الفهرس 
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Abstract
The present work was carried out at the Biotechnology Laboratory, Crop Science Department, Faculty of Agriculture, EL-Shatby, Alexandria University and at Plant Pathology Department, Faculty of Agriculture, Damanhour University in the period from 2009 to 2014.
The main objectives of this study were:
1. Study the genetic nature of the resistance to Fusarium ear rot (F. verticillioides) in two maize populations.
2. Study the genetic nature of agronomic traits under Fusarium ear rot (F. verticillioides) incidence in two maize populations.
3. Identify SSR and STS markers linked to Fusarium ear rot (F. verticillioides) resistance genes in F2 population of maize, using bulked segregate analysis.
4. Map Fusarium ear rot (F. verticillioides) resistance genes in F2 population of maize.
Two maize populations (GE440 x Sd7 and NC300 x Gm1002) were used in this study. The populations were derived from the crosses of U. S. lines (GE440 and NC300) characterized by resistant to Fusarium ear rot with poor agronomic characteristics and two adapted Egyptian maize lines characterized by susceptible to Fusarium ear rot with a good agronomic quality elite inbreeds (Sd7 and Gm1002). The parental inbred lines, F1, and F2 plants for each cross were grown at the Experimental Farm Station, Faculty of Agriculture, Alexandria University, Alexandria, Egypt, during May- October 2012. The artificial infection was done using an isolate of F. verticillioides. The primary ear of each plant (Parents, F1 and F2) was inoculated by wounding silk channel inoculation assay followed by kernel inoculation method using a conidial suspension of 1 x 106 spores/ml. Infection severity was assessed visually using a 1–7 scale. Agronomic traits were evaluated under Fusarium ear rot incidence. Statistical and genetic analysis were done to study the mechanism of the inheritance of Fusarium ear rot resistance as well as studying the inheritance of agronomic traits. After that, QTLs for resistance to F. verticillioides was described in F2 maize population (NC300 x Gm1002). The population was genotyped by SSR and STS markers.
The results obtained from the genetic analysis of Fusarium ear rot (FER) resistance in two F2 maize populations could be summarized as follows:
For GE440 x Sd7 population, the two parental lines were clearly differentiated for resistance to FER. As expected, the resistance parent; GE440 had low FER severity with a mean ear rot ratings of 2.0. On the other hand, the susceptible parent; Sd7 had high FER severity with a mean ear rot ratings of 5.42. For NC300 x Gm1002 population, the two parental lines were less differentiated for resistance to FER. The resistance parent; NC300 had score mean of 2.79; while the susceptible parent; Gm1002 had score mean of 4.81.
Severity of FER from the F1 generation was lower than the mid- parent value in both populations indicating absence of dominance for genes governing resistance (partial dominance).
Negative mid-parent heterosis was observed (-16.58 % and -16.44 % for GE440 x Sd7 and NC300 x Gm1002 populations, respectively) illustrated that the F1 hybrids were more resistance than their mid- parent.
Inbreeding depression of 13.55 % and 33.44 % was observed for the GE440 x Sd7 and NC300 x Gm1002 populations, respectively. Positive value of inbreeding depression indicates the reduction in the resistance of F2 than F1 generation.
The degree of dominance (potence ratio) for Fusarium ear rot severity of GE440 x Sd7 population was 0.36; while on NC300 x Gm1002 population, it was about 0.62, indicating partial dominance for resistance in both populations
The frequency distribution of F2 generation of GE440 x Sd7 was found to be continuous, unimodal, with a higher frequency of resistance plants (skewed toward the resistant parent, GE440) indicating a preponderance of genes dominant for resistance. In case of NC300 x Gm1002, the F2 frequency distribution was found to be continuous and slightly skewed towards increased susceptibility (susceptible parent Gm1002).
Segregation in the F2 populations derived from the GE440 x Sd7 cross best fit a 13:3 (resistant: susceptible), indicating that two dominant genes were segregating (dominant recessive gene interaction). In NC300 x Gm1002 F2 population, a 9:7 (resistant: susceptible) ratio was observed which indicated that interaction between two genes (two dominant complementary genes) were needed for FER resistance.
Estimated minimum numbers of effective factors controlling resistance to Fusarium ear rot were 2 to 5 and 1 to 4 segregating genes for GE440 x Sd7 and NC300 x Gm1002 populations, respectively.
High coefficient of genotypic variance (GCV) was observed for both populations (27.27 % and 26.04 % for GE440 x Sd7 and NC300 x Gm1002, respectively). Similar pattern was recorded for phenotypic coefficient of variance (PCV) (36.11 % and 33.38 % for GE440 x Sd7 and NC300 x Gm1002, respectively). Magnitude of phenotypic coefficients of variance was greater than those of genotypic coefficient of variance indicating the influence of environment.
Broad- sense heritability values were moderately high in both populations (54.6 % and 60.83% for GE440 x Sd7 and NC300 x Gm1002, respectively) and allowed of genetic gains through the phenotypic selection of resistance individual in segregant generations.
High values of genetic advance as percent of mean (GAM) were observed for both populations (41.12 % and 41.38 % for GE440 x Sd7 and NC300 x Gm1002, respectively).
The results obtained from the genetic analysis of agronomic traits in two maize populations under Fusarium ear rot stress could be summarized as follows:
1- GE440 x Sd7 Population
The results clarified that the inbred line Sd7 had more plant height, ear height and number of rows per ear. However, GE440 had more number of ears per plant. Moreover, Sd7 was superior with respect to ear length, number of kernels per ear, ear diameter, 100 kernel weight, grain yield per main ear and grain yield per plant
F1 means were greater than the top parents for all agronomic traits. These results indicated over dominance for all agronomic traits.
F2 mean performances were greater than the top parents for all agronomic traits except ear length and 100 kernel weight. F2 means were lower than the F1 for all agronomic traits except plant height.
Data showed that heterosis over mid parent and better parent have positive values for all the agronomic traits. Also, negative values of inbreeding depression were observed for all agronomic traits except for plant height (0.71%).
The degree of dominance for all agronomic traits were larger than unity (> +1), and ranged from 1.13 for ear length to 17.0 for ear number per plant indicating over- dominance gene effects played a major role in inheritance of these traits.
The agronomic traits assessed in this study under Fusarium ear rot severity appeared to be quantitatively inherited as shown by the nearly continuous distribution of 259 F2 plants derived from GE440 x Sd7.
The results revealed that genotypic coefficient of variation (GCV) was less than its corresponding estimates of phenotypic coefficient of variation (PCV) for all agronomic traits which indicated significant role of environment in the expression of these traits.
Relatively high estimate of GCV for grain yield/ main ear was obtained (28.44%) suggest that the selection can be effective for grain yield/ main ear trait under Fusarium ear rot severity. Grain yield per plant (19.44% and 23.99%), 100 kernel weight (16.78 and 21.57%), No. of Kernels per row (16.25 and 19.67%), and number of ears per plant (14.46 and 30.43%) showed moderate to high variability for GCV and PCV, respectively.
Broad-sense heritability estimates ranged from 22% (number of ears per plant) to 73% (ear diameter). The highest values of heritability were recorded for ear diameter (73.33%), ear height (71.48%), number of Kernels per row (68.25%), grain yield per plant (65.65%) and plant height (62.62%%), which allowed of genetic gains through the phenotypic selection of superior individual in segregant generations.
The genetic advance as percent of mean (GAM) values ranged from 12.62% (number of rows per ear) to 44.99 (grain yield of the main ear (infected ear). The GAM for grain yield of the main ear (44.99 %), Grain yield per plant (32.45 %), No. of Kernels per row (27.66%) and 100 kernel weight (24.99 %) were high.
The data for correlation studies F2 plants showed that grain yield per main ear was positively and highly significantly associated with other nine agronomic traits. Also, grain yield per plant was positively and highly significantly associated with other nine agronomic traits.
Disease severity rate exhibited negative and highly significant correlation with ear height (-0.18), 100 kernel weight (-0.37) and grain yield of the main ear (-0.24). However, it was negative and significant correlation with grain yield per plant (-0.12).
2- NC300 x Gm1002 Population.
The obtained results revealed that the inbred line Gm1002 was superior with respect to all agronomic traits except ear diameter and100 kernel weight in comparison with the inbred line NC300
F1 means were greater than the top parent for all agronomic traits except for number of ears per plant which was higher than the lowest parent. These results indicated over dominance for all agronomic traits except number of ears per plant which was partial dominance.
F2 mean performances were greater than the top parents for all agronomic traits except ear height, number of ears and 100 kernel weight.
Data showed that heterosis over mid parent and better parent have positive values for all the agronomic traits except for number of ear per plant which had a negative better parent heterosis (-8.53%). The mid parent heteosis ranged from 3.06 % for the number of ears per plant to 321.71% for grain yield per main ear. Also, the better parent heterosis ranged from -8.53% for number of ear per plant to 290.10% for per main ear.
Negative values of inbreeding depression were observed for all agronomic traits except for the number of ears per plant (2.54%). The lowest value of inbreeding depression was found for the number of rows per ear (-6.71%), while, the highest inbreeding depression was found for grain yield of the main ear (-52.08%).
The degree of dominance (potence ratio) for all agronomic traits except for the number of ears per plant were larger than unity (> +1), and ranged from 1.36 for ear height to 39.77 for grain yield of the main ear (infected ear) indicating over- dominance gene effects played a major role in inheritance of these traits.
The agronomic traits assessed in this study under Fusarium ear rot severity appeared to be quantitatively inherited as shown by the nearly continuous distribution of 280 F2 plants derived from NC300 x Gm1002. The continuous distribution of the agronomic traits indicated that those traits should be polygenic in nature.
Relatively high estimates of GCV for grain yield per plant (32.22%), grain yield of the main ear (26.73%) and number of ears per plant (23.38%) were obtained suggest that the selection can be effective for grain yield and prolific traits under Fusarium ear rot severity.
Broad-sense heritability estimates ranged from 28% (ear diameter) to 61.84% (ear length). The highest values of heritability were recorded for ear length (61.84%), ear height (48.45%) and number of ears per plant (40%), which allowed of genetic gains through the phenotypic selection of superior individual in segregant generations.
The genetic advance as percent of mean (GAM) values ranged from 0.33% (number of ear per plant) to 37.49% (grain yield per plant). The GAM for grain yield per plant (37.49%), grain yield of the main ear (29.64 %; infected ear) and ear length (23.7%) were high.
The greatest value of phenotypic coefficient of correlation was obtained between grain yield per plant and 100 kernels weight and between ear length and number of kernels per row (rp = 0.79).
Disease severity rate exhibited negative and highly significant correlation with plant height (-0.19), ear height (-0.14), ear length (-0.17), number of kernel per row (-0.20), ear diameter (-0.17), 100 kernel weight (-0.68), grain yield of the main ear (-0.44) and grain yield per plant (-0.29).
The results obtained from Identification of molecular markers linked to Fusarium ear rot resistance region could be summarized as follows:
1- SSR marker analysis
Out of 39 SSR primers, screened for polymorphisms between the two parents, NC300 (resistant) and Gm1002 (susceptible), 14 SSR primers (35.89 %) that gave polymorphic bands suitable to differentiate between the two parents were identified.
Each of these 14 markers was used to screen DNA bulks of the 14 resistant and the 14 susceptible F2 plants. SSR markers bnlg1063, umc2082, bnlg1621, umc2013, bnlg1740, umc2059 and SSR85 were, only, amplified polymorphic bands. These SSR markers were regarded as candidate markers linked to Fusarium ear rot resistance genes in maize.
The co-dominant microsatellite markers (bnlg1063, umc2082, bnlg1621, umc2013, bnlg1740, umc2059 and SSR85) were able to identify the heterozygotes. The segregation ratio was 1 (resistant homozygote): 2 (heterozygote): 1(susceptible homozygote) in the genotyping F2 plants. The ratio fitted the expected Mendalian ratio, 1:2:1.
The regression analysis for the relationship between presence of the markers, bnlg1063, umc2082, bnlg1621, umc2013, bnlg1740, umc2059 and SSR85 and Fusarium ear rot resistance phenotypes of F2 individuals were significant and they recorded, r2 = 0.46, 0.41, 0.43, 0.36, 0.42, 0.34, 0.28 and 0.30, respectively. This indicates that the SSR markers were linked with Fusarium ear rot resistance genes.
2- STS marker analysis
One of the eight STS primers; namely, STS06, revealed polymorphism. The STS06 primer generate two polymorphic fragments, one fragment at 300 bp which was present only in the resistant bulk and NC300 (resistant parent) and another polymorphic fragment at 270 bp was present only in susceptible bulk and Gm1002 (susceptible parent). These STS markers were regarded as candidate markers linked to Fusarium ear rot resistance genes in maize.
The co-dominant STS marker (STS06) was able to identify the heterozygotes. The segregation ratio was 1 (resistant homozygote): 2 (heterozygote): 1 (susceptible homozygote) in the genotyping F2 plants. The ratio fitted the expected Mendalian ratio, 1:2:1.
The regression analysis for the relationship between presence of the marker, STS06 and Fusarium ear rot resistance phenotypes of F2 individuals were significant and they recorded, r2 = 0.30. This indicates that the STS marker (STS06) was linked with Fusarium ear rot resistance genes.
The results obtained from Mapping of QTLs for Fusarium ear rot resistance could be summarized as follows:
The genetic distance between the eight markers (7 SSR + 1 STS) and Fusarium ear rot resistance genes were determined as 16.8, 20.7, 17.1, 26.7, 17.6, 26.9, 29.4 and 26.4 cM, respectively, with LOD scores of 36.4, 26.7, 33.7, 18.9, 33.2, 18.1, 13.5, and 16.5, respectively. Therefore, SSR markers bnlg1063, umc2082, bnlg1621, umc2013, bnlg1740, umc2059 and SSR85 and STS marker (STS06) were linked to the quantitative trait loci (QTL) for Fusarium ear rot resistance genes.
The ANOVA on SSR markers (bnlg1063, umc2082, bnlg1621, umc2013, bnlg1740, umc2059 and SSR85) and STS marker (STS06) and genotypes as groups for the Fusarium ear rot resistance established high significant association between SSR markers and STS marker and phenotype.
The single marker ANOVA analysis revealed that the bnlg1063-linked QTL, umc2082-linked QTL, bnlg1621-linked QTL, umc2013-linked QTL, bnlg1740-linked QTL, umc2059-linked QTL and SSR45-linked QTL accounted for 24%, 23%, 26%, 17%, 32%, 16%, 17 and 13% of the total phenotypic variation, respectively, in Fusarium ear rot resistance in the F2 population.
In all of the detected QTLs, additive effects were negative. This indicates contribution of QTL alleles in these loci from the resistant parent NC300.
The results obtained from QTL analysis for agronomic traits in the NC300 × Gm1002 population under Fusarium ear rot severity could be summarized as follows:
QTLs were detected for 100- kernel weight, grain yield/ main ear and grain yield per plant traits by CIM. On the other hand, no QTL were detected for the remaining agronomic traits.
A total of 7 QTLs were identified, ranging from 2 to 7 QTLs for each trait. Two QTLs (bins 3.06 and 6.07) were putatively consistent QTL for 100- kernel weight, grain yield/ main ear and grain yield per plant traits.
Seven QTLs were detected for 100- kernel weight, explaining from 5 to 18% of the phenotypic variance with a corresponding LOD of 3.3– 7.2. The seven QTLs were distributed on five chromosomes; one QTL on each of chromosome 3, 5 and 10, and two QTLs on each of chromosome 4 and 6.
Two QTLs, distributed on two chromosomes, were significantly associated with grain yield/ main ear.
Two QTLs; bnlg1063- 3.06 and bnlg1740- 6.07 for grain yield were detected and explained 3 to 4% of the phenotypic variance, with LOD scores of 3.0.
The present study indicated that SSR markers and STS markers, combined with bulked segregant analysis, could be used to identify molecular markers linked to Fusarium ear rot resistance genes in maize and suggested that marker-assisted selection with microsatellite and STS primers might be useful for developing improved cultivars.