Identification and Isolation of Genes Responsible for Drought Tolerance of Some Wheat Genotypes (Triticum Spp)

Fatemah Khaled AL-Ganeer
Department of Field Crops, General Commission for Scientific Agricultural Research (GCSAR)
Faculty of Agriculture, Damascus University
2017

Abstract

The research was carried out during the growing season 2012-2013 at Abu-Jerash farm, Faculty of Agriculture, Damascus, in order to evaluate the performance of nine genotypes of durum wheat (Cham3, Cham9, Douma 3, Gori, Hourani) and bread wheat (Douma 2, Douma 4, Cham 6, Cham 8), under rain-fed conditions. In addition to three types of wild gemus Aegilops namely Ae. Ovata, Ae.Triuncialis, Ae. Geniculata were used in the genetics study to study genetic diversity, and to determine the degree of genetic relationship among genotypes using SSR and ISSR techniques. The allelic variations of dihydrin genes responsible for drought tolerance were studied at the DNA level, in addition to determination the location of these genes which isolated to study
nucleotide sequence.
The results of the field study of growing season of durum and bread wheat varieties showed significant differences in the investigated traits. Bread wheat variety Douma-4was significantly superior in the mean values of number of fertile tillers/ total tillers (94.26%), length of the main spike (13.83 cm) , grain weight in the main spike (1.79 g), number of grains per plant (186 grains), and grain weight in the plant (5.46 g), while durum wheat variety Cham-9 was significantly superior in the mean values of plant height (77.31 cm), length of
the main spike (12.33 cm), number of grains (39 g), grain weight in the main spike (1.5 g), number of grains per plant (123.67), grain weight in the plant (4.12 g) and weight of 1000 kernels (31.42 g). and was closely followed by the variety Hawrani in most studied traits without significant differences among them.
The correlation between studied traits in durum wheat was studied, significant positive correlation was found between the height of the plant and the weight of the grains in the main spike, the number of grains in the plant and their weight, and the weight of 1000 grains (r = 0.753**, r = 0.883**, r = 0.828 **, r = 0.628 ** respectively). The ratio of number of fertile tillers/ total tillers was positively
and significantly correlated with the number of grains in the plant (r = 0.643 **).
The length of the main spike was positively and significantly correlated with the number of grains per spike and its weight, weight of the grains in the plant, weight of the thousand grains (r = 0.909 **, r = 0.799 **, r = 0.702 **, r = 0.918 **, respectively). Number of grains per plant was positively and significantly correlated with the weight of grains per plant (r = 0.826 **) and with the weight of 1000 grains (r = 0.660 **). The protein percentage was positively and significantly correlated with the number of fertile tillers (r = 0.594 *) and negatively correlated with length of main spike and number of grains per main spike (r = -0.807 **, r = -0.958 **, r = -0.705 **, respectively), and with the weight of the grains in the plant (r = -0.664) **), and weight of 1000 grains (r = -0.825 **). Same trends in the correlation among studied traits were recorded in bread wheat varieties.
In the genetic study conducted to determine the degree of genetic relationship between the studied wild and cultivated wheat cultivars using ISSR technique, 27 primers were used for this purpose, 13 of which proved their efficiency in giving polymorphism among studied genotypes, and 93 bands were resulted, Number of bands for each primers between 3 bands as the lowest number with
the two primers (ISSR-18, ISSR-35), and 10 bands with the primer ISSR-22, ISSR-43, with an average of 4.6 bands per primer. The meanpercentage of polymorphism was 79.6%. The lowest value of dissimilarity matrix (PDV) was 0.13 between the two cultivars (Cham 6 and Cham 8), indicating that they had a high degree of genetic relationship, while the highest value of PDV was 0.82
between the two genotypes Ae.geniculata and Douma 4, Indicating a significant genetic variation among them. The average polymorphic information content (PIC) was 0.249, where the used primers demonstrated their ability to distinguish between studied genotypes. A total of 31 unique bands were noticed,
of which 25 were found, and 6 were absent. These bands distinguished all wild and bread wheat genotypes. The genotype Douma-4 had largest number of unique bands (available and absent) with 11 bands. While the lowest number of unique bands in the genotype Cham 6 (2 bands).
In order to determine the degree of genetic relationship between wild and durum wheat cultivars, the use of these primers resulted in 90 bands, giving polymorphic percentage of 100%, and the number of bands for each primer between 2-bands as the lowest number with the two primers (ISSR-3, ISSR-37 ), to 10 bands as the highest number with the two primers (ISSR-32, ISSR-43), averaging 6.9 bands per primer. The values of the polymorphic information
content(PIC) fluctuated from 0.131 with the primer ISSR-41 as the lowest value and 0.351 with the primer ISSR-9 as the highest value with a grand mean of 0.205. It was noticed that there were 29 unique bands for the studied genotypes, of which 25 bands were found and 4 were missing. These bands distinguished all studied wild and durum wheat genotypes. The Hawrani genotype possessed the largest number of unique bands (existing and absent) with average of 6 bands, while the lowest number of unique bandswas noticed in the genotype Cham 3 (two bands).
In the study of genetic relationship between the studied genotypes by applying SSR-technique, 12 double primers were used and proved its efficiency in conferring amplification results in the wild and bread wheat varieties. The use of these primers excretes 49 alleles. These primers gave polymorphism of 97.29%.
The number of alleles for each primer fluctuated between I allele as the lowest number with the primer Dhn14, and 8 alleles with the primer Dhn9, with an average of 4.1 alleles per primer. The values of polymorphic information content (PIC) ranged from 0.30 with the primer Dhn.3 as the lowest value to 0.50 with the primers (Dhn3, Dhn6, Dhn14) as the highest values, with an overall mean of
0.45. There were 14 unique alleles (a marker) of the studied genotypes, of which 10 alleles were present and 4 absent.
The wild type Ae. geniculata had the largest number of unique alleles (existing and absent) with a rate of 5 alleles, while the lowest number of unique alleles found in the genotype Cham 8 and Douma 4 (one allele). The smallest value for PDV (0.25) was between Ae. Ovata and Ae. triuncialis, this indicate that they had a significant degree of genetic relationship, while the highest value of PDV (0.69) was between the two genotypes (Ae.geniculata and Douma 2), indicating a significant genetic variation between them.
In the determination of the degree of genetic relationship between wild and durum wheat varieties, the use of these primers resulted in 47 alleles, giving a polymorphism of 98.3%, the number of alleles for each primerranged between 1 allele as the lowest number with primer Dhn14 and 8 alleles as the highest number with the primer Dhn9, with an average of 3.9 alleles per primer. The values of polymorphic information content(PIC) ranged from 0.38 in the two primers (Dhn3, Dhn12) as the lowest value, to 0.50 in the primers (Dhn4, Dhn7, Dhn6, Dhn13) as the highest value, with an overall average of 0.46. A total of 13 unique alleles (marker) for the studied genotypes were found, out of which 9 alleles were present and 4 were absent. These alleles were distinguished all studied durum genotypes, except the genotype Douma-3 and the wild type Ae.
triuncialis, where the wild type Ae .geniculata had the largest number of unique alleles (existing and absent) with 5 bands, while the lowest number of unique alleles in the genotype Cham 9 and Cham 3 (one allele).The smallest value for PDV (0.25) was recorded between Ae. Ovata and Ae. triunciali, this indicate that
they had a great degree of genetic relationship, while the highest value of PDV (0.78) was recorded between the two genotypes (Ae. geniculata and Horani).

The comparison between SSR and ISSR techniques showed that the SSR-technique proved its efficiency in distinguishing between the studied genotypes.
In the study of allelic variations of dehydrin genes responsible for drought tolerance at the level of DNA, the study showed clear difference in these genes between the studied genotypes. The variations in molecular weight between the comparable of a single site were sometimes large and highly similar to others, and were easily discernible on the 4% metaphor agarose gel.
The PCR-reaction of the Dhn12 gene showed that there was only one pattern (A) in the genotypes Cham 6, Cham 8, Douma 4 and Douma 2. The Dhn6 gene gave two patterns (A and B) in the genotypes Cham8, Cham9, Douma4, Cham3, Horani, and Ae. Ovata. Three different patterns (A, B, C)of the Dhn3, Dhn4 and Dhn14 were observed in the studied genotypes and four patterns of the Dhn7 out of which 4 patterns were noticed in the genotype Douma3. PCR interaction also showed five patterns in the genes Dhn10, Dhn9, Dhn15, Dhn16, and 6 patterns for Dhn5 and Dhn13.
The results showed that the gene Dhn16 surpassed in the number of patterns (32)with all the studied genotypes, followed by Dhn9 with 30 patterns, while Dhn14 gave the lowest number of patterns (3 morphological patterns) with the studied genotypes. The results showed that the genotype Douma 4 was superior in the number of genetic patterns (24), followed by wild type Ae. geniculata with 20 genetic patterns, while the genotype Douma3 gave the lowest number of morphological patterns (13), The nucleotide sequence of the DNA fragment with a length of 600bp of the gene Dhn14 was also determined in the wild type Ae. Ovata, it was noticed that
they were similar to the gene Dhn14 in Triticum aestivum with a percentage of 99%, encodes a protein contained of 112 amino acid, it was similar to protein Cold shock protein in wild wheat Aegilops tauschii with a percentage of 99%.

Gene Dhn13 in the genotype Douma4 was found to be similar to the gene dehydrin HIRD11 in Aegilops tauschii with apercentage of 99%, encods a protein contained of 106 amino acid, and it was similar to protein dehydrin HIRD11 in Aegilops tauschii with a percentage of 100%. Gene Dhn12 in Horani was to be found similar to gene dehydrin Rab15 in Aegilops tauschii with a percentage of 99%, encodes a protein consisted of 149 amino acid. it was similar to protein dehydrin Rab15 in Aegilops tauschii with a percentage of 100%

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