Evaluation of Genetic Diversity in Wild Emmer Wheat (Triticum dicoccoides) and Durum Wheat (Triticum durum) Accessions Using Caat and SCoT Markers

1. Introduction

Analysis of genetic diversity within and between plant species is crucial for the success of any breeding program (Poehlman et al., 1995). Various marker technologies (RAPD, ISSR, AFLP, SSR, etc.) are widely used for genetic diversity analysis and QTL mapping (Nadeem et al.,2018). Molecular markers have been successfully used to study genetic diversity and population structure of agriculturally important crops. The assessment of genetic diversity resulting from the identification of novel genes or alleles in historical varieties, landraces, and natural populations enhances the efficacy of breeding programs by creating varieties with the necessary traits and offers new ways to locate keystone genes (Dempewolf et al., 2017; El Haddad, Kabbaj, et al., 2021; El Haddad, Sanchez-Garcia, et al., 2021). In recent years, many novel alternatives and promising marker techniques have emerged (Bertrand et al 2009). The genetic diversity in wild emmer has been thoroughly studied using diverse molecular marker techniques such as AFLP, ISSR, SSR, SNP, retransposons, etc. Start codon-targeted (ScoT), a unique molecular marker technology with high polymorphism and effectiveness, has been effectively applied in several crops, including rice, peanut, mango, potato, and grape (Collard et al 2009; Xiong et al 2011; Luo et al. 2010, 2011, 2012, Gorji et al 2011, Guo et al 2012). Studies by Joshi et al. (1997) and Sawant et al. (1999) have previously reported the existence of SCoT polymorphism, a brief conserved area that surrounds the ATG start codon in plant genes. ATG flanking the promoter initiation codon was the target of a single primer designed to amplify the genomic region containing the conserved sequence for SCoT. Recently, gene-targeted marker techniques have become an excellent and useful system for analyzing genetic diversity. CAAT box-derived polymorphism (CBDP) is also known as a novel molecular marker based on the short-conserved region of the CAAT box promoter. Singh et al (2014) reported that eukaryotic genes share a conserved sequence in the CAAT box region upstream of the start codon (GGCCAATCT). Due to their many advantages, including reproducibility, high polymorphism, and low cost, CAAT markers have been successfully used in genetic analyses (Aslan-Parviz, 2020). Additionally, the CAAT approach is anticipated to be more dependable and repeatable than random molecular markers since it uses longer primers with a higher annealing temperature (Collard et al 2009). In a similar vein, SCoT markers are widely applicable to many noteworthy but undervalued plant species. These applications include a wide range of subjects, including genetic diversity analysis, species, hybrid, and cultivar identification, gender determination, genetic relationships between different species, linkage map construction, association mapping analysis, differential gene expression investigations, and genetic evaluation of plants cultured in tissue. The information supplied includes current information on SCoT markers and their application in a variety of commercially significant plant species, encompassing both well-known and lesser-known species and cultivars (Rai, 2023). Consequently, these marker techniques may offer advantages over random techniques in QTL mapping and DNA fingerprinting applications (Andersen et al 2003, Etminan 2018). This study attempts to uncover unique genetic diversity in a panel of selected wild emmer (Triticum dicoccoides) accessions, utilizing two successful marker approaches, CAAT and SCoT.

2. Material and Method

2.1. Plant Material

In this study, forty-three genotypes selected from different populations of wild emmer wheat (Triticum dicoccoides) collected from the distinct regions of the Fertile Crescent (Turkey, Israel, Lebanon, and Syria), and five genotypes of durum wheat (Triticum durum) were used as plant material (Table1). The seeds were sown in the 2021-2022 wheat growing season. The field study was conducted at Çukurova University's Department of Field Crops.

2.2. DNA Extraction

Fresh and young leaf samples were collected at the seedling stage and dried in a lyophilizer (Dryfreezer). The dried leaf samples were stored at +4 °C until DNA isolation. Genomic DNA was extracted according to the CTAB protocol (Doyle and Doyle, 1987; Kafkas et al. 2006). The leaf samples were ground in a mortar with the help of liquid nitrogen and were transferred to 2 ml Eppendorf tubes. Afterwards, 1 ml of CTAB DNA isolation solution was added and the tubes were kept in a water bath at 65°C for 1.5 hours. The tubes were slowly shaken every 10 minutes during the water bath. The tubes were then taken out and left to cool at room temperature (approximately 5 minutes) and then 0.5 ml of chloroform: isoamyl alcohol (24:1) was added to these tubes and shaken gently by hand for 15 minutes. Afterward, the tubes were centrifuged at 135000 rpm for 10 minutes. The upper phases of the centrifuged tubes were taken and transferred to new 1.5 ml Eppendorf tubes. 500 μl of isopropanol was added to these tubes and the DNA was precipitated by slowly turning it into a single phase. All the liquid was poured so that the DNA remained at the bottom and the DNA sample was washed with 76% ethyl alcohol containing 10mM ammonium acetate and the samples were dried overnight at room temperature. After drying, 50 μl of ddH₂0 (double distilled water) was added to the DNA samples, and the dissolved DNA samples were stored at -20°C. To determine the DNA quality and the concentration of the DNA samples, 2 μl of DNA sample was taken into a 0.5 ml Eppendorf tube and 4 μl of 6X loading buffer and 14 μl of ddH₂0 were added. The mixture was vortexed and then centrifuged for a short time. 10 μl of the sample taken from these prepared samples was loaded in 0.8% agarose gel in 0.5X TBE (Tris-borate-EDTA) solution. The loaded samples run at 90 volts for 60 minutes. After the run, agarose gel was stained with ethidium bromide and visualized with the aid of gel imaging. DNA concentrations were determined with the help of λ DNA (25 ng - 50 ng - 100 ng - 200 ng) loaded in the gel. The final concentration of DNA samples was set at 10 ng/µl. DNA quality was determined by 0.8% agarose gel electrophoresis.

Table 1. Wild Emmer Wheat (Triticum dicoccoides) genotypes were collected from the fertile crescent region and durum wheat genotypes.

Accession ID	Plant Species	Genbank Code	Country Of Origin	Collsite	Lat (N)	Long (E)	Elevation
WE 270	T. dicoccoides	PI 428089	TUR	37 km NE from kilis to Gaziantep	37°20'19''	37°16'50''	830
WE 5	T. dicoccoides	PI 428023	TUR	36.2 km west of Diyarbakir in the Karacadag	37°53'00''	39°52'00''	1200
WE 230	T. dicoccoides	PI 428046	TUR	12.9 km NW from Ovadag to Pirinclik	37°47'31''	39°57'18''	1007
WE 28	T. dicoccoides	PI 428089	TUR	20.2 km east of Siverek	37°43'00''	39°30'00''	1200
WE 262	T. dicoccoides	PI 656872	TUR	34 km ESE from Narli (WSW of Karadağ)	37°20'12''	37°17'53''	780 (813)
WE 14	T. dicoccoides	PI 428051	TUR	36.2 km west of Diyarbakir in the Karacadag	37°53’00’’	39°52’00’’	1200
WE 264	T. dicoccoides		TUR	37 km NE from Kilis to Gaziantep	37°20'19''	37°16'50''	830
WE 274	T. dicoccoides		TUR	Siverek Karakeçi Road Azemi Village	37° 36' 51''	39° 20' 12''	733
WE 164	T. dicoccoides	PI 654321	TUR	4km south of Siverek on Karakecili road	37° 43’05’’	39° 19’37’’	720
WE 157	T. dicoccoides	PI 554581	TUR	25 km southwest of Diyarbakir	37° 45’00’’	40° 06’00’’	1000
WE 114	T. dicoccoides	PI 538646	TUR	36.2 km west of Diyarbakir in the Karacadag	37° 53’00’’	39° 52’00’’	1200
WE 159	T. dicoccoides	PI 554583	TUR	3 km southeast of the Junction of Karacadag Mt. road and Diyarbakir highway	37° 47’00’’	39° 47’00’’	1350
WE 154	T. dicoccoides	PI 538713	LBN	between Ain Harsch and Ain Ata	33° 26’00’’	35° 46’00’’	1192
WE 169	T. dicoccoides	TRI 18478, PI 427998	LBN	zwischen Kfarkouk und Aiha	33° 31’00’’	35° 52’00’’	1216
WE 193	T. dicoccoides	TRI 18530, PI 538706	LBN	zwischen Aiha und Kfarkouk, ca. 1 km von Aiha	33° 30’00’’	35° 52’00’’	1216
WE 150	T. dicoccoides	PI 538703	LBN	near Rashaya	33° 30’04’’	35° 50’22’’	1000
WE 41	T. dicoccoides	PI 428133	LBN	Aiha-Kfarkouk, above 'sahlet'	33° 31’00’’	35° 52’00’’	1141
WE 149	T. dicoccoides	PI 538702	LBN	near Rashaya	33° 30’04’’	35° 50’22’’	1000
WE 151	T. dicoccoides	PI 538704	LBN	near Rashaya	33° 30’04’’	35° 50’22’’	1000
WE 46	T. dicoccoides	PI 428143	LBN	between Rashaya and Aiha	33° 30’00’’	35° 50’00’’	1000
WE 1	T. dicoccoides	CItr 17675	LBN	outskirts of Rashaya	33° 30’04’’	35° 50’22’’	1000
WE 179	T. dicoccoides	TRI 18499, PI 470979	LBN	Mt. Hermon	33° 25’00’’	35° 52’00’’	2655
WE 34	T. dicoccoides	PI 428105	ISR	1 to 2 km south of Rosh Pinna towards Safad	32° 58’00’’	35° 32’00’’	549
WE 145	T. dicoccoides	PI 538696	ISR	Between 'En haShofet and Daliyya	32° 35’00’’	35° 03’00’’	122
WE 127	T. dicoccoides	PI 538670	ISR	Afula-Tiberias	32° 36’40’’	35° 17’30’’	300
WE 139	T. dicoccoides	PI 538690	ISR	near Safad on the road to Rosh Pinna	32° 58’00’’	35° 29’40’’	800
WE 134	T. dicoccoides	PI 538679	ISR	Afula-Tiberias	32° 36’40’’	35° 17’30’’	300
WE 61	T. dicoccoides	PI 466972	ISR	Bat Shelomo	32° 35’48’’	35° 00’07’’	105
WE 59	T. dicoccoides	PI 466969	ISR	Bat Shelomo	32° 35’48’’	35° 00’07’’	105
WE 62	T. dicoccoides	PI 466974	ISR	Bat Shelomo	32° 35’48’’	35° 00’07’’	105
WE 267	T. dicoccoides	PI 538696	ISR	Between 'En haShofet and Daliyya	32° 35’00’’	35° 03’00’’	122
WE 35	T. dicoccoides	PI 428112	ISR	1 to 2 km south of Rosh Pinna towards Safad	32° 58’00’’	35° 32’00’’	549
WE 87	T. dicoccoides	PI 471041	ISR	Kokhav haShahar	31° 57’00’’	35° 20’00’’	696
WE 51	T. dicoccoides	PI 466943	SYR	Kazrin	32° 59’24’’	35° 41’24’’	259
WE 80	T. dicoccoides	PI 470956	SYR	Kazrin	32° 59’24’’	35° 41’24’’	259
WE 184	T. dicoccoides	TRI 18506, PI487255	SYR	Damaskus Provinz	33° 45’00’’	36° 05’00’’	1240
WE 186	T. dicoccoides	TRI 18508, PI 487262	SYR	Damaskus Provinz	33° 40’00’’	36° 02’00’’	1300
WE 96	T. dicoccoides	PI 487260	SYR	32km from Sweida between Sale and Malah	32° 38’52’’	36° 47’24’’	1530
WE 93	T. dicoccoides	PI 487254	SYR	Nawa	32° 52’10’’	36° 01’51’’	551
WE 98	T. dicoccoides	PI 487264	SYR	Aleppo-Abeen road after Aleppo-Afrin road, Aleppo Province	36° 30’00’’	37° 00’00’’	350
WE 76	T. dicoccoides	PI 470945	SYR	Kazrin	32° 59’24’’	35° 41’24’’	259
WE 185	T. dicoccoides	TRI 18507, PI 487261	SYR	Es Suweida (Soud)	32° 38’00’’	36° 46’00’’	1450
WE 292	T.durum	PI 466947		Iran
WE 294	T. durum	Buckzafıro		Arj
WE 293	T. durum	PI 656872	TUR	112km northwest of Maras
WE 291	T. durum	PI 654317	TUR
WE 289	T. durum	Landrace		Italy

Table 1. Wild Emmer Wheat (Triticum dicoccoides) genotypes were collected from the fertile crescent region and durum wheat genotypes.

Accession ID	Plant Species	Genbank Code	Country Of Origin	Collsite	Lat (N)	Long (E)	Elevation
WE 270	T. dicoccoides	PI 428089	TUR	37 km NE from kilis to Gaziantep	37°20'19''	37°16'50''	830
WE 5	T. dicoccoides	PI 428023	TUR	36.2 km west of Diyarbakir in the Karacadag	37°53'00''	39°52'00''	1200
WE 230	T. dicoccoides	PI 428046	TUR	12.9 km NW from Ovadag to Pirinclik	37°47'31''	39°57'18''	1007
WE 28	T. dicoccoides	PI 428089	TUR	20.2 km east of Siverek	37°43'00''	39°30'00''	1200
WE 262	T. dicoccoides	PI 656872	TUR	34 km ESE from Narli (WSW of Karadağ)	37°20'12''	37°17'53''	780 (813)
WE 14	T. dicoccoides	PI 428051	TUR	36.2 km west of Diyarbakir in the Karacadag	37°53’00’’	39°52’00’’	1200
WE 264	T. dicoccoides		TUR	37 km NE from Kilis to Gaziantep	37°20'19''	37°16'50''	830
WE 274	T. dicoccoides		TUR	Siverek Karakeçi Road Azemi Village	37° 36' 51''	39° 20' 12''	733
WE 164	T. dicoccoides	PI 654321	TUR	4km south of Siverek on Karakecili road	37° 43’05’’	39° 19’37’’	720
WE 157	T. dicoccoides	PI 554581	TUR	25 km southwest of Diyarbakir	37° 45’00’’	40° 06’00’’	1000
WE 114	T. dicoccoides	PI 538646	TUR	36.2 km west of Diyarbakir in the Karacadag	37° 53’00’’	39° 52’00’’	1200
WE 159	T. dicoccoides	PI 554583	TUR	3 km southeast of the Junction of Karacadag Mt. road and Diyarbakir highway	37° 47’00’’	39° 47’00’’	1350
WE 154	T. dicoccoides	PI 538713	LBN	between Ain Harsch and Ain Ata	33° 26’00’’	35° 46’00’’	1192
WE 169	T. dicoccoides	TRI 18478, PI 427998	LBN	zwischen Kfarkouk und Aiha	33° 31’00’’	35° 52’00’’	1216
WE 193	T. dicoccoides	TRI 18530, PI 538706	LBN	zwischen Aiha und Kfarkouk, ca. 1 km von Aiha	33° 30’00’’	35° 52’00’’	1216
WE 150	T. dicoccoides	PI 538703	LBN	near Rashaya	33° 30’04’’	35° 50’22’’	1000
WE 41	T. dicoccoides	PI 428133	LBN	Aiha-Kfarkouk, above 'sahlet'	33° 31’00’’	35° 52’00’’	1141
WE 149	T. dicoccoides	PI 538702	LBN	near Rashaya	33° 30’04’’	35° 50’22’’	1000
WE 151	T. dicoccoides	PI 538704	LBN	near Rashaya	33° 30’04’’	35° 50’22’’	1000
WE 46	T. dicoccoides	PI 428143	LBN	between Rashaya and Aiha	33° 30’00’’	35° 50’00’’	1000
WE 1	T. dicoccoides	CItr 17675	LBN	outskirts of Rashaya	33° 30’04’’	35° 50’22’’	1000
WE 179	T. dicoccoides	TRI 18499, PI 470979	LBN	Mt. Hermon	33° 25’00’’	35° 52’00’’	2655
WE 34	T. dicoccoides	PI 428105	ISR	1 to 2 km south of Rosh Pinna towards Safad	32° 58’00’’	35° 32’00’’	549
WE 145	T. dicoccoides	PI 538696	ISR	Between 'En haShofet and Daliyya	32° 35’00’’	35° 03’00’’	122
WE 127	T. dicoccoides	PI 538670	ISR	Afula-Tiberias	32° 36’40’’	35° 17’30’’	300
WE 139	T. dicoccoides	PI 538690	ISR	near Safad on the road to Rosh Pinna	32° 58’00’’	35° 29’40’’	800
WE 134	T. dicoccoides	PI 538679	ISR	Afula-Tiberias	32° 36’40’’	35° 17’30’’	300
WE 61	T. dicoccoides	PI 466972	ISR	Bat Shelomo	32° 35’48’’	35° 00’07’’	105
WE 59	T. dicoccoides	PI 466969	ISR	Bat Shelomo	32° 35’48’’	35° 00’07’’	105
WE 62	T. dicoccoides	PI 466974	ISR	Bat Shelomo	32° 35’48’’	35° 00’07’’	105
WE 267	T. dicoccoides	PI 538696	ISR	Between 'En haShofet and Daliyya	32° 35’00’’	35° 03’00’’	122
WE 35	T. dicoccoides	PI 428112	ISR	1 to 2 km south of Rosh Pinna towards Safad	32° 58’00’’	35° 32’00’’	549
WE 87	T. dicoccoides	PI 471041	ISR	Kokhav haShahar	31° 57’00’’	35° 20’00’’	696
WE 51	T. dicoccoides	PI 466943	SYR	Kazrin	32° 59’24’’	35° 41’24’’	259
WE 80	T. dicoccoides	PI 470956	SYR	Kazrin	32° 59’24’’	35° 41’24’’	259
WE 184	T. dicoccoides	TRI 18506, PI487255	SYR	Damaskus Provinz	33° 45’00’’	36° 05’00’’	1240
WE 186	T. dicoccoides	TRI 18508, PI 487262	SYR	Damaskus Provinz	33° 40’00’’	36° 02’00’’	1300
WE 96	T. dicoccoides	PI 487260	SYR	32km from Sweida between Sale and Malah	32° 38’52’’	36° 47’24’’	1530
WE 93	T. dicoccoides	PI 487254	SYR	Nawa	32° 52’10’’	36° 01’51’’	551
WE 98	T. dicoccoides	PI 487264	SYR	Aleppo-Abeen road after Aleppo-Afrin road, Aleppo Province	36° 30’00’’	37° 00’00’’	350
WE 76	T. dicoccoides	PI 470945	SYR	Kazrin	32° 59’24’’	35° 41’24’’	259
WE 185	T. dicoccoides	TRI 18507, PI 487261	SYR	Es Suweida (Soud)	32° 38’00’’	36° 46’00’’	1450
WE 292	T.durum	PI 466947		Iran
WE 294	T. durum	Buckzafıro		Arj
WE 293	T. durum	PI 656872	TUR	112km northwest of Maras
WE 291	T. durum	PI 654317	TUR
WE 289	T. durum	Landrace		Italy

2.3. CAAT-PCR and SCoT-PCR Amplification

In this study, CAAT primers (24) were screened using the method developed by Etminan et al. (2018), and 7 CAAT primers with highly polymorphic bands were used in all wheat genotypes. PCR products and their concentration 10Xdream taq buffer 1.5 μl, 200 μM dNTP (1 μl), 1.5 μl primers, dream taq enzyme 0.3μl, 5.7 μl ddH2O (double distilled water) 5.7 μl were obtained in a total volume of 15 μl of PCR reactions used in the CAAT reaction. Amplification was done at 95 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 60 seconds, primer annealing at 50 °C for 60 seconds, and primer elongation at 72 °C for 120 seconds. The final extension was 7 min at 72 °C. The amplification reaction products were detected by 1.5% agarose gel stained The 10 SCoT primers with the highest number of polymorphic bands were employed following the testing of 34 primers, following Collard and David et al. (2009). All PCR reactions were performed within a total volume of 10 μl. PCR reaction mixtures contained PCR buffer (Promega; 20 mM Tris-HCl (pH 8.4), 50 mM KCl), 1.5 mM MgCl2, 0.24 mM of each dNTP, 0.5 U of Taq polymerase (Promega), and 1.6 μl of primer. Each reaction contained 25 ng of template DNA. A standard PCR cycle was used described as follows: an initial denaturation step at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min; the final extension at 72°C was held for 5 min. All PCR amplification products were separated on 1.2% agarose gels in Tris-borate buffer stained with ethidium bromide and visualized under UV light. Table 2 contains the list of PCR-amplified CAAT and SCoT markers.

2.4. Data Evaluation

Polymorphism was evaluated for the scorable bands that were evident on the gel. The information is displayed in binary form, with 1 denoting polymorphism's presence and 0 denoting its absence. A total of 48 Triticum genotypes were studied (43 Triticum dicoccoides and 5 of Triticum durum), using the CAAT and SCoT marker systems. The discriminating power (D), effective multiplex ratio (E), expected heterozygosity (H), mean heterozygosity (Hav), marker index (MI), polymorphism information content (PIC), and resolving power (R) of the genotypes were calculated. Polymorphism statistics calculated by iMEC (Online Marker Efficiency Calculator) for different primer types for wild emmer and durum wheat according to Amiryousefi et al. (2018). Binary data were generated from CAAT and SCoT primers for Triticum genotypes using DARwin software (version 6). The genetic similarity coefficients for the genotypes obtained from both marker systems were calculated using the Dice index (1945). The binary data was then analyzed following Gascuel (1997). Finally, an Unweighted Neighbor Joining clustering method was used to construct a tree.

3. Results and Discussion

Emmer wheat is one of the most important background genetic resources of domesticated wheat. It provides significant allelic diversity for various abiotic and biotic stress factors (Rasheed et al., 2017; Qiu et al., 2021; Yang et al., 2021; Tene et al., 2022). Evaluation of core collections and wild populations is a crucial step in advancing allelic variation in breeding programs. This study was conducted to evaluate the effectiveness of the CAAT and SCoT markers. Firstly, a prescreening was conducted using a total of 57 CAAT/SCoT primers on 8 wheat genotypes. Based on the results of the prescreening, 10 SCoT, and 7 CAAT primers were selected for further analysis. DNA analysis was then performed on wild emmer and durum wheat genotypes using the selected primers.

3.1. Polymorphism Values for the Entire Set of Genotypes

Genetic diversity of the whole set was evaluated by calculating the expected heterozygosity (H), mean heterozygosity (Hav), polymorphism information content (PIC), discrimation power (D), effective multiplex ratio (E), marker index (MI) and resolving power (R).

In this study, H values were determined for 48 wild emmer and durum wheat genotypes using CAAT and ScoT markers (Table 3). Among these primers, the lowest H value was observed in the CAAT12 primer with a value of 0.328, while the highest H value was observed in the CAAT14 primer with a value of 0.499. Moreover, H value was also high i.e. 0.498 and 0.491, in the primers CAAT21 and CAAT13, respectively. The lowest mean heterozygosity (Hav) of 0.001 was observed in primer CAAT22, while the highest was recorded as 0.005 in primer CAAT10.

In the case of ScoT markers, the lowest H value was observed in the SCoT9 primer with a value of 0.440, while the highest was observed in the SCoT23 with an H value of 0.500. ScoT primer SCoT16 showed the lowest Hav value of 0.001, while the SCoT19 primer had the highest Hav value of 0.002. In a similar study, Ren et al. (2013) used Single Nucleotide Polymorphism (SNP) markers to examine genetic diversity in 120 wild emmer wheat genotypes from 25 different wild wheat populations and found a Hav value of 0.184. Furthermore, Shizuka et al. (2015) reported an H value of 0.285. The H values obtained in these two studies differed from those recorded in our study. The difference could be a result of the difference in the genotypes and the types of DNA markers used.

The screening with CAAT primers revealed that the PIC value was as low as 0.348 in the primer CAAT14, while the maximum value was 0.419 in the primer CAAT12, followed by 0.401 in the primer CAAT20. The mean PIC value was found to be 0.377 in CAAT markers. In the case of ScoT markers, the lowest PIC value was seen in the primer SCoT23 with a PIC value of 0.365, while the highest PIC value (0.393) was obtained from primer SCoT9, followed by a PIC value of 0.373 in the primer SCoT16. The mean PIC value was found to be 0.373 in SCoT markers. Abouseada et al. (2023) reported that the mean number of polymorphic bands for SCoT primers in their study was 7.7, which aligns with this study, where the average number of polymorphic bands for SCoT primer was recorded as 7.6, while the average number of polymorphic bands obtained was low compared to CAAT primers (9). In addition, PIC values for the SCoT primer ranged from 0.17 to 0.37, which are similar to or lower than the values obtained in the present study. The PIC value is usually the effective factor used to compare different molecular markers and can also be used as an evaluation parameter to determine the degree of effectiveness of primers (Hongtrakul et al., 1997; Garcia et al., 2004).

The lowest D value of 0.539 was recorded in CAAT10, and the highest (0.958) was shown by the CAAT12 primer. However, the D value for SCoT primers was the lowest (0.727) in SCoT19, and the highest (0.894) was shown by the SCoT9 primer. Since allele frequencies vary between gene pools, the informativeness of a particular marker may differ between collections from different countries. However, a set containing the most informative markers identified in a collection of germplasms with high D values may also provide high discrimination power in other gene pools (Sefc et al., 2000).

The lowest and highest E value for CAAT primers was reported to be 1.361 and 6.361 in the primers CAAT10 and CAAT21, respectively. On the other hand, the lowest E value (2.361) for SCoT primers was recorded in the SCoT13, while the highest (4.191) was observed from SCoT8 primer.

When the primer MI values were examined for CAAT and SCoT molecular markers, the lowest MI (0.001) was derived from CAAT12, while the highest MI (0.006) was from CAAT10. Additionally, for ScoT markers the lowest MI value (0.003) was obtained from SCoT9, and the highest MI value (0.006) was shown by SCoT19 primer.

The R value is based on the distribution of alleles in the analyzed genotypes and correlates strongly with the ability to discriminate between these genotypes. The lowest R value for CAAT primer was recorded as 1.063 in CAAT10 and the highest R value of 7.531 was recorded from CAAT21. On the other hand, the lowest R value for ScoT markers was found to be 2.127 in SCoT11, while the highest (5.489) was obtained from SCoT8. When both markers are examined, CAAT21 and SCoT8 had the highest R values, while CAAT10 and SCoT11 had the lowest R values. Moreover, it was determined that the CAAT primers had higher R values than the SCoT primers. Therefore, the choice of CAAT and SCoT molecular markers largely depends on the level of polymorphism to be detected and their genomic coverage, rather than on the technology used to generate the markers. Gowayed et al. (2021) elaborated on some wheat (Triticum aestivum L.) genotypes that the mean values of E, MI and R values were 10.67, 7.80 and 9.15, respectively for different primers. Mean values of parameters E, MI, and R were low, medium, and high in this study. The possible reasons for these findings are whether the CAAT or the SCOT detects the polymorphism of the conserved regions around the markers.

3.2. Polymorphism Values Calculated Basen on the Collected Region of Accessions

The genetic diversity values of each collected country and region are estimated by calculating H, Hav, PIC, D, E, MI and R values for wild emmer wheat from the Fertile Crescent (Turkey, Israel, Lebanon, and Syria), as well as durum wheat (Table 4).

3.3. Parameters Obtained Using CAAT Molecular Markers

The expected genetic diversity values in wild emmer wheat genotypes collected in Turkey were found to be the lowest 0.278 with CAAT12 and the highest 0.500 with CAAT14. The expected H mean was found to be 0.428, and the calculated Hav was the lowest at 0.002 in the CAAT22 primer, while the highest was recorded to be 0.005 in CAAT14. The expected genetic diversity value was obtained as the lowest at 0.329 with CAAT12 and the highest at 0.500 with CAAT13 in Israel, and the calculated Hav was lowest at 0.018 in the CAAT10 primer, while the highest was recorded to be 0.006 in CAAT14. The lowest expected genetic diversity value was 0.368 with CAAT12, while the highest was 0.500 with CAAT13/CAAT21. The expected H mean was determined to be 0.444 for wild emmer wheat genotypes collected from Lebanon, and the calculated Hav value was the lowest at 0.004 in CAAT22 primer, and the highest at 0.021 in CAAT10. The expected genetic diversity value in wild emmer wheat genotypes obtained from Syria was determined as the lowest 0.320 with CAAT10/CAAT20 and the highest 0.498 with CAAT21. The expected H mean was determined to be 0.408, and the calculated Hav value was found to be the lowest 0.003 in the CAAT20/CAAT22 primer and the highest 0.016 in the CAAT10 for Syria. The expected genetic diversity value in durum wheat genotypes was determined as the lowest 0.095 with CAAT22 and the highest 0.500 with CAAT10, and the calculated Hav value was found to be the lowest 0.002 in the CAAT22 primer and the highest 0.050 in the CAAT10 for durum wheat.

CAAT analysis on wild emmer wheat genotypes collected from Turkey produced the lowest PIC value of 0.349 from the CAAT14 primer, the highest of 0.435 from the CAAT12 primer, and the mean PIC value of 0.379. The PIC value ranged from 0.364 with CAAT13 primer to 0.435 with CAAT12 primer, with a mean of 0.391 collected from Israel. Among the wild emmer wheat genotypes collected in Lebanon, the PIC value was the lowest (0.365) in CAAT13/CAAT21 primers and the highest (0.423) in primer CAAT12, with a mean PIC value of 0.391. According to CAAT analysis on wild emmer wheat genotypes collected from Syria, the lowest PIC value of 0.331 was recorded from the CAAT21 primer, and the highest (0.403) was obtained from the CAAT10/ CAAT20 primer with a mean PIC value of 0.369. The PIC value in durum wheat genotypes was determined as the lowest 0.171 with CAAT10 and the highest 0.292 with CAAT22, with a mean PIC value of 0.243. Ren et al. (2013), studied Single Nucleotide Polymorphism in 120 wild emmer wheat genotypes and recorded the PIC value as 0.153.

The mean MI showed a high value of 0.024 (CAAT21) and a low of 0.004 (CAAT12), with a mean MI value of 0.015 in wild emmer wheat samples from Turkey. The wild emmer wheat genotypes from Israel had a high value of 0.026 (CAAT10/CAAT21) and a low of 0.006 (CAAT12), with a mean MI value of 0.018. Similarly, the wild emmer wheat genotype from Lebanon also exhibited a high value of 0.030 (CAAT14) and a low MI value of 0.009 (CAAT12), with a mean MI value of 0.021. The MI exhibited a high value of 0.026 (CAAT10) and a low of 0.006 (CAAT20), with a mean MI value of 0.016 in wild emmer wheat harvested from Syria. The minimum and maximum MI values for durum wheat were 0.001 from CAAT22 and 0.050 from CAAT10, with a mean of 0.016.

This study evaluated the D values of wild emmer wheat from Turkey and Israel. The results showed the lowest D value of 0.653 with CAAT21 (Israel) and the highest D value of 0.974 with CAAT12 primer (Turkey), yielding a mean D value of 0.823 for wild emmer genotypes collected in Turkey. The mean discriminating power of wild emmer wheat from Israel was 0.774, with a high D value of 0.959 by using CAAT12 and a low D value of 0.481 with CAAT10. The mean D value in wild emmer wheat from Lebanon was 0.767, with a high of 0.944 for CAAT12 and a low of 0.521 for CAAT10. Similarly, wild emmer wheat from Syria had a mean D value of 0.811, with a high D value of 0.962 in CAAT20 and a low of 0.368 in CAAT10. D showed a high value of 0.995 for CAAT20 and a low value of 0.778 for CAAT10, with a mean value of 0.940 in durum wheat.

The markers had a mean R value of 3.785, ranging from 1.166 for CAAT10 to 6.500 for CAAT21 in wild emmer wheat from Turkey. For wild emmer wheat from Israel, the markers had a mean R value of 3.870, ranging from 1.090 with CAAT10 to 7.636 with CAAT22. The markers demonstrated a mean R value of 3.914 with a range from 1.200 (CAAT10) to 8.200 (CAAT21) in wild emmer wheat from Lebanon, whilst the mean R was 3.914. In wild emmer wheat from Syria with a range of 0.600 (CAAT12) to 4.800 (CAAT21). In durum wheat, the mean resolving power of the markers was 1.714, with a range from 0.001 (CAAT10) to 3.600 (CAAT13). Aslan-Parviz et al. (2020) reported that the used primers can detect the genetic variation in the investigated durum wheat genotypes. The R value of the 12 primers varied between 5.73 (CBDP12) and 12.60 (CBDP1) with a mean of 9.62. The lowest and highest value of MI was recorded for CBDP12 (3.68) and CBDP6 (5.99), respectively. The R value of the 12 primers ranged from 5.73 (CBDP12) to 12.60 (CBDP1), with a mean value of 9.62, the marker parameters possibly correlate with the frequency of allelic variance, which may be affected by different climatic conditions, these variations could potentially be attributed to the genotypes utilized in this investigation. The markers' informative parameters, such as the expected heterozygosity, Polymorphism Information Content, effective multiplex ratio, mean heterozygosity, marker index, discriminating power, and resolving power, indicate that the applied primers possess adequate discriminating power. Therefore, the genetic diversity and structure in the wheat germplasm can be analyzed effectively by implementing this technique.

3.4. Parameters Obtained Using SCoT Molecular Markers

Parameters obtained using SCoT molecular markers for the wild emmer and durum wheat genotypes collected from different countries are shown in Table 5. The mean H value in wild emmer wheat genotypes collected from Turkey was 0.467, with a minimum of 0.413 for SCoT9/SCoT16, with a maximum of 0.499 for SCoT7. Calculated Hav was found to be the lowest (0.003) in SCoT16 primer, while the highest was 0.008 in SCoT19 for Turkey. The mean H value in wild emmer wheat genotypes collected from Israel was 0.484, with a minimum value of 0.452 for SCoT19 and a maximum of 0.499 for SCoT13/ SCoT17. Calculated Hav value was the lowest (0.004) in SCoT8 primer, while the highest (0.008) was in SCoT19 for Israel. The mean H value in wild emmer wheat genotypes collected from Lebanon was 0.478, with a minimum of 0.420 for SCoT13, with a maximum of 0.499 for SCoT7. Calculated Hav was recorded to be the lowest 0.0047 in SCoT8 primer, and the highest (0.010) was in SCoT19 for Lebanon. The mean H value in wild emmer wheat genotypes collected from Syria was 0.454, with a minimum of 0.353 for SCoT23, with a maximum of 0.500 for SCoT10. Calculated Hav found the lowest 0.004 in SCoT8 primer, while the highest (0.010) was recorded in SCoT19 for Syria. The mean H value in durum wheat genotypes was 0.359, with a minimum of 0.180 for SCoT9, with a maximum of 0.496 for SCoT10. The lowest calculated Hav was 0.005 in the SCoT9 primer and the highest was 0.015 in the SCoT19 primer for durum wheat.

The mean PIC value per primer was 0.379, with a maximum of 0.400 for SCoT9/SCoT16 and a minimum of 0.360 for SCoT7 among wild emmer wheat genotypes collected in Turkey. The mean PIC value per primer was 0.382, with a maximum of 0.397 for SCoT19 and a minimum of 0.376 for SCoT13/ SCoT17 in wild emmer wheat genotypes collected from Israel. The mean PIC value per primer was 0.385, with a maximum of 0.411 for SCoT13 and a minimum of 0.375 for SCoT11/ SCoT7 in wild emmer wheat genotypes collected from Lebanon. The mean PIC value per primer was 0.363, with a maximum of 0.405 for SCoT23 and a minimum of 0.342 for SCoT10 in wild emmer wheat genotypes collected from Syria. The mean PIC value per primer was 0.329, with a maximum of 0.384 for SCoT9 and a minimum of 0.277 for SCoT10 in durum wheat genotypes.

Effective multiplex ratio (E) had a high value of 4.333 (SCoT7) and a low of 2.250 (SCoT16), with a mean E value of 3.216 in wild emmer wheat collected from Turkey. E had a high value of 5.272 (SCoT16) and a low of 2.818 (SCoT11), with a mean E value of 3.709 in wild emmer wheat from Israel. The E values ranged from 6.100 (SCoT8) to 2.100 (SCoT13), with an average of 3.700 for wild emmer wheat from Lebanon. The lowest and highest E values for wild emmer wheat from Syria were 1.600 from SCoT23 and 4.000 from SCoT16 markers, with an average of 2.820. The lowest and highest E values for durum wheat were obtained as 0.80 from SCoT9/SCoT13/17 and 3.80 from SCoT8/SCoT10, with an average of 1.86.

The MI had a high value of 0.024 (SCoT17/ SCoT19) and a low of 0.010 (SCoT9/ SCoT16), with an average marker index of 0.017 in wild emmer wheat from Turkey. MI had a high value of 0.027 (SCoT19) and a low of 0.016 (SCoT8/SCoT9), with an average marker index of 0.022 in wild emmer wheat collected from Israel. MI had a high value of 0.029 (SCoT23/SCoT17) and a low of 0.013 (SCoT13), with a mean marker index of 0.023 in wild emmer wheat from Lebanon. MI had a high value of 0.027 (SCoT11) and a low of 0.008 (SCoT23), with an average marker index of 0.018 in wild emmer wheat from Syria. The lowest and highest MI values for durum wheat were 0.036 from SCoT9 and 0.054 from SCoT10, respectively, with an average of 0.022.

For the D the lowest value was 0.663 with SCoT17, the highest 0.917 with SCoT9 primer mean D of 0.802 for wild emmer wheat collected from Turkey. D had a high value of 0.862 in SCoT9 and a low of 0.576 in SCoT19, with an average discriminating power of 0.746 in wild emmer wheat from Israel. D had a high value of 0.913 on SCoT13 and a low of 0.626 on SCoT17/SCoT23, with an average discriminating power of 0.755 in wild emmer wheat from Lebanon. D had a high value of 0.950 SCoT23 and a low of 0.720 SCoT11, with a mean D value of 0.853 in wild emmer wheat from Syria. D had a high value of 0.990 SCoT11 and a low of 0.713 SCoT10, with an average discriminating power of 0.915 in durum wheat. The mean R value of the markers was 2.566 with a range of 1.333 (SCoT10) to 5.333 (SCoT8) for wild emmer wheat from Turkey. The mean R of the markers was 2.872 with a range of 1.636 (SCoT11) to 4.727 (SCoT8) for wild emmer wheat from Israel. The mean R of the markers was 2.32 with a range of 1.00 (SCoT10/SCoT17) to 4.200 (SCoT8) for the wild emmer wheat from Lebanon. The mean R of the markers was 2.480 with a range of 1.00 (SCoT17) to 3.800 (SCoT8) for the wild emmer wheat from Syria. The mean R of the markers was 1.440 with a range of 0.400 (SCoT10/SCoT13) to 2.800 (SCoT7) to durum wheat.

3.5. Mean Genetic Diversity Parameters for CAAT and SCoT Markers

Table 6 displays several iMEC characteristics estimated to assess the mean genetic diversity in CAAT and SCoT molecular markers for wild emmer and durum wheat genotypes collected from different countries. The Hav with CAAT primers in durum wheat was found to be the lowest at 0.012, while the highest was recorded from SCoT primers in durum wheat at 0.010. The H value was the lowest 0.29 with CAAT primers in durum wheats, while the highest was 0.48 with SCoT primers in populations from Israel. Shizuka et al. (2015) reported mean genetic diversity of 0.28 and 0.29 in the two populations they studied, respectively, and the difference revealed in the current study can be attributed to differences in marker selection and genotype origin. It was discovered that the lowest mean PIC value was 0.243 (CAAT primers) in durum wheat, while the highest mean value was 0.391 (CAAT primers) obtained from Israel/Lebanon. The mean D was measured and found to be the lowest at 0.746 (SCoT primers for wild emmer wheat from Israel) and the highest at 0.940 (CAAT primers for durum wheat). The mean E in durum wheat was the lowest at 1.628 (CAAT primers), while the highest was found in Israel at 3.844. Furthermore, the mean primer MI was lowest (0.015) in CAAT primers for Turkish wild emmer wheat and highest (0.023) for wild emmer wheat from Lebanon. The mean R value was the lowest (1.440) for durum wheat (SCoT primers) and highest (3.914) for wild emmer wheat from Lebanon (CAAT primers). These traits were used to evaluate the efficiency of primers in wild emmer wheat genotypes, revealing degree and genotype differences.

3.6. Principal Coordinate Analysis (PCoA)

To obtain further information about the wild emmer wheat and durum wheat genotypes, principal coordinate analysis (PCoA) was used to identify the variance in SCoT primers (Figure 2 and Figure 3). Turkey, Israel, Lebanon, and Syria are represented by accessions from the known wild emmer region (Triticum dicoccoides), while durum corresponds to wheat genotypes. For the majority of the comparable GPS source coordinates, many identical accessions were detected. Cluster analysis has been used extensively to describe genetic diversity and clustering based on similar characteristics. Based on the Unweighted Neigbor-joining clustering method (UNJ), the 43 wild emmer wheat and 5 durum wheat genotypes were grouped into three major groups with SCoT primers (Figure 4). Cluster A consisted of 27 genotypes from Turkey, Lebanon, and Israel. Cluster B included 16 genotypes from Syria, wild emmer wheat from Lebanon, and durum wheat. The remaining genotypes formed a third cluster originating from Israel, Syria, and Lebanon. According to the UNJ, the 48 genotypes of wild emmer and durum wheat were grouped into three distinct clusters for CAAT primers (Figure 5). Cluster I consisted of 27 genotypes of wild emmer from Turkey, Lebanon, Israel, and Syria. Cluster II included 19 genotypes of wild emmer from four different countries and five durum wheat. The last group, Cluster III had two genotypes within Israel and Syria. The dendrogram of 38 durum wheat landraces in Iran, based on the dissimilarity matrix developed with SCoT markers using the UNJ method of Seyedimoradi et al (2016), showed that SCoT and CDDP markers formed three main groups each, whereas SCoT markers formed three main groups and CAAT markers formed two main groups in the wild emmer and durum wheat studied. Genetic diversity and relationships between wild emmer and durum wheat genotypes revealed three clustering patterns according to the UNJ dendrogram, implying that CAAT-SCoT markers are an acceptable marker system.

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4. Conclusions

Maintaining the highest possible level of genetic diversity is one of the main objectives of genetic resource conservation programs, and assessing genetic diversity using reliable methods provides useful information for genetic resource management and crop improvement programs. The results revealed a substantial degree of variation in the wheat germplasm examined in various Triticum species. The current study found that both SCoT and CAAT marker systems can effectively estimate genetic diversity in wild emmer and durum wheat genotypes, as demonstrated by the calculated via iMEC to compare the mean of genetic diversity parameters in CAAT and SCoT molecular markers. In general, SCoT/CAAT markers generated high polymorphism and PIC values. In conclusion, two marker methods, which targeted certain parts of the plant genome or candidate genes by primer design were used to estimate the genetic diversity of durum wheat. This information is valuable for germplasm classification and defining various/ heterotic groups, which is particularly important in hybrid/ cross-breeding programs for wheat. The results obtained revealed a significant genetic diversity in wild emmer populations of different countries. These results were supported by various statistical analyses such as iMEC online marker efficiency, UNJ, PCoA, and cluster analyses showing a high difference between populations. The study showed that the new-generation SCoT and CAAT marker systems are powerful tools for exploiting the genetic diversity of wheat wild relatives and cultivars. Thus, it can be suggested that these DNA-based systems can be used in combination with other molecular markers for genetic analyses such as association mapping studies and the construction of linkage maps.

Availability Statement: The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.