The Usage of Specific Markers for Some Major Traits in Egyptian Wheat (Triticum aestivum L.) and Their Wild Relatives

Wheat (Triticum aestivum) is one of the world’s major cereal crops. As the unique molecular make up of its grain allows its use as a primary structural ingredient of breads, pastas, tortillas, and other products worldwide. To achieve the food production levels needed to supply worldwide demands, plant breeders have focused on the development of agricultural varieties possessing two characters: high yield potential and high end-usequality. In order to meet the demands of the future populations, there is a need to develop new methods not only for increasing wheat yield, but also for increasing the utility and reliability of the resultant grain (Collard et al., 2005).

Wheat is a major crop for human consumption. Its importance hinges upon unique rheological properties of wheat flour which allow for the production baked goods. In recent years, wheat production has been increasing rapidly enough to keep pace with population growth, and is predicted to continue increasing at an average yearly rate of 1.9%, rising from 609 million tons in 1997 to a projected 641 million tons.

Genetic markers represent genetic differences between individual organisms. Generally, they do not represent the target genes them selves but act as ‘signs’or ‘flags’. Genetic markers that are located in close proximity to genes (i.e. tightly linked) may be referred to as gene ‘tags’. Such markers themselves do not affect the phenotype of the trait of interest because they are located only near or ‘linked’ to genes controlling the trait.

All genetic markers occupy specific genomic positions within chromosomes (like genes) called ‘loci’ (singular ‘locus’)(Hammer et al., 2000). Simple seqance repeat (SSR) markers are abundant, highly polymorphic, evenly distributed throughout the genome and require only small amounts of genomic DNA for analysis (Nicot et al., 2004).Further, SSR markers were shown to be successfully able across different wheat species, making them a powerful tool for population genetics and mapping studies in wild and cultivated wheat (Fahima et al., 2002). Recent studies on SSR markers published a high level of polymorphism among diploid wheat species (Hammer et al., 2000), tetraploid wild wheat accessions (Fahima et al., 2002) and hexaploid wheat varieties (Plaschke et al., 1995). Nader (2014) used 312 microsatellite markers to analyze DNA polymorphism of three Egyptian wheat aiming to develop specific molecular markers useful in future Egyptian wheat breeding programs.

A total of 477 fragments were detected and among 312 simple sequences repeat markers 162 were proved to be polymorphic. The percentage of genetic polymorphism ranged from 33% to 100 % and fragment size from 112 to 535 bp. The present reasarch aims to usage of some specific markers for major traits such as drought, aluminum tolerance, quality of gluten (Low and high molecular weight), fungal disease resistance (stem, stripe, leaf rust, pre harvest sprouting resistance and Fusarium head blight resistance in some Egyptian wheat (Triticum aestivum L.) and their wild relatives, and detect the genetic distance and similarity between the Egyptian wheat studied varieties based on SSR markers.

MATERIALS AND METHODS

The present research was carried out at the Faculty of Agriculture (Saba Basha), Alexandria University, Egypt during the seasons of 2014 up to 2016 to study the implementation of specific markers on some major traits in Egyptian wheat (Triticum aestivum L.)

Plant materials:

Five Egyptian bread wheat genotypes, Egypt 1, Gemmeiza 9 & 11, Sakha 93, Sids 1 and one wild wheat Aegilops ventricosa Tausch were used in the current experiment. Grain samples were obtained from Field Crops Research Institute, Agriculture Research Center, Giza.

Molecular analysis:

DNA Extraction:

Twenty known wheat DNA from USDA Genotyping Lab, Manhattan KS, USA were used as controls for different genes. Leaf tissue was collected from 14 d old seedlings into 96-well plate (1mL), dried for two d in a freeze drier (Thermo Fisher, Waltham, MA), and ground by shaking the plate containing a 3.2 mm metal bead in each well for 3 min at 25 times per second using a Mixer Mill (Retsch GmbH, Haan, Germany).

Genomic DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method (Saghai-Maroof et al., 1984). The quantity and quality of DNA were evaluated by running it in 0.8% agarose gel. Twenty SSR markers associated with important wheat genes were selected based on the previous reports (Tables 1 and 2).

Polymerase cycle reaction (PCR) amplifications were performed in a Tetrad Peltier DNA Engine (Bio-Rad Laboratories, Hercules, CA). A 13μl PCR mixture contained 1.0 μl of 10×NH₄ buffer (Bioline Inc. Taunton, MA), 2.50mM MgCl₂, 200μM each dNTP, 50nM forward-tailed primer, 90 nM reverse primer, 40 nM M13 fluorescent-dye-labeled primer, 1.0U of Taq DNA polymerase and 40ng of template DNA. Briefly, the reaction was incubated at 95°C for 5 min, and then continued for 5 cycles of 1 min at 96°C at 68°C with a decrease of 2°C in each subsequent cycle, and 1 min at 72°C. For another five cycles, the annealing temperature started at 58°C for 2 min, with a decrease of 2°C for each subsequent cycle.

Reactions then went through an additional 40 cycles of 1 min at 96°C for 2 min at 58°C, and 1 min at 72°C with a final extension at 72°C for 5 min. PCR products were separated in an ABI 3730 DNA analyzer (Applied Bio systems, Foster City, CA) and data were scored using Gene Marker (version 1.6; Soft Genetics LLC. State College, PA).

Table (1). SSR markers associated with important traits selected for the current study.

Table (2). Primers name and sequences of the SSR loci reaction.

(source:USDA Genotyping Lab, Manhattan KS, USA)

Data analysis:

Specific peaks for target genes were scored across all genotypes in addition to other unique peaks for susceptible genotypes of all the traits. Fragments scored as present/absent. Fragment scoring and lane matching performed automatically on digital images of the gels, using geneMarker programe.

RESULTS AND DISCUSSION

Specific peaks for target genes were scored across all genotypes in addition to other unique peaks for susceptible genotypes of all the traits. Polymorphic level in this study is high (90%) across all wheat accessions, especially between wild and domesticated wheat used in this study (Table 3). The high level of polymorphism could be attributed to selection of genotypes with diverse characteristics. These genotypes will be useful for developing mapping populations between wild and domesticated wheat. The polymorphism observed in this study represents inherent variability among genotypes at the DNA level. Microsatellite markers are becoming the markers of choice due to the level of polymorphism, as well as higher reliability (Plaschke et al., 1996 and Fu et al., 2005). In wheat, abundant wheat genomic SSR markers are now available and mapped, making them a useful resource for further studies.

A total of 110 alleles were detected (Table 3) among which 21 alleles (19%) were polymorphic and 89 alleles were specific for target genes (81%). For SSR, it is very common that each primer set amplifies multiple fragments and they are either different alleles in one locus or different loci. The result reveals significant differences in allelic diversity among wheat accessions studied. The fragment sizes ranged from 103 to 440bp. The study indicated the presence of specific markers in cultivated wheat using SSR markers. This opens up a possibility to apply marker-assisted selection (MAS) in developing new Egyptian wheat cultivars. The results showed one specific allele per locus, except some markers showed both resistant and susceptible allele (Table 3) while (Fahima et al., 1998) reported an average of 10 alleles per locus on some wild wheat accessions; also, Zeb et al. (2009) reported an average of 5.2 alleles per cultivar.

In addition, Salem et al. (2008) reported an average of 3.2 alleles from seven wheat cultivars. These allelic variations in different studies are mostly attributed to the kind of wheat genotypes for the mentioned studies (Abdel Tawab et al. 2003). The introduction of some traits into plants can be very difficult and expensive. Some important markers can be considered as a useful marker for screening some biotic and a biotic stress trait in the Egyptian wheat genotypes.

In many studies they are mostly attributed to the kind of wheat genotypes. Considerable amount of natural out crossing that occurs in wild wheat accessions and also the landraces which are selected from local germplasm have a wide range of diversity and thus will result in higher alleles (Salem et al. 2008). However, cultivars which are product of repeated inbreeding would have lower alleles than both of wild genotypes or landraces. The size of the detected alleles produced from using the SSR primer sets ranged from 59 to 635 bp (Table 3) which reflects not a large difference in the number of repeats between different alleles.

While, Salem et al. (2008) obtained an allelic size range between 77 to 266 bp on using 15 microsatellite markers on some wheat genotypes. In addition, Moghaieb et al. (2011) reported an allelic size range between 82 to 1620bp on using SSR markers associated with salt tolerance in Egyptian wheats. It should be noted that SSR markers can not only show different allelic variations in the same species but they are also able to assess even monoallelic differences in subspecies specifically (Naghavi et al. 2007).

The microsatellite variation is thought to be due to slippage of the DNA polymerase during replication of unequal crossing over resulting in differences in the copy number of the core nucleotide sequence. Data in Table 4 indicated that the six Egyptian bread and wild wheat were resistant for most traits especially for Fusarium head blight, Stripe rust resistance and Stem rust resistance. The results also showed that these genotypes have high protein content (HGPC). While in some traits such as pre harvest sprouting, all the genotypes were susceptible, on the other hand both resistant and susceptible were showed for drought tolerance, aluminum tolerance and leaf rust resistance (Table 4). These kinds of data can be very important for wheat breeders in Egypt.

The advantage of molecular markers for researchers is that they can test for a particular trait as early as in seeds of plants before they are planted. There is no longer a need for the plant to develop to a stage at which the trait can be observed, delay that in some cases can take many months. DNA markers have gradually been integrated into breeding programs, not as a big revolution replacing conventional breeding, but as an additional tool. This integration is only possible through a close interaction between breeders and molecular laboratory so that there is a mutual understanding of what is required for an optimised use of markers within the breeding schemes (Reynolds and Borlang, 2006). Several studies of molecular assisted-selection markers on wheat using different methods such as Abdel Tawab et al. (2003) detected five positive and negative RAPD markers for drought tolerant in Egyptian bread wheat. Moreover, the results are in line with those reported by Bruckner and Fraberg, (1987), Abdel–Bary et al. (2005), Rampino et al. (2006) and Alan, (2007) who assigned RAPD markers to drought stress tolerance in wheat genotypes using molecular markers. The present results also agree with those of Rashed et al. (2010), who indicates that there are potential markers to be used as marker assisted selection to improve drought stress tolerance by molecular breeding. Marker-assisted selection based on genotype mean performance will greatly increase breeding efficiency (Manavalan et al. 2009; Irada and Samira, 2010). Data in Figure (1) showed with using Glu-A3 marker for screening about the Gluten strength (Low Molecular Weight) for five Egyptian wheat and one wild type that Egypt 1, Gemmeiza 9 and 11 showed the specific peak at 108 bp comaring with with the published work. While with using Glu-A3d/Glu-A3 marker for Gluten strength (Low Molecular Weight) detect the specific peak on 128 bp for Egypt 1, Sakha 93 and gemmeiza 9 & 11, except the wild wheat Ae. verntricosa (Figure, 1). According to the data in Figure (5) with GWM0614/Lr17 marker, Gemmeiza 11, Sakha 93, Ae. ventricosa showing specific peak for leaf rust resistance at 168 bp. Gemmeiza 9 & 11, Sakha 93, Ae. ventricosa showed one specific peak at 363 bp with TSM0120/1RS Rye marker for Drought tolerance (Figure 2)

Figure (1). GeneMarker analysis for some wheat cultivars using Glu-A3 abd Glu-A3d/Glu-A3 markers showing specific peak for Gluten strength (Low Molecular Weight) and pblished in Theor Appl Genet (2004) 108:1409–1419.

Figure (2). GeneMarker analysis for some wheat cultivar such as Gemmeiza 11, Sakha 93, Ae. ventricosa using GWM0614/Lr17 marker showing specific peak for leaf rust resistance and pblished in Genetics 1998,149:2007–2023.

Figure (3). GeneMarker analysis for some wheat cultivar such as Sakha 93 and Ae. ventricosa using UMN25/Glu-D1 marker showing specific peak for Gluten strength (HMW) and pblished in Theor Appl Genet (2008) 118:177–183.

Table (3). Primers, chromosome locations, and specific SSR markers in some Egyptian wheat and their relatives

*Ae.vent: Aegilops ventricosa Tausch

Table (4). Resistance and susceptible genotypes for some traits in Egyptian wheat.

            *R: Resistance peak *S: susceptible peak *R/s: both peaks

Conclusion

The present research will be a useful reference and initial step for conventional plant breeders, physiologists, pathologists and other plant scientists in Egypt to decrease the cost and time on selecting the markers in the future studies.  This study aimed to develop molecular markers associated with some different traits in wheat using simple sequence repeat (SSR) markers and usefulness of these markers to detect possible specific markers to be utilized in the wheat future breeding programs in Egypt.