Genetic diversity and relationships among Iris aucheri genotypes determined via ISSR and CDDP markers

doi:10.21203/rs.3.rs-4836588/v1

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Genetic diversity and relationships among Iris aucheri genotypes determined via ISSR and CDDP markers

https://doi.org/10.21203/rs.3.rs-4836588/v1

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published 10 Sep, 2024

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Iris aucheri, which belongs to the Iridaceae family, is one of the most important wild ornamental plants distributed widely throughout the Iraqi Kurdistan Region (IKR) (north of Iraq). The genetic diversity of this plant species is partly known. Thus, ten ISSR markers and ten CDDP markers were utilized to evaluate the genetic diversity and perform population analysis of forty-eightwild Iris aucheri genotypes from five locations in the IKR. The results revealed 108 and 134 polymorphic bands for the ISSR and CDDP markers, respectively. The mean values of the number of observed alleles (Na), effective number of alleles (Ne), Shannon's information index (I), expected heterozygosity or gene diversity (He), unbiased expected heterozygosity (uHe), and polymorphic information content (PIC) were 1.71, 1.43, 0.39, 0.26, 0.27, and 0.32 for the ISSR primers and 1.53, 1.37, 0.34, 0.22, 0.23, and 0.26 for the CDDP primers, respectively. All the genotypes were classified into two main clades and two populations on the basis of the UPGMA dendrogram and population structure analysis derived from the marker data. The variation within populations was 89.59%, 90.64%, and 90.31% for ISSR, CDDP, and combinations of both markers, respectively. Among all the data, population 2 presented the highest values of the majority of diversity indices. Our results revealed the efficacy of both markers in determining the genetic variability among iris genotypes. This is the first attempt to use these markers to elucidate genetic diversity among I. aucheri plants. These findings can be used in germplasm conservation and future breeding plans.

Iris

Molecular markers

Genetic variation

Diversity indices

Clustering

Iris aucheri (Baker) Sealy is an attractive bulbous species of the subgenus Scorpiris Spach (synonym genus Juno Tratt.) belonging to the family Iridaceae. The subgenus Scorpiris, known colloquially as ‘the Junos,’ is the largest in the genus Iris, with approximately 55 species (Mathew 2000). In Iraq, Scorpiris represents one section of Juno comprising six distinct species: I. aucheri, I. persica, I. pseudocaucasica, I. caucasica, I. heylandiana, and I. gatesii (Townsend and Guest 1985). This subgenus is different from other Iris subgenera because of its distinct flower morphology, reduced flower standards, well-developed falls, large bulbs, fleshy rigid roots, arils present on the seeds, and falcate leaf shapes (Kandemir and Yakupoğlu 2016; Kandemir 2019; Mathew 2001; Rudall and Mathew 1994). Iris aucheri has many variable colors and is common in the forest zone in Iraq and northern Syria, southeastern Turkey, and western Iran (Townsend and Guest 1985). Morphologically, I. aucheri has a strong structure and a large bulb size with the ability to grow, reproduces quickly in dry and wet habitats, and has stunning colorful flowers that can be used in rock gardens and on house balconies (Usta 2002; Güner 2012).

The genus Iris consists of more than 262 species that are native to and widely distributed across the Northern Hemisphere, including in Asia, Europe, and North America (Xu et al. 2019); in Iraq, 13 species have been described (Majeed 2021; Majeed and Salih 2023; Townsend and Guest 1985). Species of Iris are ornamental and medicinal plants, many of which are notably utilized in landscaping because of their simple cultivation, extensive management, and low maintenance costs. These species play significant roles as ornamental plants, cut flowers, and potted plants as a symbol of beauty because of their large, colorful, attractive flowers and wide range of colors. In addition to their ornamental importance, irises have been given other uses throughout time, including being considered valuable medicinal plants and being used in the cosmetic, perfume, and food industries (Kiran et al. 2022). Among the 260 species of the genus Iris, only 53 have published genome sizes (Samad et al. 2020) that are estimated to range from 1.05–28.2 pg/1C (Pellicer and Leitch 2020), and the genus Iris has exceptionally high diversity of chromosome numbers (n = 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 36, 54) (Rice et al. 2015); among them, the number of chromosomes of Iris aucheri is 2n = 2x = 24 (Kiran et al. 2022).

The classification of the genus Iris has been controversial because of the focus on botany and horticulture, and morphological classification also has certain disadvantages, often leading to an incorrect evaluation (Kiran et al. 2022). Therefore, several multidisciplinary studies, such as anatomy, palynology, molecular phylogeny, and karyology, are needed to clarify the taxonomy of the genus. In addition, I. aucheri accessions for economic and industrial purposes must be selected on the basis of the development of agronomic, biochemical, and database genetic programs.

To manage the plant conservation process, understanding the genetic variation of populations is needed. Understanding the genetic diversity and relationships among plant species and varieties is important for breeding and intellectual property rights. PCR-derived markers, which are highly effective tools for studying population genetics, could be developed (Kalendar and Glazko 2002). Because of the diversity of their natural geographical distributions, only a few species have been studied. Morphological marker analysis is a marker technique in which the phenotypic traits of a plant are used to describe its growth and development; these traits are generally affected by the environment (Zhao et al. 2020).

Consequently, molecular markers are more reliable for assessing genetic diversity on the basis of DNA sequence polymorphisms to determine plant genotypes within populations (Karapetsi et al. 2021). Hence, many molecular markers, such as ISSR, CCDP, RAPD, SSR, SRAP, RFLP, and AFLP, have recently been utilized efficiently to adjust genetic diversity among plant species (Amar and El-Zayat 2017; Maeruf et al. 2024). ISSRs are preferred because of their codominant nature, genome redundancy, high polymorphism, and repeatability (Madadi et al. 2017) because the availability of information on the genetic relationships between accessions is significant (Karapetsi et al. 2021).

Since the use of molecular markers, i.e., CCDP and ISSR markers, for analyzing the genetic diversity of iris plants in the Iraqi Kurdistan region (IKR) has not been explored, only a small amount of molecular data are available, especially in Iraq. The present study investigated forty-eight accessions of I. aucheri and determined the overall degree of genetic polymorphism via CCDP and ISSR markers. The knowledge provided will be valuable in hybridizing and improving cultivar-breeding approaches and potential commercial production.

Plant materials

During the bloom period, from the middle of February to the end of April 2021, forty-eight Iris aucheri genotypes were collected from five different locations in Iraqi Kurdistan: Haibat Sultan, Tawella, Bestana, Qaradakh, and Warte. Table 1 and Fig. 1 show the genotype codes, global positioning system (GPS) coordinates, and geographical distribution map. Taxonomists thoroughly examined the samples after collection and compared them to those found in Iraq's national herbarium and flora. The samples were deposited as vouchers at the University of Sulaimani, College of Agricultural Engineering Science Herbarium (SUFA), and are identified with the code SUFA: HOMS#.

DNA isolation

The protocol described by Ahmed et al. (2022) and Valizadeh et al. (2021) was followed for extracting DNA from freshly harvested leaves and storing the samples at -20 °C. The isolated DNA was visualized on a 1.2% agarose gel, and a NanoDrop spectrophotometer (NanoPLUS-MAANLAB AB, Sweden) was used to assess the quality and quantity of the isolated DNA.

PCR amplification of ISSR and CDDP markers

The genetic diversity of Iris aucheri genotypes was analyzed via ten primers for ISSR (Faraj 2023; Rasul et al. 2022) and ten primers for CDDP (Rasul 2023; Tahir et al. 2023a) (Table 2). The PCR mixture consisted of 10 µL of master mix buffer (AddStart Taq Master, Addbio, Korea), 5 µL of extracted DNA (50–100 ng), 5 µL of primer (10 µM) (Addbio, Korea), and 3 µL of deionized water, for a total volume of 23 µL. PCR amplification was performed via a standard PCR machine (MultiGene OptiMax Thermal Cycler, Labnet Company). The program was set to one cycle of initial denaturation at 94 °C for 10 minutes, 36 cycles of denaturation, annealing, and elongation for 1 minute at 94 °C, 1 minute at a specific temperature (depending on the primer), and 2 minutes at 72 °C, and a final extension of 10 minutes at 72 °C. The PCR products were electrophoresed on a 1.5% agarose gel using 1X TBE (tris-base, boric acid, and EDTA) buffer and 0.15 µg/mL ethidium bromide; the gels were exposed to a steady voltage of 85 V for 90 minutes. The stained gel was visualized using a UV transilluminator and photographed using digital imaging equipment.

Statistical analysis and data scoring

The scorable bands were manually identified by assigning 1 and 0 to the presence and absence of bands, respectively. PowerMarker 3.25 software was used to compute the polymorphic information content (PIC) (Ahmed et al. 2021). The unweighted pair-group technique (UPGMA) in XLSTAT version 2020.1.3 (Tahir et al. 2023b) was used to define the accession groups and calculate genetic distances. GenAlEx software, version 6.5, was used to calculate the molecular variance among and within populations along with the diversity indices. To identify ancestry, the model for population structure was analyzed via the STRUCTURE program version 2.3.4 (Pritchard et al. 2000). The population structure was investigated via an admixture model, correlated allele frequencies, and 50,000 Markov chain Monte Carlo (MCMC) repeats followed by 500,000 Markov chain Monte Carlo (MCMC) runs. In ten different simulations, K varied from 1 to 10. The genotypes were clustered using the run with the highest probability (Mekonnen et al. 2020).

ISSR and CDDP marker polymorphisms and discriminating features

The genetic diversity of the forty-eight Iris aucheri genotypes was assessed via ten ISSR primers and ten CDDP primers. The ISSR and CDDP primers generated 108 polymorphic bands (with an average of 10.8 bands per primer) and 134 polymorphic bands (with an average of 13.4 bands per primer), respectively (Table 3). For the ISSR primers, the total number of polymorphic bands (TPB) ranged from 7.00 (UBC-813, UBC-847, and UBC-849) to 15.00 (UBC-811), whereas the total number of polymorphic bands (TPB) varied from 7.00 (ERF1) to 21.00 (ABP1-1) for the CDDP primers.

Among the ISSR markers, UBC-813 and UBC-847 presented the highest number of observed alleles (Na) of 1.80, whereas UBC-834 presented the lowest value (1.49). The maximum values of the effective number of alleles (Ne), Shannon’s information index (I), expected heterozygosity or gene diversity (He), unbiased expected heterozygosity (uHe), and polymorphic information content (PIC) were recorded for UBC-823, with values of 1.57, 0.47, 0.32, 0.34, and 0.40, respectively. The lowest values of Ne, I, He, and uHe were found for UBC-812, with values of 1.30, 0.32, 0.20, and 0.21, respectively. The minimum PIC value was 0.22 for UBC-834. In the case of the CDDP markers, the highest values of Na, Ne I, He, and uHe were recorded with the Myb2 primer at 1.68, 1.47, 0.41, 0.28, and 0.29, respectively. The maximum PIC value was recorded at 0.37 with the KNOX3 primer. The lowest values of Na, Ne, I, He, and uHe were observed for the MADS-1 marker, with values of 1.26, 0.28, 0.17, and 0.18, respectively. The lowest PIC value was indicated by the Myb1 primer. The mean values of Na, Ne, I, He, uHe, and PIC for the ISSR markers were 1.71, 1.43, 0.39, 0.26, 0.27, and 0.32, whereas those of the CDDP markers were 1.53, 1.37, 0.34, 0.22, 0.23, and 0.26, respectively.

Cluster analysis using ISSR, CDDP, and ISSR+CDDP data.

The dendrogram generated via UPGMA demonstrated that the ISSR markers categorized all Iris aucheri genotypes into two primary clusters (Fig. 2A). The first cluster (in blue) contained six genotypes, and two subclusters were produced; each subcluster included three genotypes: G43, G44, and G45. G43, G44, and G45 produced the first subcluster, whereas the second subcluster comprised G42, G46, and G47. The second cluster (in red) had forty-two genotypes, and eight subclusters were generated: the first subcluster included only two genotypes (G36 and G41), and the second subcluster included five genotypes (G21, G34, G35, G38, and G39). The third subcluster included seven genotypes, whereas the fourth subcluster contained three genotypes. The fifth, sixth, seventh, and eighth subclusters comprised 6, 8, 3, and 8 genotypes, respectively. UPGMA was used to cluster the Iris aucheri genotypes on the basis of the CDDP data, resulting in two major clusters (Fig. 2B). The first cluster (in blue) included twenty-two genotypes, and four subclusters were generated, whereas the second cluster (in red) included twenty-six genotypes, with four subclusters. Two main clusters were generated from the combined ISSR and CDDP marker data (Fig. 2C). The first cluster (blue) included eight genotypes, and two subclusters were produced. The second cluster (red) comprised forty genotypes, and seven subclusters were generated.

Structural analysis of the Iris aucheri genotypes on the basis of marker datasets

Structural analysis was utilized to study the population stratification of the forty-eight Iris aucheri genotypes via ISSR markers. The number of genotype clusters was calculated in terms of the K value on the basis of genotypic data from the whole genome. The optimal K value was determined by graphing the number of clusters (K) versus K, with the largest peak occurring at K = 2 (Fig. 3A). The optimal K value suggested that two populations (Pop 1 in red and Pop 2 in green) presented the greatest possibility of population clustering (Fig. 3B). The criterion for membership probability was 0.80. Each subgroup of accessions was determined individually, with fractions less than 0.20 indicating admixture. The first population included forty-two I. aucheri genotypes, whereas the second population included only five I. aucheri genotypes: G16, G17, G18, G19, and G40. Only one genotype (G1) was identified as an admixed genotype between the two populations, which indicates that this genotype is not pure.

On the basis of the CDDP data, the optimal K value was determined via structure–harvester analysis, and the number of clusters (K) was plotted versus K, with a maximum peak occurring at K = 2. (Fig. 3C). The forty-eight genotypes of I. aucheri were divided into two genetic groupings (Fig. 3D). The first group (red) consisted of just four genotypes (G16, G17, G18, and G19), whereas the second group consisted of forty-one genotypes (green). All forty-five genotypes investigated were from pure populations, whereas the remaining three genotypes (G1, G40, and G43) were from an admixed population. The combined ISSR and CDDP genotyping data were used in STRUCTURE software to conduct population structure analysis on forty-eight I. aucheri genotypes via an admixture model. According to the Evano method, the optimal number of clusters is K=2 (Fig. 3E). A genotype was considered a pure member of a cluster if its chance of membership, in that cluster, was greater than 80%. Forty-six genotypes were classified as pure, whereas the remaining two genotypes (G1 and G40) were labeled admixed (Fig. 3F).

Analysis of molecular variance and diversity indices of Iris aucheri populations

Molecular variance analysis (AMOVA) was used to assess genetic differences within and among populations via ISSR, CDDP, and ISSR+CDDP marker data (Table 4). The significant PhiPT values for the ISSR markers, CDDP markers, and their combination were 0.10, with a p value of 0.001; 0.09, with a p value of 0.001; and 0.10, with a p value of 0.001, respectively. The ISSR, CDDP, and ISSR+CDDP marker data indicated that the variance among populations accounted for 10.41%, 9.36%, and 9.69%, respectively, of the total variation. However, the greatest variation occurred within the population, with 89.59, 90.64, and 90.31% for ISSR, CDDP, and their combined markers, respectively.

The diversity indices (Na, Ne, I, He, uHe, PPL, and NPB) of the I. aucheri populations are shown in Table 5. According to the ISSR data, Pop 5 had the greatest Na and PPL diversity indices of 1.81 and 88.89, respectively, whereas Pop 2 had the highest Ne, I, He, uHe, and NPB values of 1.47, 0.42, 0.28, 0.29, and 1.00, respectively. Pop 4 had the lowest values of these indices, with values of 1.42, 1.36, 0.33, 0.22, 0.23, 64.81, and 0.00. Among the CDDP markers, Pop 3 presented the greatest values of Na and PPL, with values of 1.69 and 80.60, respectively. Pop 4 presented the highest value of NPB (5.00 bands). Pop 2 presented the highest values for Ne, I, He, and UHe, i.e., 1.44, 0.39, 0.26, and 0.28, respectively. Pop 4 presented the lowest values of Na, Ne, I, He, uHe, and PPL, with values of 1.25, 1.29, 0.26, 0.17, 0.18, and 55.22, respectively, whereas Pop 5 presented the lowest value of NPB (0.00). On the basis of the combined ISSR and CDDP marker data, Pop 2 presented the greatest values of Ne, I, He, and uHe, with values of 1.45, 0.41, 0.27, and 0.28, respectively, whereas Pop 5, Pop 3, and Pop 4 presented the highest values of Na (1.69), PPL (82.57), and NBP (5.00), respectively. Pop 4 had the lowest Na, Ne, I, He, uHe, and PPL values, i.e., 1.32, 1.32, 0.29, 0.19, 0.20, and 59.34, respectively, whereas Pop 5 had the lowest NBP value (0.00). The mean values of Na, Ne, I, He, uHe, NPB, and PPL were 1.71, 1.43, 0.39, 0.26, 0.27, 82.00, and 0.40 for the ISSR markers; 1.52, 1.37, 0.34, 0.22, 0.23, 71.05, and 2.20 for the CDDP markers; and 1.61, 1.39, 0.36, 0.24, 0.25, 75.93, and 2.60 for the combined markers, respectively.

Juno irises are considered ornamental groups and have been used in gardening for many years. In Iraq, many plants can be considered members of this group, including I. aucheri. This species is morphologically characterized by a wide variety of flower colors, e.g., ranging from white to creamy, dark blue, light blue, and a mix of light and creamy colors. Genetic diversity and population structure are proven effective methods for illustrating these kinds of variability and revealing the level of variation and the relationships between and among populations (Majeed et al. 2024).

With respect to individual molecular properties, specific statistical methods or combinations of methods for the classification of genotypes are usually used in genetic diversity research. The assessment of genetic disparity in plant germplasms can be conducted via a potent technique for identifying superior breeding resources and improving breeding efficacy (Ahmed et al. 2023; Aziz and Tahir 2023). Recently, molecular markers, including RAPD, RFPL, SRAP, and SCoT, have been widely utilized to explore genetic variations among different species of irises (Bublyk et al. 2013; Wang et al. 2009; Wroblewska et al. 2003; Xu et al. 2019).

To preserve genetic resources, it is important to assess the genetic diversity of wild plant species, and molecular marker techniques can be used to identify genetic differences. In contrast to conventional methods, DNA fingerprinting is a novel methodology that may reveal genetic variants in plants with consistent data and authenticity, which is necessary for assessing genetic diversity (Lamare and Rao 2015). Nonetheless, these previously mentioned molecular markers have not yet been applied to Iris aucheri to clarify the genetic differences that exist both within and between populations for this alluring class of bulbous plants.

There is a clear indication that there is an increase in genetic diversity on the basis of the abundance of alleles in the accessions. This approach considers the properties of powerful molecular markers for discriminating the appropriate population for conservation, breeding, and selection. In addition, the trustworthiness of data should be significantly improved via the use of different, high numbers of molecular markers (Kushanov et al. 2021). The ISSR and CDDP markers amplified 242 bands, of which 134 and 108 bands were identified by ISSR and CDDP, respectively. These variations in the detection of bands between the ISSR and CDDP markers indicate the polymorphisms in the genome of I. aucheri. Whereas ISSR markers are concentrated in microsatellite-flanked genomic regions, CDDP markers amplify the conserved sites of functional genes (Ahmed et al. 2021; Aziz and Tahir 2022). These high variations in bands generated by both markers (ISSR and CDDP) are strong indications of high genetic variation in I. aucheri populations on the one hand and are strong indicators of the ability of these markers to reveal the relationships between individuals among these populations on the other hand (Allan and Max 2010; Xu et al. 2019).

Polymorphic information content (PIC) is a significant index for revealing the efficiency of molecular markers for determining the genetic diversity of organisms. According to previous research (Botstein et al. 1980; Santhosh et al. 2009; Wei et al. 2005), a PIC equal to or less than 0.25 is a low-efficiency marker, and a PIC of 0.5 or more is a very powerful marker (Aziz and Tahir 2023; Guo et al. 2014; Haerinasab et al. 2021; Majeed et al. 2024). Accordingly, both markers’ PIC values are above the minimum (0.25), i.e., 0.32 and 0.26 for the ISSR and CDDP, respectively. The greater PIC values in our study aided in selecting the optimal ISSR and CDDP markers for genetic divergence analysis by suggesting a greater degree of ISSR and CDDP marker polymorphism.

Statistical metrics known as diversity indices are employed to illustrate population variation, whereby individual genotypes are allocated to discrete groups. Indices with lower values yield less diversity, whereas indices with higher values yield greater diversity (Majeed et al. 2024). Most of the ISSR indices in our study are superior to those of the CDDP markers except for the number of polymorphic bands. This finding explains why ISSRs are more applicable for revealing the genetic diversity of I. aucheri. The degrees of mean genetic heterozygosity (He) of the ISSR and CDDP markers utilized in this study were 0.26 and 0.22, respectively, indicating that these marker types are effective for genetic diversity analysis in Aucheri irises and confirming the presence of significant genetic variation. These results led to the discovery of a moderate level of genetic variation among samples in a population. The average Shannon's information index (I) values of the ISSR and CDDP markers were 0.39 and 0.34, respectively, indicating their effectiveness in determining the degree of diversity among the accessions.

Cluster analysis is used here as an appropriate scheme for classifying the groups on the basis of their similarities and differences. The topology of the dendrogram here reveals the high scattering of the individuals between and within populations. The first clade in the ISSR, colored blue, consists of six accessions from the Warte site, and the second clade includes the remaining individuals from all the studied sites. In contrast, the topology of the CDDP markers was divided into two clades with nearly the same number of genotypes from all locations. The topology of the combined markers is close to that of the ISSR marker. This proliferation pattern of I. aucheri accessions can be explained by the greater ability of ISSR markers than CDDP markers to identify ancient intra- and interspecific hybridization.

UPGMA clustered all the genotypes into two main groups, with some subgroups. On the basis of both types of marker data, clustering cannot distinguish all genotypes according to location, while the genotypes of these two main groups of each marker also differ. These discordances between the dendrograms may be related to the nature of each marker, which are present in different locations on the iris genome, and the level of polymorphisms obtained. The structural analysis of these two markers classified all the genotypes into two main genetic populations and interspecific crosses, as well as allele introgression between these wild irises; thus, the majority of the genotypes were pure.

According to the AMOVA statistics, genetic variation between and among the populations was observed, and the variation within the populations was greater than the variation between the populations. These results may be related to gene drift and gene flow affecting the studied populations of these iris genotypes. Wroblewska et al. (2003) indicated that natural crossing, clonal propagation, or long-lived species cause high variation within populations, and harsh environments lead to low genetic variation (Arnold 2000).

By introducing molecular markers that are highly capable of revealing interpopulation and intrapopulation genetic variation, it is possible to develop breeding systems that are successful and comprehensive in their conservation efforts. By elucidating the genetic variation in I. aucheri, the present study's utilization of both the ISSR and CDDP molecular markers proved to be successful throughout the initial stages of the investigation. The high variance within the population of I. aucheri suggests that the genetic structure was not affected by the diverse sites. Horticulturists and breeders should consider these variances to establish effective breeding programs to produce new forms and colors of ornamental plants to satisfy the requirements of the market and the horticulture industry.

Author contribution statement: Kamaran Salh Rasul: Conceived and designed the experiments; performed the experiments; wrote the paper; and contributed reagents, materials, analysis tools or data. Hoshman Omar Majeed: Collected the plant materials and wrote the paper. Jamal Mahmood Faraj: Performed the experiments; contributed reagents, materials, analysis tools or data. Djshwar Dhahir Lateef: Conceived and designed the experiments. Nawroz Abdul Razzak Tahir: Conceived and designed the experiments; analyzed and interpreted the data; wrote the paper.

Conflicts of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments: The authors are grateful to the people who worked at the College of Agricultural Engineering Sciences at the University of Sulaimani for their assistance and cooperation throughout the research process.

Funding: This research received no external funding.

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Genetic diversity and relationships among Iris aucheri genotypes determined via ISSR and CDDP markers

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