Genomic variation and genetic structure prole of Iraqi barley accessions using ISSR and arbitrary functional gene-based molecular markers

Few attempts have been made in Iraq to investigate the genetic variation in barley accessions using molecular markers. In this context, the objective of this study was to investigate the diversity of 59 accessions of barley using inter simple sequence repeat (ISSR), conserved DNA-derived polymorphism (CDDP), and start codon targeted (SCoT) markers. A total of 391 amplied polymorphic bands were generated using 45 ISSR, 9 CDDP, and 12 SCoT primers that produced 255, 35, and 101 polymorphic bands, respectively. The average values of gene diversity were 0.77, 0.67, and 0.81 for ISSR, CDDP, and SCoT markers, respectively. The mean values of polymorphism information content for ISSR, CDDP, and SCoT markers were 0.74, 0.63, and 0.80 respectively. The discrimination power of the three approaches for assessing allelic diversity in barley accessions was as follows: SCoT > ISSR > CDDP. The barley accessions were classied and clustered into two main groups. Molecular variance analysis revealed 15, 9, and 14% variability among populations with ISSR, CDDP, and SCoT markers, respectively. The mantel test results revealed that the three molecular marker matrices had signicant positive relationships. The SCoT markers have the potential to be useful tools for selecting appropriate parents for breeding program. of to in supports Our evaluating the genetic diversity reported by et al. who scored 145 polymorphic bands using 11 ISSR primers.


Introduction
World Food Security announced serious concern about the growing world population. According to recent estimates, the world population will reach 9.8 billion or more, and meeting the needs of this estimate of the global population will likely require a doubling in food production. It is critical to improve the productivity of not only food crops, but also crops used for livestock fodder to meet the food demands of the near future (Qaim 2020). For this purpose, in different plant species, inclusive genome and population structure explorations of genetic diversity have received considerable attention. To adapt to different environmental conditions, including uctuation in climate conditions, the genetic diversity in a given species allows plants to overcome these obstacles in an orderly manner. The genetic variation in a population of a plant species, containing landraces, cultivars, as well as wild individuals, is an essential resource for sustainable agricultural practices and increasing food production (Latutrie et al. 2019). For that reason, to discover useful genes in crops, the assessment of genetic diversities in the available genetic resources is the main step in conducting the plant research project.
Barley (Hordeum vulgare L.) is considered the oldest and most important cereal crop cultivated by humans, and is the fourth largest cereal plant after maize, wheat, and rice worldwide. In general, the genome of barley is a diploid that contains 14 chromosomes. This crop requires low cultivation inputs, including water demand, fertilizer, and toleration of abiotic stress (Baum et al. 2015). From this point, the distribution and cultivation of barley are largely perceived in regions of arid and semiarid rainfall. It is used as human food as the feed of livestock as malt industries. During the evolution from growing landraces to cultivation, barley is the suitable model crop to study phenotypic differences and genome diversity (Brantestam et al. 2007). Many researchers have established signi cant contributions through traditional techniques to barley improvement in a breeding program that depends on phenotypic traits, and many researchers apply it (Hadado et al. 2009;Hagenblad et al. the high cost of AFLP, as polyacrylamide gels are required for detecting polymorphism and need technical skill (Meudt and Clarke 2007). In the RAPD marker, no information on heterozygosity can be provided (Jiang 2013). For detecting polymorphism in the genome by using the SSR technique, it is essential to identify the anking sequences and develop speci c primers (Kaur et al. 2015). In addition, SNPs markers seem exciting, but it could be expensive by researchers with limited resources, as it is in most developing countries, and require speci c equipment, like next-generation sequencing.
Among different types of molecular markers available for barley, ISSR markers have proven suitable markers inbreeding and genetic diversity studies due to high polymorphism and repeatability across the whole genome. This type of marker-based on polumerase chain reaction (PCR) ampli cation of repeated DNA nucleotides to target multiple locations in the genome. For their use, no previous genomic information is required, and small amounts of DNA are needed (Adhikari et al. 2017).
Conserved DNA-derived polymorphisms (gene family sequences discernible in many copies within plant genomes) are powerful and cost-effective molecular approaches for accessing polymorphisms (variability) in plant species. However, there are other newly derived molecular marker systems, conserved DNA-derived polymorphism (CDDP), start codon targeted polymorphism (SCoT) markers, and CAAT box-derived polymorphism (CBDP) markers, which have curtailed conserved gene sequences and present at multiple sites within plant genomes, and hence provide various primer binding positions (Collard and Mackill 2009).
Worldwide, modern crop breeding has resulted in a rapid reduction in genetic diversity over time due to concentrated selections on targeted genes or quantitative trait loci. Consequently, knowing the current condition of genetic diversity and the extent of the possible genetic decline in Iraq's germplasm could be bene cial for barley breeding in terms of effectively improving important traits and correctly predicting genetic relatedness and diversity. Up to this point, there have been limited initiatives in Iraq to explore genetic variation in barley accessions using molecular markers. (Al_Hadeithi 2015;Al-Hadeithi 2016). In this context, the objective of this study was therefore to investigate the diversity of 59 accessions of barley plants collected from different geographical regions in Iraq using ISSR, CDDP, and SCoT markers.

Materials And Methods
Plant material and DNA isolation A total of 59 barley accessions were considered in the present work, which originated in almost all research centres in Iraq (Table 1). These accessions are the most widely cultivated in Iraq. The complete genomic DNA was extracted from young leaves (2-week old seedlings), which were grown in greenhouse plants from each accession according to the cetyltrimethylammonium bromide (CTAB) protocol (Tahir 2015). The quality of DNA was estimated and examined by using a 1.5% agarose gel. Then, DNA samples were diluted to about 50 ng/µL using ddH2O, and the extracted genomic DNA was saved in the freezer (-20oC) until used in a polymerase chain reaction.

ISSR, CDDP, and SCoT assay
In the present investigation, 45 ISSR primers were selected mostly from the set of Biotechnology Laboratory, University of British Columbia, Canada (Table 2). Nine CDDP and 12 SCoT primers were also tested in our study (Tables 3 and 4). PCR reactions were performed in a quantity of 25 µL. The reaction mixture contained 4 µL of isolated DNA from each sample, 10 µL master mixes (GoTaq® Green Master Mix, Promega, USA), 2 µL of used primer, then the nal volume of 25 µL completed with de-ionized water.
The PCR was carried out in accordance with this procedure. : 1 cycle of 94ºC for 10 min, followed by 35 cycles of 1 min denaturing step at 94°C, 1 min annealing temperatures which ranged between (42-60°C) depending on the different ISSR, CDDP, and SCot primer sequences, and 2 min extension at 72°C. Finally, the post-extension was set up at 72°C for 7 min. The ampli cation reaction products were detected and separated by 1.5% agarose gels (1xTBE buffer) stained with ethidium bromide and visualized under UV light.
Scoring and statistical data analysis After ampli cation of the fragments, the scorable bands were manually coded by recording 0 and 1 for the absence and presence of bands, respectively. To calculate the similarity coe cient of Jaccard, the scored data matrices were subjected to statistical analysis using the XLSTAT 2016 computer software. To perform cluster analysis between accessions, the Jaccard coe cient was converted into a dissimilarity matrix using the unweighted pair-group technique with arithmetic averages (UPGMA). The binary data (0 and 1) was converted to A and T to create the dendrogram tree using CLC Sequence Viewer version 8. Polymorphism information content (PIC) allele frequency and gene diversity were calculated using Power Marker version 3.25 software to measure the e ciency of the markers and distinguish between accessions. To infer the genetic structure and clarify the numbers of sub-populations for the population structure, a model analysis was conducted with the help of the software STRUCTURE, version 2.3.4 (Pritchard et al. 2000). The numbers of supposed populations (K) were set from one to ten, and the analysis was repeated three times. The burn-in and MCMC were xed to 50,000 each for each category, and iterations were set down to 5000. The run with the maximum likelihood was engaged to set accessions into populations. All ISSR primers produced scorable and well-de ned ampli cation products, and showed polymorphisms among the 59 analyzed barley accessions ( Table 2). The 45 ISSR primers used in this study generated 255 scorable polymorphic bands. The numbers of ampli ed bands detected in our study ranged between 1 and 11 for the ISSR markers UBC-813 and ISSR-9, respectively. The major allele frequency reached from 0.10 to 0.81, with 0.36 as an average allele per marker. Major alleles with the highest frequency (81%) were observed for the ISCS20 marker. The gene diversity values were observed in the variety of 0.32-0.96 with an average value of 0.77 per ISSR marker. PIC revealed complete consent of discriminating power of ISSR primers, suggesting a high e ciency of this DNA marker to discover genetic diversity among barley accession used in this investigation. Our results showed that the PIC values ranged from 0.96 (ISSR-8 and UBC-823) to 0.29 (ISCS20) with a mean of 0.74 (Table 2). This represents the positive capability of ISSR-8 and UBC-823 primers to assess genotyping in barley germplasm, and therefore provides a useful tool for analyzing population genetics on diverse plants and recognizing population. In the current study, 26 ISSR markers had PIC values larger than the average PIC value (0.74), which could be helpful for trait mapping and tagging studies in Iraqi barley accessions. The ability to determine genetic differences among different genotypes may be more directly    In the present investigation, the SCoT marker technique revealed a higher mean of PIC (0.80) than ISSR (0.74) and CDDP (0.63), which explains the precise application of the SCoT technique in the assessment of accessions diversity. Based on the above results, the discrimination power of the three approaches for assessing allelic diversity in barley accessions was as follows: SCoT > ISSR > CDDP, exhibiting that SCoT functional gene-based markers were both informative and effective at assessing genetic diversity. In addition to SCoT and ISSR performance, CDDP markers demonstrated a poor ability to differentiate barley accessions. Finally, it was suggested that genetic analyses based on SCoT and ISSR markers would be extremely useful for crop improvement programs, including QTL mapping, genetic diversity estimation, linking maps, and genotype identi cation, as SCoT markers were derived from the functional region of the genome.

Cluster analysis of different barley accessions
Multivariate statistical approaches are critical in studying genetic diversity. Cluster analysis, one of the multivariate statistical techniques, separates individuals into graphs based on intervals. A marker pro le data aims to maintain a genetic relationship between genotypes under investigation, using distance measures that express accessions' relationship. The dendrogram was constructed based on 45 ISSR markers to better estimate the genetic distance among 59 Barley accessions (Fig. 1). The UPGMA method and Jaccard coe cient dissimilarity were performed for analyzing the ISSR markers data set. Two major clades within 7 subgroups at the dissimilarity threshold were indicated (Fig. 1A). The rst clade included almost all barley accessions, which comprised 38 tested accessions. This group contains 5 subgroups including the barley accessions: Bhoos-912, BA4, BN6, Bhoos-244, BN2R, CANELA, ABN, A1, MORA, MSEL) grouped in the rst subgroup, and the second subgroup consisted of 10 barley accessions named Al-warka, 16 HB, GOB, Numar, Furat 9, Boraak, Radical, Arivat, Ra dain-1, Al-khayr, which mostly spread and cultivated in South of Iraq. In addition, 13 barley accession clustered in the third subgroup, including Black-Bhoos-B, Arabi aswad, Victoria, Scio/3, Acsad strain, Acsad strain, Samr, Irani, White-Halabja, Abiad, White-Zaxo, Black-Zaxo, Black-Bhoos Akre, while the fourth subgroup had 4 barley accession: Al-amal, IBAA-99, IBAA-265, White-Akre. However, Bhoos-H1 was isolated in the fth subgroup, demonstrating its genetic divergence from the other accessions. Whereas, in the second clade, two main sub-clusters were exhibited. The rst sub-cluster comprised 5 accessions (Rehaan, Sameer, Warka-B12, Al-Hazzar, IBAA-995), which are mainly distributed over the South of Iraq, and the rest of the barley accessions were grouped in the second sub-cluster. The clustering pattern for ISSR data showed that barley accessions can be separated into two major groups according to geographical origin. Therefore, to enhance the appearance of heterosis, the breeder may use genetic distance data to make informed decisions about crossing accessions from distinct groups or subgroups for population development, or to promote the analysis of various parents to cross in hybrid combinations. The present outcome supports previous reports on the correlation between ISSR markers and eco-geographical distribution of the accessions Regarding the clustering patterns for CDDP primers, two main groups were optioned, but with different sub-clusters. In general, two main sub-clusters were determined in the rst group. The rst sub-cluster included only Ukranian-Zarayan accession, while the second sub-cluster in the same group separated the barley accessions to the two sets of arrangements. The rst sub-sub-cluster was composed of three accessions (Black-Akre, Black-Garmiyan, Black-Chiman), which were cultivated by the farmer in the North of Iraq, and the remaining barley accessions were arranged in the second sub-sub-cluster based on genomic similarity. However, group 2 included only two barley accessions, namely Al-Hazzar and IBAA-995, indicating their genomic differences from the rest of the accessions (Fig. 1B). The number of groups detected by CDDP markers in this study was lower than in the reporting of Ahmed et al. (2021), who exhibited three clusters in 82 Iranian barley accessions. This could be due to the type of markers and the number of accessions used.
Clustering of barley accessions using the SCoT molecular dataset revealed two major classes (Fig. 1C). The rst class included most barley accessions categorized into two different sub-clusters. One of them comprised three barley accessions, namely (A1, Arabi Aswad, and Clipper), while the other sub-cluster distributes the most barley accession into three sub-subclusters. The rst sub-sub-cluster comprised four barley accessions (16 HB, Furat 9, Al-warka, and Black-kalar), which were mostly distributed and cultivated in the South of Iraq. The second sub-sub-cluster included ve barley accessions (Black-Akre, Black-Garmiyan, Black-Chiman, White-Zarayan, and Bujayl 2-Shaqlawa). The farmer widely cultivated these barley accessions in North of Iraq, while forty-two barley accessions based on genomic dissimilarity were arranged in the third sub-sub-cluster in this particular group. However, the second class involved only ve distinct barley accessions, namely Rehaan, Sameer, Warka, B12, Al-Hazzar, and IBAA-995, which originated from the South of Iraq. The number of clusters formed by the SCoT approach in this study was smaller than that previously reported by Ahmed et al. (2021).
Intriguingly, the general dendrogram constructed using the combined data of all molecular markers used in our investigation (ISSR, CDDP, and SCoT) (Fig. 1D) divided barley accessions into two major clusters. Almost all barley accessions were well distributed in the rst cluster, which included fty barley accessions. In this particular cluster, it is clear that some barley accessions based on genetic distance were grouped, especially for the accessions of Arabi aswad and Clipper, while the rest of the forty-eight accessions were grouped in six sub-sub-clusters. Conversely, the second cluster consisted of nine barley accessions, namely Black-Akre, Black-Garmiyan, Black-Chiman, Bujayl2-Shaqlawa, Rehaan, Sameer, Warka-B12, Al-Hazzar, IBAA-995. Accordingly, our nding supported the available suggestion that many molecular techniques could either be applied individually or in combination with other molecular marker techniques to nd reliable information about genetic relationships and assess the genetic variation, which would support strategies for effective collection of barley germplasm and knowing their conservation. Insu cient genetic differentiation could imply a high level of gene ow. Stimulatingly, taking a close look at the dendrogram using two different marker systems, a similar pattern of alignments for most barley accessions can be found. Similarly, a group of researchers, Naceur et al. (2012), worked on 31 barley accessions to reveal genetic distance, and obtained 9 classes demonstrating wide diversity among studied accession. This is probably due to the collection of barley germplasm from three countries (Egypt, Algeria, Tunisia). Whereas, compared to our previous work, seven clusters were obtained using the SSRs marker technique (Tahir et al. 2020). This type of molecular technique was now widely used by another research groups to determine the genetic diversity of plant species (Liu et al. 2020;Saidi et al. 2018;Talebi et al. 2018). For many plants, all three markers have been effectively used to determine genetic relationships and diversity. In addition, DNA analysis using three methods has proved to be an inexpensive and e cient way to provide molecular data for evaluating genetic differences ( has been noted the larger the distance between accessions, the greater the likelihood of accumulating wider genetic diversity, which also de nes their places on clusters (Skroch and Nienhuis 1995).
Population structure pro le in barley accessions STRUCTURE software and the Bayesian statistical index were used to perform effective population structure assessment, reliable population grouping, and identi cation of mixed genotypes. Separate and combined data of all molecular markers ISSRs, CDDP, and SCoT were used to estimate the population structure of 59 barley accessions. To measure the level of genetic strati cation in a multi-locus data set, the program STRUCTURE has become one of the most commonly used programs. However, this program has its limitation, as it cannot interfere with the number of clusters (k) that best t the data set (Pritchard et al. 2000). To solve this limitation, the STRUCTURE harvester, a web-based program, has been generated for quickly analyzing and summarizing the data outcome from the STRUCTURE program. Therefore, detecting numbers of K groups that best t the population will be selected (Evanno et al. 2005). In this investigation, to evaluate the likely number of inferred populations, the likelihood of K equals 1 to 10, with each K-value replicated three times (Earl 2012).
The optimal value of k is 2 in all three used markers (Figs. 2 and 3). The STRUCTURE outcomes suggested the population could be divided into two main populations in all case scenarios. The grouping was mildly following the geographic background of the barley accessions. Although two populations may be practical for our panel, based on the size of the population and the difference in numbers of barley accessions representing three main areas in Iraq from which they were collected. The optimal value of k = 2 occasionally misrepresents the true structure in the population, and could mean that either the STRUCTURE program unsuccessfully identi ed the underlying genetic structure of the collection, or there is no de nite population structure (Cullingham et al. 2020). Therefore, cluster analysis was performed, as mentioned previously, to truly understand the genetic structure of this population. Based on the results of these analyses, it was observed that there are no differences between the two analyses and completely match the cluster analysis obtained by the molecular markers data set. This meets our expectations better, as it was observed that the accessions from the neighboring locations were mostly clustered together compared with the populations derived by STRUCTURE.
Remarkably, the result from STRUCTURE Harvester for all molecular markers demonstrated that (k) value had the maximum peak at K = 2, inferring that the probable number of genetic clusters in the population incorporates all individuals from 59 accessions with the highest likelihood (Figs. 2 and 3). This was observed when the mean of the log of posterior probability was graphed, demonstrating that two populations can be observed, which were visualized in two distinct colors (Green and Red). Based on the membership fractions, the accessions with a probability of 80% or above were assigned to matching populations. with others characterized as an admixture, and indicating the purity of tested materials. The combination of the two mentioned colors represents barley accessions in which they possess different genetic structures.
Concerning the separate and combined analysis of structure for all marker data set conducted in this investigation, the rst population indicated in red color including individuals of pure genetic make-up (with the probability of ≥ 80% ), which populations in numbers of individuals was much higher than in the admixture form, which showed the uniformity of tested accessions. This nding con rms that the lemma and palea remain tightly closed during the period of pollen release in barley.
This phenomenon is known as cleistogamy (Nair et al. 2010). However, admixture probably occurred due to breeding lines developed through random mating by the breeders, for it is a speci c trait improvement (Hernandez et al. 2020).

Genetic differences within and among populations
The analysis of molecular variance (AMOVA) based on the results achieved from the molecular data from three different marker systems was suggested that 15,14, 9, and 14% of total variation were among the three populations (North, Middle, and South of Iraq) in which the sample was grown and collected (Table 1) based on the molecular data set from ISSR, CDDP, SCoT and the combined data of all three marker systems, respectively, while the analysis was revealed the variation was much higher within the studied individuals, which were 85, 86, and 91% depending on the data obtained from ISSR, CDDP, and SCoT, respectively (Table 5), whereas, the collective data from all three marker sets propose 86% of total differences were inside the population. These outcomes suggest that barley accessions from Iraq shared a common ancestry and are highly admixed, with high variation within populations from all marker systems in which this investigation was conducted. This result revealed a distinct genetic base for the 59 barley accessions. The AMOVA results con rmed clustering and structure analyses. The partitioning of molecular variance reported that the greatest divergence was detected among individuals in the same population. Genetic diversity within and between populations improves the selection of populations that account for the vast majority of extant variations. If genetic diversity is largely found within a population, it means fewer populations are needed to conserve and sustain the differences in accessions or populations. Conversely, if genetic diversity across populations is preserved, a greater number of preserved across populations, more populations should be emphasized for maintenance and utilization (Igwe et al. 2021). Correlation analysis between genetic dissimilarity achieved by three sets of markers data To compare the genetic distance matrices produced by three marker systems (ISSRs, CDDP, and SCoT), the mantel test was performed. However, this analysis has its limitation, especially in the case of using two different markers (Vieira et al. 2007). In the case of our investigation, 45 ISSR, 9 CDDP, and 12 SCoT primers were conducted. Remarkably, mantel test correlation values revealed a positive signi cant correlation between and among all three different marker systems in which clusters with a general dendrogram were present ( Table 6). The highest mantel value was observed between ISSR and CDDP markers, while the minimum was displayed between ISSR and SCoT markers. This demonstrates the novelty of current work and the possibility of composing reference collections of tested barley accessions using the information attained from genetic pro les tested by three different molecular methods. Natural selection could also clarify the appropriate relationship between the diverse patterns of these markers in the regions exacerbated by ISSR, CDDP, and SCoT markers. These ndings are consistent with previous research of Ahmed et al. (2021), who detected signi cant associations between CDDP and SCoT markers. In any plant genetic resource conservation program, the main goal is to obtain the highest possible level of genetic diversity.
The three different marker systems revealed a comprehensive pattern of genetic diversity among the barley accessions collected. Our ndings revealed a high level of genetic diversity among barley accessions. From all the above analyses, it is possible to conclude that selecting an ideal primer with high information content from all studies would improve the e ciency of future studies. SCoT and ISSR primers were considered more effective primers to distinguish between barley accessions. Besides, the CDDP markers could be used to determine the genetic variations among tested accessions. Dendrogram and structural analysis of accessions with different genomic statutes indicated considerable accessions grouping. Furthermore, these results could allow future insights into barley breeding programs, and thus the crossing between more genetically distant individuals increases the chance of segregation in their offspring. Consequently, the SCoT-selected primers could be effective tools for selecting desirable hybrids for enhanced breeding and germplasm preservation. Assessment of SCoT marker linkages with signi cant agronomic variables in barley can develop marker-assisted selection strategies using these functionally gene-based molecular markers. Speci c alleles/bands for various gene-based markers may also be used to clone and design competitive allele-speci c PCR (KASP) markers in barley.

Declarations
Author contributions DL and NT performed the experiments and analyzed the data. NT and KM planned the experiments and wrote the manuscript.