https://orcid.org/0000-0002-2319-005X
Figures
Abstract
The genus Agropyron has an important role in soil protection and forage production in rangelands. The investigation utilized 37 ISSR primers, resulting in the detection of 956 loci within the A. elongatum genome and 705 loci within the A. cristatum genome. The findings revealed a high level of polymorphism, with 97% of loci in A. elongatum and 84% of loci in A. cristatum exhibiting variability. Notably, the primer (AC)8GCT emerged as a promising candidate for evaluating genetic diversity due to its ability to amplify numerous loci in both species. Using both the UPGMA algorithm and Bayesian analysis, the examined Agropyron accessions were categorized into two subgroups based on their respective species. The Q values associated with these subgroups suggested that certain accessions, namely "G16," "G19," "G20," "G21," "G22," "G23," "G24," and "G25," displayed potential admixture genomes. An analysis of molecular variance (AMOVA) underscored the significance of within-species variability, which accounted for 69% of the overall diversity, compared to between-species variability at 31%. Various genetic diversity parameters, including Na, Ne, I, He, and the number of private loci, were found to be higher in A. elongatum when compared to A. cristatum. Furthermore, Jaccard similarity coefficients ranged from 0.33 to 0.66 in A. cristatum and from 0.25 to 0.7 in A. elongatum, indicating the extent of genetic relatedness among these species. Intriguingly, the study identified two and three heterotic groups in A. cristatum and A. elongatum, respectively, which could be harnessed in the development of synthetic varieties to exploit heterosis. The results also indicated that a small proportion of ISSR loci pairs (5.2% in A. elongatum and 0.5% in A. cristatum) exhibited significant levels of linkage disequilibrium (LD) (P≤0.05), suggesting the potential utility of LD-based association mapping in Agropyron species. In conclusion, this research sheds light on the genetic diversity of Agropyron species and provides valuable insights into their potential applications in soil protection and forage production, as well as the prospects for enhancing genetic variability and heterosis in these species.
Citation: Hatami Maleki H, Mohammadi R, Firouzkuhi F, Darvishzadeh R, Zeinalzadeh-Tabrizi H (2023) Molecular evidence depicts genetic divergence among Agropyron elongatum and A. cristatum accessions from gene pool of Iran. PLoS ONE 18(11): e0294694. https://doi.org/10.1371/journal.pone.0294694
Editor: Aimin Zhang, Institute of Genetics and Developmental Biology Chinese Academy of Sciences, CHINA
Received: September 1, 2023; Accepted: November 6, 2023; Published: November 30, 2023
Copyright: © 2023 Hatami Maleki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Zeinalzadeh-Tabrizi, Hossein, 2023, "Agropyron ISSR Dataset", https://doi.org/10.7910/DVN/BA6XKU, Harvard Dataverse, V1.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The genus Agropyron, from the family Poaceae, is one of the important pasture plants that is native to Europe and Asia. Agropyron spp. is a perennial herb with diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), and hexaploid (2n = 6x = 42) genomic formulas [1]. It is shown to be a good supply of animal feed as well as a suitable habitat for household animals and wildlife. These genus’ plants are useful for weed control, soil stability, and watershed management, as well as providing a rich gene pool for breeding bread wheat varieties [2, 3]. Moreover, Agropyron plants have beneficial traits such as resistance to biotic and abiotic challenges such as low temperature, salt, drought, diseases, and pests, which protect the plant’s gene pool and lead to enhanced pastures and greater fodder production [4, 5].
The genus Agropyron contains several species, 19 of which are found in Iran [6]. The identified species of Agropyron are allopolyploids with the P genome. This genome can be combined with the St genome from Pseudoroegneria spicata and the Y genome from an unknown donor in various allopolyploid combinations, including the StP genome from Douglasdeweya wangii and the StYP genome from the genus Kengyilia [7]. A. cristatum (2n = 28, genome PPPP), known as crested wheatgrass, is a perennial plant with medium height that is known as a cool-season plant and is resistant to drought as well as cold stresses [4]. A. cristatum has a divergent and deep root system, which makes it a suitable choice for soil stabilization. There are some reports about crossing A. cristatum with bread or durum wheat with the aim of introducing desirable genes like disease resistance as well as grain yield from A. cristatum. By acquiring wheat-A. cristatum disomic addition lines, it is possible to utilize these desired genes for improving wheat varieties. Till now, wheat-A. cristatum 1P, 2P, 3P, 4P, 6P, and 7P disomic addition lines have been successfully created, and many excellent genes were located in specific chromosomes and transmitted into wheat [8]. For example, it has been found that the A. cristatum 6P addition line confers gene clusters related to yield, such as multiple florets and grains per spike, and the 2P addition line possesses gene clusters related to disease resistance, including powdery mildew, leaf rust, and stripe rust [9–11]. Nevertheless, it’s worth noting that the wheat-A. cristatum 5P disomic addition line has not been documented. Another notable species belonging to the Agropyron genus is A. elongatum, commonly referred to as tall wheatgrass, renowned for its robust resistance to salinity stress and its ability to endure challenging environmental conditions [5]. It is an essential natural source for wheat breeding because of its high seed protein content and resilience to plant disease. Many different types of hybrid plants have been developed by sexually crossing common wheat with A. elongatum or A. cristatum [5, 12]. Disease resistance genes, along with traits related to plant characteristics like dwarfism, photosynthetic efficiency, and yield, have been effectively incorporated into wheat from A. elongatum. As a result, several valuable wheat genotypes have been released, including Xiaoyan 6, Xiaoyan 759, Xiaoyan 22, Gaoyou 504, and Shanrong 3 in China, as well as Oasis and Seri in Mexico [3]. In detail, Lr24/Sr24, Lr19, Sr26, and Sr43 genes controlling rust resistance and the Fhb7 gene controlling Fusarium head blight resistance from Thinopyrum ponticum and Thinopyrum elongatum, as well as powdery mildew resistance genes including Pm21, Pm62, and Pm55 from Dasypyrum villosum, have been introgressed into wheat [13].
In order to guarantee future food and nutritional security, plant genetic resources are crucial [14]. Thus, evaluating genetic diversity and understanding the relationships between germplasm collections improves germplasm management and genetic advancement. Several anatomical and molecular attributes have been introduced for the evaluation of genetic variation in selected germplasm. The examination of divergence and grouping of individuals may be performed in a straightforward, precise, and expeditious way using genetic markers regarding their stability across tissues and growth stages, impenetrability by environmental effects, and fast techniques for declaring genetic variability [15]. The study of genetic diversity in native Agropyron spp. accessions is relevant for genetic resource conservation, widening the genetic base, and practical applications in breeding programs. Several molecular marker systems, such as simple sequence repeats (SSR) [4, 16], genotyping by sequencing (GBS) [17], and functional markers related to the P genome of Agropyron [18] have been used to evaluate the genetic variability of Agropyron spp. germplasm. Nowadays, because of the advent of molecular markers based on repeated sequences of the genome, such as ISSR markers [19], the genetic diversity of plant germplasm may be assessed in greater depth. These marker systems offer distinct advantages and have found widespread use in assessing genetic variation and population structure in forage crops [20].
Given the limited reports regarding the genetic variability of Agropyron spp. in Iran, it is hypothesized that significant genetic diversity will be exhibited by the domestic accessions of Agropyron spp. when they are assessed using ISSR markers. Furthermore, it is expected that distinct patterns in the population structure of these accessions will be revealed, which could contribute to the conservation of genetic resources and provide information for breeding programs concerning this important pasture plant.
Materials and methods
Plant material, genomic DNA extraction, and ISSR assay
In this study, 32 native accessions of Agropyron spp. from two species, A. cristatum (7 accessions) and A. elongatum (25 accessions), were generously provided by the Agricultural and Natural Resources Research and Education Center’s Gene Bank, Isfahan, Iran (https://esfahan.areeo.ac.ir/). Regarding the plant material, the collection of plant specimens adhered to the pertinent national guidelines and regulations. Table 1 shows the code and origin of the accessions investigated. Regarding Table 1, some accessions have the same code but differing numbers, for example, accessions 1000/116-1 and 1000/116-2, which show that this genotype originated from the common population. Because Agropyron is a cross-pollinated plant, each of the derived plants may have different characteristics. In the greenhouse, seedlings from each accession are grown in individual plastic pots. Once the plants had matured sufficiently, leaves from all of the pots were collected and combined to obtain genomic DNA. The genomic DNA was isolated using the technique described by Doyle and Doyle [21]. The DNA quality was determined by running 1μl DNA in 0.8% agarose (w/v) gels in 0.5X TBE buffer (45mM Tris base, 45mM boric acid, 1mM EDTA pH 8.0). In the next step, a NanoDrop spectrophotometer (ThermoFisher Scientific, USA) was used to measure the concentration of the DNA samples. Samples of DNA were then prepared for amplification by polymerase chain reaction (PCR) at a concentration of 20 ng/mol of genomic DNA. In this study, molecular diagnostics was applied by using 37 ISSR primers [22] presented in Table 2. For ISSR analysis, the polymerase chain reaction was carried out in a volume of 13 μl using the Biometra UNO II thermocycler (Analytik Jena, Germany). The reaction mixture contains 20 ng DNA, 6 μL master mix (dNTP, Mgcl2, Taq DNA polymerase), 5 μL ddH2O, and 1 μL primer. The manufacturing of mater mixes and the synthesis of primers are accomplished by Sina Clone Company, Tehran, Iran. The PCR program was as follows: an initial step of 4 min at 94°C, followed by 35 cycles of 94°C for 45 s, 52°C to 54°C for 45 s (annealing temperature, which differed depending on the primer type), 72°C for 2 min, and 10 min at 72°C. The reaction products were mixed with an equal volume of formamide dye (98% formamide, 10 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol) and resolved in 2% (w/v) agarose gel (0.5X TBE). Then, they were stained with ethidium bromide (1.0μg ml-1) and photographed under UV light by the Gel Documentation System (Bio-Rad, Canada). Here, all of the presented chemical reagents were prepared by Sigma-Aldrich.
Data analysis
A binary data matrix was constructed by assigning a value of 0 (absence) or 1 (presence) to each band in the PCR amplification products for each of the 32 accessions. The POPGENE version 1.31 software [23] (University of Alberta, Canada) was then used to compute a variety of statistics, including the total number of loci, the number of polymorphic loci, the effective number of alleles (ne), the Shannon’s Information Index (I) [24], and the Nei’s genetic diversity (h) [25]. A model-based Bayesian approach in the program STRUCTURE 2.3.4 [26] (University of Stanford, USA) was used to infer population structure among the examined Agropyron spp. accessions. In this regard, 10 independent runs were performed with the setting the number of subpopulations (K) from 1 to 10, burn in time and MCMC (Markov Chain Monte Carlo) replication numbers both set to 500,000, and a model for admixture and correlated allele frequencies. Delta K (ΔK) based on the second-order rate of change in the likelihood [27] was used to represent the K value. Inferred ancestry estimates of individuals (Q matrix) were derived for the selected population [28]. In order to classify and visualize specimen dispersion, the principal coordinate analysis as well as neighbor-joining clustering were implemented by GenAlEx [29] and Darwin (CIRAD, France) software, respectively. The linkage disequilibrium (LD) was estimated with TASSEL 2.1 [30] (Cornell University, USA). In this study, Jaccard similarity coefficients were calculated among accessions belonging to each species separately. Then, using a similarity matrix, the UPGMA algorithm has been applied to classify accessions of A. cristatum as well as A. elongatum in Darwin software [31].
Results
The molecular evidence presented in this study clearly demonstrates the presence of genetic diversity among the cultivated Agropyron accessions from Iran. The DNA marker analysis revealed a total of 956 loci in the genome of A. elongatum and 705 loci in the genome of A. cristatum (Table 2). A sample output of germplasm fingerprinting is shown in Fig 1A. In the species A. elongatum, the highest and lowest values of amplified loci were calculated for ISSR23 ((AC)8GCT) and ISSR2 ((GA)8YT) respectively. Also in the species A. cristatum, ISSR23 ((AC)8GCT) and ISSR3 ((GA)8YG) had the maximum and minimum values of total loci, respectively. Results showed that 97% of amplified loci in A. elongatum and 84% of amplified loci in A. cristatum are polymorphic (Table 2). Using the Jaccard similarity matrix and UPGMA algorithm, the studied Agropyron spp. accessions were partitioned into two main groups, including groups I and II (Fig 1B). Group I involved accessions that belonged to the species A. cristatum, while all accessions from the species A. elongatum were located in group II. Here, the highest value of the calculated cophenetic correlation coefficient (r = 0.93) verified the suitability of the distance measurement method and also the classification algorithm. A model-based Bayesian technique in the program STRUCTURE was utilized to reveal the genetic structure of the analyzed germplasm. After using Evanno’s approach, the most likely value of K was found to be two. Table 1 shows the membership percentage of each accession to recognized subgroups (Q values). Accordingly, 32 Agropyron spp. accessions were partitioned into two main subgroups, including “green” and “red” which corresponded to two species (Fig 2A–2C). Some accessions, including "G16", "G19", "G20", "G21", "G22", "G23", "G24" and "G25" were identified in the “Mix” subgroup (Table 1 and Fig 2A) in order to have Q values lower than %70 in the group [32] and therefore, these genotypes have a more likely admixture genome.
A: Genotyping profile of Agropyron spp. accessions using the "ISSR16" primer (the scoring district in gel was illustrated with a red box, and a 1kb DNA ladder was used in this study); B: Classification of the studied plant germplasm based on the UPGMA algorithm.
A: Genetic relatedness of studied Agropyron germplasm based on 1013 ISSR markers (The color of the bars indicates the two subpopulations identified through the STRUCTURE program); B: Principal coordinate analysis for visualization of Agropyron germplasm based on ISSR data; C: Classifying studied Agropyron genotypes based on identified ISSR loci using neighbour joining clustering method.
Here, analysis of molecular variance by assuming the existence of two identified subgroups (Fig 3A), revealed that 31% of genetic variability belonged to between-groups (between species), whereas 69% of variation was related to within-groups (within species). As shown in Fig 3B and Table 3, the mean values of the Na, Ne, I and He parameters at A. elongatum were higher than those found at A. cristatum (Table 3). Also, the number of private loci detected for A. elongatum was higher than that for A. cristatum (Fig 3B). Regarding the r2 value [33] 5.2% and 0.5% of all possible pairs of ISSR loci showed a significant level of LD (P≤0.05) in A. elongatum and A. cristatum, respectively (Fig 4A and 4B). The high level of LD opens an avenue for studying genome-wide association analysis on this inspected association panel. In the following, the inspection of genetic variability inside each species was done by calculating Jaccard similarity coefficients (Tables 4 and 5) accompanied by the UPGMA clustering algorithm (Fig 5A and 5B). Overall, the Jaccard similarity coefficient had low values for both Agropyron species. In detail, the Jaccard similarity coefficient varied between 0.33 and 0.66 in species of A. cristatum (Table 4) and 0.25 and 0.7 in species of A. elongatum (Table 5). Moreover, two and three heterotic groups were identified for each of the A. cristatum (Fig 5A) and A. elongatum (Fig 5B) species, respectively. In the species A. cristatum (Fig 4A), three accessions were located in the same group, and others established a second group, whereas the arrangement of A. elongatum accessions in heterotic groups was as follows: three groups with one, nine, and fifteen accessions.
Analysis of molecular variance (A) and ISSR genotyping pattern across studied Agropyron species (B). No. Bands = No. of Different Bands, No. Bands Freq. > = 5% = No. of Different Bands with a Frequency > = 5%; No. Private Bands = No. of Bands Unique to a Single Population; He = Expected Heterozygosity = 2×p×q.
Identification of heterotic groups in studied A. cristatum (A) and A. elongatum (B) accessions using the UPGMA algorithm.
Linkage disequilibrium calculated for A. cristatum (A) and A. elongatum (B). above and below diagonals depict r2 and P-values, respectively.
Discussion
Beside the feeding application of the genus Agropyron, some species, such as A. cristatum [34] and A. elongatum [3] possess promising novel genes that can be used in wheat improvement. Iran is considered a rich resource for Agropyron spp. [35], and thus collection and diversity analysis of domesticated Agropyron accessions are critical in order to accelerate wheat breeding programs and achieve sustainable agriculture through rangeland management. In this study, 32 domesticated Agropyron accessions from the species Agropyron elongatum and Agropyron cristatum were examined. Our finding showed the existence of genetic variability among accessions based on ISSR markers, which was in line with reports by Mohammadi, Panahi [36] and Mohammadi, Amiri [20]. As inferred by the represented ISSR assay, the genetic variability among Agropyron spp. accessions can be inferred and clarified by focusing on the genomic sequence between two SSRs. Also, similar to previous reports [20, 37] each studied ISSR primer could amplify numerous loci across the genome of Agropyron spp. and so this marker system could be implemented for the construction or saturation of the genetic linkage map of Agropyron spp. [38] in addition to diversity analysis. Interestingly, the majority of amplified loci in two species were polymorphic, and this item may result from the old history of the genus Agropyron and the accumulation of mutation and genetic recombination within this period. In this research, the highest value of amplified loci achieved by primer ISSR23 ((AC)8GCT) could be effectively applied for the evaluation of genetic variability among Agropyron accessions.
The categorization of the examined accessions, employing both the UPGMA method and Bayesian analysis, unveiled the presence of two distinct groups, with each group comprising accessions belonging to the same species. So, it can be exploited that these species (A. elongatum and A. cristatum) have different genetic structures. Hence, it is mandatory to exert population structure and kinship relationships in association mapping studies of Agropyron spp. to avoid false positive results in identifying significant trait-marker relationships [39]. It is concluded that by applying ISSR markers, a breeder can distinguish two species from each other at the primary growth stage and save time. Agropyron spp. differentiation is important because each species has its own growth habit; for example, A. elongatum has a longer days to flowering period as well as a shorter plant height than A. cristatum [4, 40]. In the following, an ANOVA for ISSR data manifested the magnitude of within-species genetic variability, which was paralleled with the findings of Che, Yang [16] and Absattar, Absattarova [2]. Here, Nei [25] and Shannon [24] diversity indices also verified a high level of diversity within A. elongatum from Iran. Among the studied Agropyron species, A. elongatum had more private alleles than A. cristatum, which implies promising selection potential in A. elongatum. Evidence of private alleles in both analyzed accessions demonstrates their evolutionary importance [41]. On the other hand, both studied species have notable conservation value except for A. elongatum, which has a higher proportion. The population structure, a tool employed to elucidate the connections between individuals within and among populations [28], offered valuable insights into the evolutionary relationships among the individuals studied within the provided germplasm. As a benefit of this method, genotypes with an admixture genome could be recognized. For instance, in the present study, in addition to the identified subgroups (Green and Red), eight accessions ("G16", "G19", "G20", "G21", "G22", "G23", "G24" and "G25") are admixtures. Such mixed accessions may be a consequence of selection pressure or seed transfer between regions [42]. However, the cross-pollinated nature of Agropyron spp. could also lead to this phenomenon. Close to population structure, the existence of LD is a prerequisite for a given plant population to be utilized in association mapping analysis [33]. In this study, two studied species had different sample sizes, but generally, the LD value belonged to the species A. elongatum was higher than the LD value calculated for A. cristatum. Such a level of LD could guarantee the success of marker-trait association identification through an association mapping approach in Agropyron spp. [17] especially A. elongatum.
These additional analyses, involving the calculation of Jaccard similarity coefficients and species-specific classification, were undertaken to pinpoint unique genotypes within each species. This is crucial because the genetic distance between parental genotypes stands as the foremost factor influencing the manifestation of the heterosis phenomenon [43]. Here, the calculated Jaccard coefficients between accessions inside each species had lower values, implying a vast within-species diversity. So, it is possible to achieve genetic progress through simple selection in Agropyron germplasm. Likewise, identified genotypes from different heterotic groups can be implemented for the production of synthetic varieties, which is the best breeding method for forage crops like Agropyron spp. [44] to achieve heterosis.
Conclusion
In summary, the genetic diversity within Agropyron species from Iran, particularly A. elongatum and A. cristatum, stands out significantly, especially in the context of their utilization as fodder crops. This germplasm has been effectively characterized and differentiated using ISSR markers. Both of the studied Agropyron species exhibit distinct historical evolutionary patterns, as demonstrated through population structure analysis. Our research underscores the presence of substantial genetic diversity and the identification of distant heterotic groups within each of these species, which underscores the potential for leveraging heterosis in the development of forage cultivars. Additionally, concerning linkage disequilibrium, our study suggests that the studied Agropyron panel, as well as other panels from Iran, holds promising potential for application in genome-wide association studies aimed at mapping specific desirable traits.
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