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Genetic Diversity Robinia pseudoacacia L. Growing in Drought Conditions using ISSR

P.A. Kuzmin1, P.A. Krylov1,*
1Laboratory of Molecular Breeding and Laboratory of Genomic and Postgenomic Technologies, Federal State Budget Scientific Institution “Federal Scientific Centre of Agroecology, Complex Melioration and Protective Afforestation of the Russian Academy of Sciences” 400062, Volgograd, pr-t Universitetskij, 97, Russia.

Background: The aim of this study was to evaluate the genetic polymorphism of two populations of introduced R. pseudoacacia growing under drought conditions using ISSR markers. The individuals of R. pseudoacacia from two populations grow in the Ergeninskaya Upland, where a low level of moisture supply is observed. The difference between the populations lies in the conditions of growth, with the first population having a less favorable hydrothermal regime.

Methods: ISSR markers were used to analyze the genetic structure of R. pseudoacacia. During this analysis, 6 out of 18 ISSR primers with a high information content were selected. Polymorphism and genetic diversity of ISSR loci in R. pseudoacacia were evaluated in two populations based on the obtained electropherograms using POPGENE v 1.31.

Result: The study revealed that individuals of R. pseudoacacia growing in less favorable conditions have the greatest polymorphism of ISSR loci. Among individuals of the first population of R. pseudoacacia, the highest proportion of polymorphism was observed when using ISSR primers UBC 808 and UBC 826. Comparison of the obtained results of genetic structure analysis and the vital state showed that individuals with higher polymorphism, particularly using UBC 808 and UBC 826, have a more pronounced resistance to soil moisture deficit. The obtained results can be further used for selection and developing new resistant forms of R. pseudoacacia.

Currently, one of the tasks of population genetics is to identify the genetic diversity and features of intraspecific differentiation of woody species under arid conditions (Andablo-Reyes et al., 2023; Santos et al., 2023; Firmat et al., 2022; Pathirana et al., 2022). Genetic diversity evaluation is carried out using various molecular markers (Sun et al., 2023; Yan et al., 2022; De Mori et al., 2023), which allow to identify the features of intraspecific population structure and develop effective conservation measures based on them in the future (Zakhozhiy et al., 2020). Moreover, based on the evaluation of genetic intraspecific diversity, individuals with economically valuable traits, including resistance to various types of stress, can be identified and used in selection. This, in turn, reduces the time required for selection activities.
Various non-specific markers, such as ISSR (Ramzan et al., 2021; Abd-Dada et al., 2023), RAPD (Kader et al., 2022, Matsiakh et al., 2023), SSR (Paliwal et al., 2022; Zhao et al., 2023, Nezami et al., 2023; Hamm et al., 2023) and others (Müller et al., 2023; Vaishnav et al., 2023), are used to evaluate genetic diversity of tree species, DNA barcoding (Panigrahy et al., 2024) and GWAS (Banerjee et al., 2023). This is due to the fact that most of woody species growing in arid areas do not have sequenced and annotated genomes (Belyaev et al., 2023). A feature of marker-mediated analysis is its ability to be conducted at any stage of plant development (Novikova et al., 2012). ISSR markers are used for a more informative determination of genetic diversity. ISSR markers are non-specific multi-locus markers that amplify DNA fragments of various lengths up to 5000 base pairs. These markers are located in the genome between microsatellites. ISSR markers are best suited for genotyping and are effective in the absence of specific markers (Luo et al., 2018).
R. pseudoacacia is a promising introduced species that enhances species diversity of woody species in arid regions. This species has several advantages in terms of adaptation to dry conditions, good vital state and functionality compared to other indigenous and introduced species (Lazarev 2020). Given the justification of the chosen research topic, the aim of this study was to evaluate the genetic polymorphism of two populations of R. pseudoacacia, growing in the Ergeninskaya Upland under drought conditions using ISSR markers.
Laboratory and field studies were conducted on the basis of the FSC of agroecology RAS in the laboratory of molecular breeding from October 2022-November 2023. The research objects were two populations of R. pseudoacacia with 30 individuals from each population, growing in the northern part of the Ergeninskaya Upland. The study was carried out based on cluster dendrological collections (cadastre ¹34:34:070010:21-Kirov Forestry, ¹34:34:000000:122- arboretum of the FSC of agroecology RAS). The two populations differ in terms of moisture supply, geological history and relief.
The first population (RPK) was localized in the Kirov Forestry on the upland, which contributed to the formation of specific local growing conditions characterized by a low level of moisture supply. The average height of trees was 6.5 m. The second population of R. pseudoacacia (RPP) grew in the arboretum of the FSC of agroecology RAS, in a low relief area. The average height of trees in this population was around 8 m.
The vital state of R. pseudoacacia was determined visually based on the degree of damage to the assimilation apparatus and tree crowns. According to the method of V.S. Nikolaevsky (1999), the following parameters were evaluated on a ten-point scale: the number of living branches in the tree crowns (P1); the degree of foliage in the crowns (P2); the number of living (without necroses) leaves in the crowns (P3); the average amount of living leaf area (P4).Then the total score (maximum 40 points) of the condition of R. pseudoacacia was determined and the distribution was carried out on a scale of categories: 39-40 points-good condition; 35-38-satisfactory; less than 35 points-weakened plants.
The genomic DNA was extracted from 50 mg leaf samples using the modified cetyltrimethylammonium bromide (CTAB) method with 0,2% β-mercaptoethanol (Shestibratov et al., 2021). Spectrostar-Nano (BMG Labtech, Germany) was used for measuring extracted DNA concentrations in the samples. The concentrations of DNA were set up to 50 ng·μL-1 in TE-buffer and DNA was stored at -20°C for the next amplification experiments. ISSR-PCR-based reaction was performed to detect the polymorphism of R. pseudoacacia genotypes using the synthesized primers presented in Table 1 (CJSC Evrogen, Russia).

Table 1: Codes and sequences of ISSR.

The ISSR-PCR-based amplification was conducted using kit qPCRmix-HS (cat: #PK145L) (CJSC Evrogen, Russia). The ISSR-PCR-based amplification was performed using kit qPCRmix-HS (cat: #PK145L, CJSC Evrogen, Russia) according to the manufacturer’s instructions. The Applied Biosystems QuantStudio 5 (Thermo Fisher Scientific, USA) was used to amplify genomic DNA with ISSR markers. The conditions of ISSR-PCR amplifications included initial denaturation at 95°C for 10 min followed by 40 cycles at 95°C for 30 sec, annealing temperature for the various primers at 55°C for 30 sec (extension) and final extension at 72°C for 4 min.
The 2% agarose gel electrophoresis with TAE buffer was used to separate the results of the ISSR-PCR reactions reactions (Al-Khayri et al., 2022). The size of DNA bands on the gel was determined using a 100+bp DNA ladder (cat: #NL002, CJSC Evrogen, Russia). Ethidium bromide (Helicon, Russia) was used to stain the agarose gel, which was then visualized using a UV illuminator (VilberLourmat, France). The frequencies of polymorphisms and the number of bands produced by each primer were calculated individually.
Data were generated based on the triplicate amplification of each studied primer with consistent bands. The ISSR-based PCR loci were scored as present (1) or absent (0), each of which was treated as independent. Data were compiled in a binary (0/1) matrix using MS Excel. The binary generated data were used to calculate primer binding characteristics using POPGENE Version 1.31 (Louati et al., 2019). The following parameters of genetic variability were used to estimate the population: na = Absolute number of alleles, Ne = Effective number of alleles Kimura and Crow (1964), h = Nei’s (1973) gene diversity, I = Shannon’s Information index Lewontin (1972). Nei’s Original Measures of Genetic Identity and Genetic distance See Nei (1972) was also calculated between 2 populations R. pseudoacacia.
Quantitative data were processed using Statistica 10.0 (StatSoft, Inc. USA) and presented as median (Me) and upper and lower quartiles (Q1-Q3). Statistical significance between samples was assessed using the Mann-Whitney nonparametric test with a confidence level of p<0.05.
Assessment of the vital condition of trees
The evaluation of the vital state of the studied populations of R. pseudoacacia was conducted according to the method of Nikolaevsky, which allows determining the condition of aboveground shoots, leaf blades and the plant photosynthetic apparatus (Fig 1). The analysis of the vital state showed that individuals from the RPP population scored 39.5 points, corresponding to a good vital state. Individuals from the RPK population had a satisfactory vital state. The high scores of vital state in the RPP population were due to a greater number of living branches in the tree crowns, better foliage in the crowns, a larger number of living leaves and a larger leaf area (Fig 1A, 1B).

Fig 1: The appearance of individuals and the analysis of the vital state of R. pseudoacacia.

A decrease in the vital state of plants in the RPK population was noted due to a significant decrease in foliage coverage by 4-6 points (p<0.05, n = 30) and the number of living leaves in the tree crown by 5.5-6 points (Fig 1C). The parameter of the amount of living leaf area also showed a significant decrease for plants from the RPK population by 1.2-1.5 points, compared to this parameter for plants from the RPP population (p<0.05, n = 30) (Fig 1C). This indicates the presence of a morpho-physiological adaptation strategy elements for plants growing in arid conditions. The total viability score of RPP population individuals of R. pseudoacacia was 39.5, which is 3.5 points higher than in the RPK population (p<0.05, n = 30) (Fig 1D).
To identify the intraspecific genetic diversity of R. pseudoacacia, 18 ISSR primers were tested in both populations. As a result of the testing, 6 ISSR primers were selected for their high efficiency in amplifying a larger number of genomic DNA fragments (Table 2).

Table 2: Evaluation of PCR amplification product of ISSR markers in populations R. pseudoacacia.

During the analysis, ISSR primer UBC808 showed differences in locus polymorphism between the two R. pseudoacacia populations. The other 5 primers, UBC818, UBC823, UBC826, UBC857, UBC860, had similar polymorphic loci ranging from 20% to 45.5%. DNA marking of the first and second population using selected ISSR primers allowed for the identification of 33 amplified genomic DNA fragments, 14 of which were polymorphic. The number of amplified fragments varied from 3 to 11 depending on the primer. The size of the fragments ranged from 125 to 1250 bp. The highest number of identified fragments was observed with primer UBC826. The remaining 12 primers did not amplify with the samples of isolated genomic DNA and were therefore excluded from further research. For a more detailed evaluation of the effectiveness of the used ISSR primers, the frequency of occurrence of each locus was evaluated in two populations of R. pseudoacacia, obtained through electrophoresis of amplicons (Table 3). Each analyzed locus of ISSR marker formed a specific spectrum of amplification products in each individual of R. pseudoacacia in the populations (Fig 2).

Table 3: The frequency of ISSR markers of the two studied populations of R. pseudoacacia.


Fig 2: Example of an electrophoregram of amplification products of two populations of R. pseudoacacia with ISSR primer UBC826, Std marker of molecular weight (100-1000 bp), 2-10 – RPK, 11-18 - RPP.

Based on the obtained amplicon spectra shown in Fig 1, binary matrices were compiled for further mathematical processing in POPGENE to evaluate the genetic diversity in two populations of R. pseudoacacia growing in different territories. The results of the evaluation of genetic variability parameters are presented in Table 4.

Table 4: Indicators of genetic diversity in two populations of R. Pseudoacacia.

In population RPK, the average absolute number of alleles and effective number of alleles per locus were higher by 4% compared to population RPP, indicating greater individuality in genetic structure. We also observed a 12% decrease in the Ne parameter compared to Na in both populations. The results of genetic distance evaluation according to Nei showed that the two populations have high genetic similarity. The Shannon index showed low values for both populations, indicating low inter-population diversity, while the proportion of polymorphic loci was higher in the RPK population at 72%.
Thus, 6 of the 18 primers tested are suitable for the analysis of R. pseudoacacia DNA polymorphism, since they can be used to identify a large number of loci and evaluate their polymorphism (Guo et al., 2006). Only 30% of the primers showed effective performance. This may indicate high intraspecific polymorphism in the genetic structure of R. pseudoacacia. Comparative analysis of multi-locus spectra of amplified products of the studied markers in populations revealed high genetic similarity among individuals. However, plants growing in more arid conditions showed a higher degree of polymorphism, potentially enhancing their survival (Shaban et al., 2022). At the same time, there is evidence that less adapted genotypes are eliminated under the pressure of stress factors (Mollashahi et al., 2023). The growing conditions for the first populations of R. pseudoacacia, growing at a higher geographical point on the Ergeninskaya Upland, were more arid and therefore the vital state was worse than that of the second population.
The features of the relief of the Ergeninskaya Upland have influenced the formation of specific habitat conditions (Melikhova, 2022), which in turn have contributed to the formation of populations of various tree and shrub species with different degrees of genetic polymorphism. The research confirms the theory that plants with higher genetic polymorphism are more resilient and capable of surviving in arid conditions. Therefore, it is promising to use more genetically polymorphic individuals in populations for selection of tree and shrub species, especially R. pseudoacacia, to solve the problems of agroforestry and protective afforestation in areas with arid climates.
As a result of the research on the genetic structure of R. pseudoacacia using ISSR markers, it was found that individuals with a higher proportion of polymorphic loci UBC808 and UBC826 may have high resistance to water deficit. The vital state of R. pseudoacacia individuals is satisfactory, while populations in less arid conditions show lower polymorphism of loci and good vital state. The obtained data can be used in the future for selecting more drought-resistant R. pseudoacacia individuals for agroforestry and protective afforestation in arid territories.
The work was carried out within the framework of the State task “Search for breeding valuable genetic material for the creation of new genotypes of tree and shrub species by molecular breeding methods” (No. FNFE-2022-0009).
All authors declare that they have no conflicts of interest.

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