Screening the Promising Wild Emmer Wheat Genotypes Containing the Yr-15 Gene in Türkiye and Syria Through Molecular Markers

Background Stripe rust also known as yellow rust is a most devastating fungal disease of wheat caused by Puccinia striiformis f. sp. tritici (Pst). The possibility of the breakdown of important stripe rust resistance genes and the threat of the emergence of aggressive new races remains a concern in all wheat-growing areas around the world. The presence of Yr15 resistant gene in the wild emmer wheat (Triticum turgidum ssp. dicoccoides) confers strong broad-spectrum resistance to Pst isolates. The aim of this present study was to investigate at molecular level to con�rm the presence or absence of the major resistance gene Yr15in tested samples. Methods and Results A total of 140 wild emmer wheat genotypes originating from Türkiye and Syria were screened using gene speci�c molecular markers Y15K1_F2/uhw30_1R. Ten promising wild emmer wheat genotypes Türkiye (Diyarbakır) and Syria (Al Qunay ţ irah) containing the Yr15 gene were identi�ed. Conclusion These �ndings can be bene�cial in wheat breeding programs to be conducted for resistance to stripe rust. As well, this study contributes to the evolutionary process studies related to Yr15 gene in wild emmer wheat populations of Fertile Crescent.


Introduction
Wheat is an important staple food crop of the peoples in various countries which is cultivated nearly 220 million hectares area of the world with 760 million tonnes of production (FAO, 2020).Due to its bene cial amino acid content, high commercial volume, role in crop rotation systems, sociocultural importance, and high adaptability to different climates, wheat has been a dispensable food for all growing countries.Today, Türkiye has 17.5 million tonnes of production and more than 67 million hectare of wheat-growing area (TUIK 2021).
Various biotic and abiotic stresses affect the yield of wheat every year, speci cally; the fungal pathogens that threaten the wheat cropss.Stripe rust of wheat is a serious disease caused by Puccinia striiformis f.sp.tritici, a biotrophic and heteroecious fungal pathogen.Severe losses in wheat due to this pathogen is predicted to be more than ve million tonnes every year which cost is counted to be nearly one billion US Dollars (Beddow et al. 2015).
Because the pathogen spread very rapidly, consequently the new emergence of Pst (Puccinia striiformis f.sp.tritici) races, studies on the development of resistant varieties have been prompted (Klymuk et al. 2018).Breeding stripe rust tolerant cultivar is one of the most e cient methods to combat this pathogen.The use of fewer fungicides due to tolerant cultivars will be environmentally friendly, also the costs for the fungicides will diminish (Turgay et al. 2022).
Currently, 83 designated, 63 provisionally designated genes and 61 quantitative trait loci (QTLs) have been described in wheat and its close relatives (Liu 2017, Jan et al. 2021).However, the changes in virulences in stripe rust pathogen and the epidemics occurring in genotypes/varieties containing a single major gene resulted in catastrophic losses (Ali et al. 2017; Chen and Khan 2017).This research explores the new resistance genes in wheat for rust.In particular, the use of wild relatives that consists of diverse valuable traits in breeding studies is getting widespread ( Many of the diverse wild emmer wheat and all other relatives in the rst tier of gene pools that constitute a modern wheat originated from Türkiye (Harlan, 1995;Van Zeitz and De Roller, 1995;Karagöz et al. 2010).However the studies of wild emmer wheat populations have been intensively done in the south Fertile Crescent (Israel, Lebanon, Jordan, and Syria).Therefore, this study has been conducted with materials-mostly from the northern Fertile Crescent (southeast Türkiye) and from Syria.A total of 140 genotypes were screened with molecular markers to check the presence of the Yr15 resistant genes.

Material And Method 1. Plant materials and isolation of DNA
The wild emmer wheat accessions were obtained from the Türkiye Seed Gene Bank of Field Crops Central Research Institute, Ankara, Türkiye (TGB), and the National Small Grains Collection (USDA), USA.The obtained seed from these gene banks were sown in the rst year for taxonomic veri cation.
The seeds were selected and separated morphologically by spike color, awns, and single spikes was obtained from these selected seeds.These single spikes collected isolated from the accessions grown for 2 years and 140 genotypes composing the materials of this study were acquired (Table .

88-2 Türkiye
Leaf tissue samples were collected from 140 genotypes frozen in liquid nitrogen and stored at -80°C.DNA was extracted according to the method described by Rogers and Bendich (1985).The quality and quantity of extracted DNA were checked by using 1% of agarose gel and compared to it with 50ng and 100ng lamda DNA for dilution preparations.1% of agarose gel showed clear bands of extracted DNA of wild emmer wheat (Figure1).

PCR analysis
DNA samples of 140 genotypes were used for the detection and to check the presence of Yr15 resistance gene s using gene-speci c primer sets (Y15K1_F2 5-GGAGATAGAGCACATTACAGAC-3 and uhw30_1R 5-TTTCGCATCCCACCCTACTG-3) of Yr15 (Klymiuk et al. 2018).Ampli cation of PCR was done by using the MyTaq TM 'Red DNA Polymerase' (Cat.No. BIO-21108) Bioline.A total of 10µL of PCR reaction mixture was prepared including 2µl of 5x Taq Red Reaction Buffer, 0.4µl of 10 µM each (F/R) primers, 0.05 µl of MyTaq Red DNA Polymerase, 1µl of DNA Template (80-100ng) and remaining water to complete the total volume of PCR reaction.Successful ampli cation of Yr15 resistant gene was done by using the PCR thermocycler conditions according to the manufacturing company of MyTaq TM 'Red DNA Polymerase including initial denaturation at 95 °C for 3min, followed by 35 cycles of 95°C for 15sec, 56°C for 15sec and 72°C for 10sec and there was no nal extension.The ampli ed product was con rmed by gel electrophoresis in 2% of agarose with the expected 993 bp band size of Yr15.

Results And Discussion
One hundred-forty (140) extracted DNA samples were tested to check the presence of Yr15 resistant gene.The rst two-lane after the ladder was positive control and the further 1 st arrow showed TGB DNA 84-1 and the 2 nd arrow showed USDA DNA 3 in 2% of agarose gel.Thirty-one out of 140 DNA samples were obtained from TGB and only one sample TGB genotypes number 84-1obtained from Türkiye/Diyarbakır showed Yr15 resistant gene with 993 bp amplicon (Figure2) (Table2).
In this study, we screened a total of 140 genotypes for the presence of Yr15 resistant gene using gene-speci c primers.The materials showed successful ampli cation of the expected fragment size (993 bp) in the PCR.The obtained result shows that 7.1% of the screening wild emmer wheat genotypes were positive for Yr15 resistant gene and the rest of the 92.9 % showed no ampli cation for Yr15 resistant gene.In total, 10 out of 140 genotypes was detected in Yr15 resistant gene and 5 of the10 genotypes with Yr15 resistant gene were determined from Türkiye/Diyarbakır and the rest of the 5 genotypes with Yr15 resistant gene were determined in Syria Al Qunayţirah (Table1and Table2 ).
Molecular study showed that wild emmer wheat is known as a species that is about 360,000 years old that was found somewhere in Fertile Crescent (Dvorak and Akhunov 2005).The rst yellow rust resistance Yr15 gene was discovered in1989 in accession G25 (G25) a wild emmer accession G-25 by Gerechter-Amitai.According to the current studies, the presence of Yr15 resistant gene was found in populations of Israel, Lebanon, Jordan, and Syria.However, while a majority of the materials in those studies belonged to Israel accessions, the number of materials from Türkiye, Iran and Iraq was limited.Most of the materials in this study is from Türkiye and there is no study done with a great number of material from Türkiye and Syria.Moreover, no previous record has been found that Yr15 resistant genes were detected from these regions.In conclusion, more genotypes needed to be screened in these regions, and as a result of these screenings, the results could be compared with sequence information related to Israel accessions, which detected Yr15 gene before.Therefore, this comparison will contribute to the evolutionary process of wild emmer wheat populations in Fertile Crescent.
In light of the promising results obtained from this study, genotypes found to have the Yr15 resistance gene can be considered as new genetic resources in breeding programs for the management of stripe rust disease.Breeding for stripe rust resistance will continue to be based on the current knowledge of stripe rust's variability, a commitment to research and commercial development of new and effective resistance combinations, and their use as the best way to control the disease.

discovered in Israel rst and then was widely found in Fertile Crescent (i.e. Israel, Jordan, Lebanon, Syria, east Türkiye, West Iran, and North Iraq) (Nevo et al. 2012) with reported various signi cant traits (Nevo 1983). Especially, G-25 accession of wild emmer wheat was tested on 20 different isolates from six countries and conferred a high resistance. Further studies concluded that this endurance was controlled by a gene called Yr15, found by Gerechter- Amitai et al. (1989).
Migicovsky and Myles 2017; McCouch et al. 2020) and of these, wild emmer wheat (Triticum turgidum ssp.dicoccoides) is known for important disease resistance traits (Klymiuk et al. 2022; Li et al. 2020; Oliver et al. 2007; Rahman et al. 2020).The genes detected in wild emmer wheat have been used to develop new durum and bread wheat varieties (Rahman et al. 2020; Yaniv et al. 2015; Mukhtar et al. 2015; Ipek 2022).The wild emmer wheat was 1and Table.2).Of 140 genotypes, 31 from TGB and 109 from USDA were obtained.Table1.Wild emmer wheat genotypes obtained from Green Global, USDA screened for Yr15 resistant gene Klymiuk et al. (2018)oseman et al. 1985;Nevo et al. 1991) Fertile Crescent including southeastern Türkiye(Ozkan et al. 2002;Luo et al. 2007).İntrogression of wild emmer disease-resistant genes to wheat and the development of resistant wheat varieties have an important role in today's wheat breeding studies(Perugini et al. 2008).It is also used as a valuable resource against many important diseases of wheat as well as yellow rust disease (Grama and Gerechter-Amitai 1974;Moseman et al. 1984;Moseman et al. 1985;Nevo et al. 1991).He et al. (2020) conducted a study to nd the Yr15 resistant gene from 361 wild emmer wheat accessions, 19 were originating from Türkiye, one from Lebanon, and one from Syria and rest of the materials were originating from Israel that screened using three pairs of gene-speci c primers for Yr15 gene.As a result of the study, only 13.6% of 361 wild emmer wheat accessions were positive for the Yr15 gene-speci c markers, and accessions from Lebanon, Syria, and Türkiye populations were Yr15 negative.According toKlymiuk et al. (2018), Yr15 was detected in 18% of the accessions from Israel, Lebanon, Jordan, but was not detected in Türkiye and Iran.They used as a material 19 wild emmer wheat originating from Türkiye and Iran, and 162 wild emmer wheat originating from Israel, Lebanon, Jordan, or Syria.