A. Gallais

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 F2:3 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 (R2 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.

Genomic regions involved in response to grain yield selection at high and low nitrogen fertilization in maize

In order to validate the role of genomic regions involved in nitrogen use efficiency and detected in a population of recombinant inbred lines (RIL), we have applied from the same population a recurrent selection for adaptation to low N-input (N0) and to high N-input (N1). Variation of allele frequency at neutral marker during the two cycles of recurrent selection may provide information about markers linked to QTLs. Significant temporal variation of allele frequency was investigated using the test of Waples, which tests the hypothesis of genetic drift versus selection. Most genomic regions (12/19) responding to selection were detected for selection at high N-input and only two were common to selection at high and low N-inputs. This was consistent with the greater grain yield response to selection observed for the population selected under high N-input compared with the population selected under low N-input, when they were evaluated at high N-fertilization. In contrast, when they were evaluated at low N-input both types of selection gave similar yield. As was expected, in the first cycle we observed selection of markers linked to grain yield QTLs. In the course of the second cycle three situations were observed: the confirmation of most regions already selected in C1 including all C1 regions overlapping with grain yield QTLs; the non-confirmation of some C1 regions (2/9); and the identification of new genomic zones (10/17). The detected marker–QTL associations revealed the consistency of the involvement of some traits, such as root architecture and glutamine synthetase activity, which would be of major importance for grain yield setting whatever the nitrogen fertilization.

Genetic and Physiological Analysis of Germination Efficiency in Maize in Relation to Nitrogen Metabolism Reveals the Importance of Cytosolic Glutamine Synthetase

We have developed an approach combining physiology and quantitative genetics to enhance our understanding of nitrogen (N) metabolism during kernel germination. The physiological study highlighted the central role of glutamine (Gln) synthetase (GS) and Gln synthesis during this developmental process because a concomitant increase of both the enzyme activity and the amino acid content was observed. This result suggests that Gln is acting either as a sink for ammonium released during both storage protein degradation and amino acid deamination or as a source for amino acid de novo synthesis by transamination. In the two parental lines used for the quantitative genetics approach, we found that the increase in Gln occurred earlier in Io compared with F2, a result consistent with its faster germinating capacity. The genetic study was carried out on 140 F6 recombinant inbred lines derived from the cross between F2 and Io. Quantitative trait locus mapping identified three quantitative trait loci (QTLs) related to germination trait (T50, time at which 50% of the kernels germinated) that explain 18.2% of the phenotypic variance; three QTLs related to a trait linked to germination performance, kernel size/weight (thousand kernels weight), that explain 17% of the phenotypic variance; two QTLs related to GS activity at early stages of germination that explain 17.7% of the phenotypic variance; and one QTL related to GS activity at late stages of germination that explains 7.3% of the phenotypic variance. Coincidences of QTL for germination efficiency and its components with genes encoding cytosolic GS (GS1) and the corresponding enzyme activity were detected, confirming the important role of the enzyme during the germination process. A triple colocalization on chromosome 4 betweengln3 (a structural gene encoding GS1) and a QTL for GS activity and T50 was found; whereas on chromosome 5, a QTL for GS activity and thousand kernels weight colocalized withgln4, another structural gene encoding GS1. This observation suggests that for each gene, the corresponding enzyme activity is of major importance for germination efficiency either through the size of the grain or through its faster germinating capacity. Consistent with the possible nonoverlapping function of the two GS1 genes, we found that in the parental line Io, the expression ofGln3 was transiently enhanced during the first hours of germination, whereas that of gln4 was constitutive.

Genetic variation for N-remobilization and postsilking N-uptake in a set of maize recombinant inbred lines. 3. QTL detection and coincidences

The objective of this study was to map and characterize QTLs for traits related to nitrogen utilization efficiency (NUE), grain N yield, N-remobilization and post-silking N-uptake. Furthermore, to examine whether QTLs detected with recombinant inbred lines (RILs) crossed to a tester are common to those detected with line per se evaluation, both types of evaluations were developed from the same set of RILs. The material was studied over two years at high N-input, and one year at low N-input. We used 15N-labelling to evaluate with accuracy the proportion of N remobilized from stover to kernels and the proportion of postsilking N-uptake allocated to kernels. With 59 traits studied in three environments, 608 QTLs were detected. Using a method of QTL clustering, 72 clusters were identified, with few QTLs being specific to one environment or to the type of plant material (lines or testcross families). However, considering each trait separately, few QTLs were common to both line per se and testcross evaluation. This shows that genetic variability is expressed differently according to the type of progeny. Studies of coincidences among QTLs within the clusters showed an antagonism between N-remobilization and N-uptake in several QTL-clusters. QTLs for N-uptake, root system architecture and leaf greenness coincided positively in eight clusters. QTLs for remobilization mainly coincided in clusters with QTLs for leaf senescence. On the whole, sign of coincidences between QTLs underlined the role of a “stay-green” phenotype in favouring N-uptake capacity, and thus grain yield and N grain yield.

Experimental evaluation of several cycles of marker-assisted selection in maize

A program was initiated in 1994 to compare the efficiency of marker-assisted selection (MAS) and conventional phenotypic selection. A population of 300 F3:4 families was generated from the cross between two maize inbred lines F2 and F252 and selected on an index combining grain yield and grain moisture at harvest. This population was characterised for 93 RFLP markers and evaluated as testcrosses in a large range of environments. Three methods of selection were applied (i) two cycles of conventional phenotypic selection; (ii) two cycles of MAS based on an index combining phenotypic values and QTL genetic values and (iii) one cycle of combined MAS followed by two cycles of selection based only on the QTL effects estimated in the first generation. The different populations were characterised for RFLP markers. The evolution of allele frequencies showed that selection on only-markers was very efficient for fixing QTL alleles found favourable in the initial population. This evolution was quite different from that observed for phenotypic selection or combined MAS. Genetic gain was evaluated and found significant for each method of selection. Nevertheless, the difference between phenotypic selection and combined MAS was not significant. The two additional cycles of MAS on only-markers did not improve significantly the genetic value of the population. Moreover, the genetic variance of this population remained high, despite most of the QTL initially detected were almost fixed. The results suggest that the QTL effects estimated in the initial population were not stable due to epistasis and/or QTL by environment interactions.

The genetic basis of heterosis: multiparental quantitative trait loci mapping reveals contrasted levels of apparent overdominance among traits of agronomical interest in maize (Zea mays L.).

Understanding the genetic bases underlying heterosis is a major issue in maize (Zea mays L.). We extended the North Carolina design III (NCIII) by using three populations of recombinant inbred lines derived from three parental lines belonging to different heterotic pools, crossed with each parental line to obtain nine families of hybrids. A total of 1253 hybrids were evaluated for grain moisture, silking date, plant height, and grain yield. Quantitative trait loci (QTL) mapping was carried out on the six families obtained from crosses to parental lines following the “classical” NCIII method and with a multiparental connected model on the global design, adding the three families obtained from crosses to the nonparental line. Results of the QTL detection highlighted that most of the QTL detected for grain yield displayed apparent overdominance effects and limited differences between heterozygous genotypes, whereas for grain moisture predominance of additive effects was observed. For plant height and silking date results were intermediate. Except for grain yield, most of the QTL identified showed significant additive-by-additive epistatic interactions. High correlation observed between heterosis and the heterozygosity of hybrids at markers confirms the complex genetic basis and the role of dominance in heterosis. An important proportion of QTL detected were located close to the centromeres. We hypothesized that the lower recombination in these regions favors the detection of (i) linked QTL in repulsion phase, leading to apparent overdominance for heterotic traits and (ii) linked QTL in coupling phase, reinforcing apparent additive effects of linked QTL for the other traits.

Use of trial clustering to study QTL × environment effects for grain yield and related traits in maize

A population of 300 F3:4 lines derived from the cross between maize inbred lines F2 and F252 was evaluated for testcross value in a large range of environmental conditions (11 different locations in 2xa0years: 1995 and 1996) in order to study (1) the magnitude of genotypexa0×xa0environment and (2) the stability of quantitative trait loci (QTL) effects. Several agronomic traits were measured: dry grain yield (DGY), kernel weight, average number of kernels per plant, silking date (SD) and grain moisture at harvest. A large genotypexa0×xa0environment interaction was found, particularly for DGY. A hierarchical classification of trials and an additive main effects and multiplicative interaction (AMMI) model were carried out. Both methods led to the conclusion that trials could be partitioned into three groups consistent with (1) the year of experiment and (2) the water availability (irrigated vs non-irrigated) for the trials sown in 1995. QTL detection was carried out for all the traits in the different groups of trials. Between 9 and 15 QTL were detected for each trait. QTLxa0×xa0group and QTLxa0×xa0trial effects were tested and proved significant for a large proportion of QTL. QTL detection was also performed on coordinates on the first two principal components (PC) of the AMMI model. PC QTL were generally detected in areas where QTLxa0×xa0group and QTLxa0×xa0trial interactions were significant. A region located on chromosome 8 near an SD QTL seemed to play a key role in DGY stability. Our results confirm the key role of water availability and flowering earliness on grain yield stability in maize.

Detection of marker-QTL associations by studying change in marker frequencies with selection

The value of selective genotyping for the detection of QTL has already been studied from a theoretical point of view but with the assumption of a negligible contribution

A method of line development using doubled haploids: the single doubled haploid descent recurrent selection

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