markers. The f _ij estimated from pedigree, RFLP, and SSR data were highly correlated (r=0.87–0.97), although significant differences were found among the three sets of f _ij estimates. We concluded that pedigree and marker data often lead to different estimates of parental contribution and f _ij , and that SSR markers are superior to RFLP markers for estimating genetic relationship. A relevant question is whether or not the inbreds previously genotyped with an older marker system (e.g., RFLP) need to be re-analyzed with a newer marker system (e.g., SSR) for the purpose of estimating genetic relationship. Such re-analysis seems unnecessary if data for the same type of marker are available for a given inbred and both of its parents. Received: 2 June 1999 / Accepted: 30 July 1999     

Identification of opaque-2 genotypes in segregating populations of Quality Protein Maize by analysis of restriction fragment length polymorphisms

Quality Protein Maize (QPM) is a name given to genetically modified opaque-2 maize with hard endosperm. The opaque-2 mutation conditions a reduction in the amount of zein seed storage protein; zeins are deficient in the essential amino acids lysine and tryptophan, and mutant seed have a higher nutritional value. To utilize the potential of opaque-2 maize, elite inbreds can be converted to o2/o2 forms and subsequently to hard endosperm opaque-2. Since opaque-2 is recessive and endosperm specific, conventional backcross procedures to convert elite inbreds to opaque-2 forms are inefficient. To alleviate this problem, a marker-assisted selection procedure was developed for the Texas A&M University Quality Protein Maize breeding program. Hybridization of an O2 cDNA probe to blots of DNA from plants carrying O2 and o2 alleles showed that restriction fragment length polymorphisms (RFLPs) exist between the W64A o2 allele and O2 alleles of Mo17 and TX5855 inbred lines. To identify the opaque2 genotypes in segregating populations, an RFLP marker assay combining the O2 cDNA probe and HindIII-digestion of genomic DNA was developed. The effectiveness of the O2 RFLP marker assay was tested under field conditions using F₂ and backcross populations of several hard endosperm opaque-2 lines. A comparison of the genotypes identified by RFLP analysis with the seed phenotypes of the next generation indicated that this procedure is accurate and can be used for identifying O2/O2, O2/o2, and o2/o2 genotypes of individual juvenile plants in breeding populations.     

Marker-based estimates of identity by descent and alikeness in state among maize inbreds

Molecular markers are useful for determining relationships and similarity among inbreds, especially if the proportion of marker loci with alleles common to inbreds i and j is partitioned into: (1) the probability that marker alleles are identical by descent (_Mf_ij); and (2) the conditional probability that marker alleles are alike in state, given that they are not identical by descent ( _ij). Our objectives were to: develop a method, based on tabular analysis of restriction fragment length polymorphism marker data, for estimating _Mf_ij, _ij, and the parental contribution to inbred progeny; validate the accuracy of the method with a simulated data set; and compare the pedigree-based coefficient of coancestry (f_ij) and _Mf_ij among a set of maize (Zea mays L.) inbreds. Banding patterns for 73 probeenzyme combinations were determined among 13 inbreds. Iterative estimation of _Mf_ij, _ij, and the parental contribution to progeny was performed with procedures similar to a tabular analysis of pedigree data. Deviations of _Mf_ij from pedigree-based f_ij ranged from 0.002 to 0.288, indicating large effects of selection and/or drift during inbreeding for some inbreds. Differences between marker-based estimates and expected values of parental contribution to inbred progeny were as large as 0.205. Results for a simulated set of inbreds indicated that tabular analysis of marker data provides more accurate estimates of _Mf_ij and _ij than other methods described in the literature. Tabular analysis requires the availability of marker data for all the progenitors of each inbred. When marker data are not available for the parents of a given inbred, _Mf_ij and _ij may still be calculated if parental contributions to the inbred are assumed equal to their expectations.     

Some practical considerations for using RFLP markers to aid in selection during inbreeding of maize

Summary If molecular markers are to be routinely used in maize (Zea mays L.) breeding for selection of quantitative trait loci (QTL), then consistent marker-trait associations across breeding populations are needed, as are efficient methods for weighting information from different markers. Given 15 restriction fragment length polymorphism (RFLP) markers associated with grain yield in testcrosses of 220 [BS11(FR)C7 x FRMol7] F₂ individuals to FRB73, separate weighting schemes were attempted in order to maximize the frequency of favorable marker genotypes associated with increased grain yield in selected F₂ individuals and F₂:S₄ Unes. The following principles were apparent: (1) Differential weighting among markers, in addition to weighting individual marker genotypes on the basis of associated mean effects, should be emphasized when using markers to select in breeding populations. This is due to limited population sizes that can readily be handled. (2) Relatively few markers may need to be used to screen segregating populations (e.g., F₂) of limited size for loci affecting complex traits, such as combining ability for grain yield, assuming prior knowledge of marker-QTL associations. Markers given greatest weight (largest estimates of associated effects) will determine most selections. (3) When marker-based selection is among individuals at higher levels of inbreeding (e.g., S₄) within selected families, more markers need to be used in screening because those associated with relatively small effects have an increased chance of affecting selection.These results suggest a qualitative approach for utilizing RFLP markers to aid in selection of complex traits in commercial hybrid maize breeding programs. Commercial research programs produce thousands of crosses each year aimed at inbred line development. Discovery of molecular markers with consistent QTL associations across breeding populations and close QTL linkages would allow for rapid screening of new F₂ populations at a few key markers. Early elimination of individuals with undesirable genotypes would reduce the extent of hybrid performance testing necessary during later stages of inbreeding.     

Prediction of testcross means and variances among F3 progenies of F1 crosses from testcross means and genetic distances of their parents in maize

 Prediction of the means and genetic variances in segregating generations could help to assess the breeding potential of base populations. In this study, we investigated whether the testcross (TC) means and variances of F₃ progenies from F₁ crosses in European maize can be predicted from the TC means of their parents and F₁ crosses and four measures of parental genetic divergence: genetic distance (GD) determined by 194 RFLP or 691 AFLP^{TM 1} markers, mid-parent heterosis (MPH), and absolute difference between the TC means of parents (∣P₁−P₂∣). The experimental materials comprised six sets of crosses; each set consisted of four elite inbreds from the flint or dent germplasm and the six possible F₁ crosses between them, which were evaluated for mid-parent heterosis. Testcross progenies of these materials and 20 random F₃ plants per F₁ cross were produced with a single-cross tester from the opposite heterotic group and evaluated in two environments. The characters studied were plant height, dry matter content and grain yield. The genetic distance between parent lines ranged between 0.17 and 0.70 for RFLPs and between 0.14 and 0.57 for AFLPs in the six sets. Testcross-means of parents, F₁ crosses, and F₃ populations averaged across the six crosses in a particular set generally agreed well for all three traits. Bartlett’s test revealed heterogeneous TC variances among the six crosses in all sets for plant height, in four sets for grain yield and in five sets for dry matter content. Correlations among the TC means of the parents, F₁ crosses, and F₃ populations were highly significant and positive for all traits. Estimates of the TC variance among F₃ progenies for the 36 crosses showed only low correlations with the four measures of parental genetic divergence for all traits. The results demonstrated that for our material, the TC means of the parents or the parental F₁ cross can be used as predictors for the TC means of F₃ populations. However, the prediction of the TC variance remains an unsolved problem. Received: 4 August 1997 / Accepted: 17 November 1997     

Quantitative trait locus analysis of a recombinant inbred line population derived from a Lycopersicon esculentum x Lycopersicon cheesmanii cross

Quantitative trait loci influencing fruit traits were identified by restriction fragment length polymorphism (RFLP) analysis in a population of recombinant inbred lines (RIL) derived from a cross of the cultivated tomato, Lycopersicon esculentum with a related wild species Lycopersicon cheesmanii. One hundred thirty-two polymorphic RFLP loci spaced throughout the tomato genome were scored for 97 F₈ RIL families. Fruit weight and soluble solids were measured in replicated trials during 1991 and 1992. Seed weight was measured in 1992. Significant (P<0.01 level) quantitative trait locus (QTL) associations of marker loci were identified for each trait. A total of 73 significant marker locus-trait associations were detected for the three traits measured. Fifty-three of these associations were for fruit weight and soluble solids, many of which involved marker loci signficantly associated with both traits. QTL with large effects on all three traits were detected on chromosome 6. Greater homozygosity at many loci in the RIL population as compared to F₂ populations and greater genomic coverage resulted in increased precision in the estimation of QTL effects, and large proportions of the total phenotypic variance were explained by marker class variation at significant marker loci for many traits. The RIL population was effective in detecting and discriminating among QTL for these traits previously identified in other investigations despite skewed segregation ratios at many marker loci. Large additive effects were measured at significant marker loci. Lower fruit weight, higher soluble solids, and lower seed weight were generally associated with RFLP alleles from theL. cheesmanii parent.     

Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize

The basic prerequisite for an efficient breeding program to improve levels of resistance to pathogens in plants is the identification of genes controlling the resistance character. If the response to pathogens is under the control of a multilocus system, the utilization of molecular markers becomes essential. Stalk and ear rot caused by Gibberella zeae is a widespread disease of corn: resistance to G. zeae is quantitatively inherited. Our experimental approach to understanding the genetic basis of resistance to Gibberella is to estimate the genetic linkage between available molecular markers and the character, measured as the amount of diseased tissue 40 days after inoculation of a suspension of Fusarium graminearum, the conidial form of G. zeae, into the first stalk internode. Sensitive and resistant parental inbreds were crossed to obtain F₁ and F₂ populations: the analysis of the segregation of 95 RFLP (restriction fragment length polymorphism) clones and 10 RAPD (random amplified polymorphic DNA) markers was performed on a population of 150 F₂ individuals. Analysis of resistance was performed on the F₃ families obtained by selfing the F₂ plants. Quantitative trait loci (QTL) detection was based either on analysis of regression coefficients between family mean value and allele values in the F₂ population, or by means of interval mapping, using MAPMAKER-QTL. A linkage map of maize was obtained, in which four to five genomic regions are shown to carry factors involved in the resistance to G. zeae.     

Molecular marker diversity among current and historical maize inbreds

Advanced-cycle pedigree breeding has caused maize (Zea mays L.) inbreds to become more-elite but more-narrow genetically. Our objectives were to evaluate the genetic distance among current and historical maize inbreds, and to estimate how much genetic diversity has been lost among current inbreds. We selected eight maize inbreds (B14, B37, B73, B84, Mo17, C103, Oh43 and H99) that largely represented the genetic background of current elite inbreds in the U.S. seed industry. A total of 32 other inbreds represented historical inbreds that were once important in maize breeding. Cluster analysis of the inbreds, using data for 83 SSR marker loci, agreed well with pedigree information. Inbreds from Iowa Stiff Stalk Synthetic (BSSS), Reid Yellow Dent, and Lancaster clustered into separate groups with only few exceptions. The average number of alleles per locus was 4.9 among all 40 inbreds and 3.2 among the eight current inbreds. The reduction in the number of alleles per locus was not solely due to sample size. The average genetic distance (D _ij ) was 0.65 among the eight current inbreds, 0.67 among the 32 historical inbreds, and 0.67 among all 40 inbreds. These differences were statistically insignificant. We conclude that genetic diversity among current inbreds has been reduced at the gene level but not at the population level. Hybrid breeding in maize maintained, rather than decreased, genetic diversity, at least during the initial subdivision of inbreds into BSSS and non-BSSS heterotic groups. We speculate, however, that exploiting other germplasm sources is necessary for sustaining long-term breeding progress in maize. Received: 21 August 2000 / Accepted: 5 January 2001     

Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize

The basic prerequisite for an efficient breeding program to improve levels of resistance to pathogens in plants is the identification of genes controlling the resistance character. If the response to pathogens is under the control of a multilocus system, the utilization of molecular markers becomes essential. Stalk and ear rot caused by Gibberella zeae is a widespread disease of corn: resistance to G. zeae is quantitatively inherited. Our experimental approach to understanding the genetic basis of resistance to Gibberella is to estimate the genetic linkage between available molecular markers and the character, measured as the amount of diseased tissue 40 days after inoculation of a suspension of Fusarium graminearum, the conidial form of G. zeae, into the first stalk internode. Sensitive and resistant parental inbreds were crossed to obtain F₁ and F₂ populations: the analysis of the segregation of 95 RFLP (restriction fragment length polymorphism) clones and 10 RAPD (random amplified polymorphic DNA) markers was performed on a population of 150 F₂ individuals. Analysis of resistance was performed on the F₃ families obtained by selfing the F₂ plants. Quantitative trait loci (QTL) detection was based either on analysis of regression coefficients between family mean value and allele values in the F₂ population, or by means of interval mapping, using MAPMAKER-QTL. A linkage map of maize was obtained, in which four to five genomic regions are shown to carry factors involved in the resistance to G. zeae.     

A ca. 40 kDa maize (Zea mays L.) embryo dehydrin is encoded by the dhn2 locus on chromosome 9

Dehydrins (LEA D11 proteins) are the products of multigene families in a number of higher plants [5]. To date, however, only one dehydrin locus, dhn1 (a major embryo and drought-induced protein of ca. 18 kDa) has been placed on chromosome 6L of the genetic linkage map of maize. The presence of a larger, ca. 40 kDa embryo protein that is also specifically detected by anti-dehydrin antibodies had been observed in some maize inbreds, including B73, suggesting that other dhn loci may exist. The ca. 22 kDa and ca. 40 kDa immunopositive proteins were purified from B73 and their amino acid compositions determined. The two proteins' amino acid compositions are typical of dehydrins, yet they differ from each other, indicating that they are distinct dhn gene products. Different size alleles for both proteins, or presence/absence in the case of the ca. 40 kDa protein, were evident from comparisons of embryo proteins of various maize inbreds. Analysis of segregating F₂ progeny derived from self-pollination of F₁ hybrids from four crosses (B73 × OH43, Mo17 × A632, AHO × A632, Latente × A632) revealed that alleles of the two genes assort independently. Map positions of the two dhn loci were then determined using two maize recombinant inbred line (RIL) mapping populations. The predicted map position of the gene controlling production of the ca. 22 kDa protein confirmed that this protein is the product of the dhn1 gene. The gene encoding the ca. 40 kDa dehydrin-like protein maps to a new locus on chromosome 9S near wx1, which we have named dhn2.     

Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of <Emphasis Type="Italic">crtRB1</Emphasis> and <Emphasis Type="Italic">lcyE</Emphasis> governing higher accumulation of provitamin A in maize

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC₁F₁, BC₂F₁ and BC₂F₂ revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.