Genetic Diversity Assessment of Saltcedar (Tamarix ramosissima Ledeb.) Populations in Isfahan Province Using CDDP Molecular Marker

Abstract

Tamarix species are tolerant to adverse environmental conditions and have a significant distribution in dry and salty areas. Tamarix ramosissima is the most widely distributed species among the Tamarix in Isfahan province and is mostly located in dry and desert areas. Due to the high morphological diversity of this species, the lack of population differentiation, and to investigate the diversity in the populations of this species, 55 populations from 15 cities in Isfahan province were identified, and one individual was sampled from each population. After extracting their genomes, 10 CDDP marker primers were used to investigate the genetic diversity of these plant populations. NTSYS-pc, GenAlex, and PowerMarker software were utilized for data analysis. The results showed that 102 bands (80.168%) were polymorphic among the 124 bands produced. On average, each primer created 7.85% polymorphic bands. The WRKY-F1 primer identified more gene loci than other primers by producing 16 bands. The average polymorphic information content (PIC) was calculated to be 0.308. The indicator index for each primer was, on average, 3.865. Molecular analysis of variance indicated that intra-population variation was higher than inter-population variation. Cluster analysis and PCoA revealed the existence of two separate populations and three subpopulations among them. In general, the effectiveness of the CDDP marker in differentiating the populations of Tamarix ramosissima and the high genetic diversity within the populations, which is a valuable trait for breeding purposes, were confirmed. Introduction The genus Tamarix, included in the Tamaricaceae family, is one of the most taxonomically challenging tree genera. It is not possible to separate and differentiate species within this genus using vegetative traits alone. Additionally, hybridization between species of the genus, followed by introgression, has further complicated the classification of this genus. Tamarix species are tolerant to adverse environmental conditions and have significant distribution in dry and salty areas. T. ramosissima is the most widely distributed species within this genus in Isfahan province and is predominantly found in dry and desert areas. The high morphological diversity of this species, coupled with the lack of population differentiation, has underscored the necessity of a careful study of its populations. The genetic diversity of T. ramosissima has not yet been studied in Iran. Furthermore, detailed information about the taxonomy, genetic structure, and population genetics of the species that grow in Iran is scarce. Therefore, this research aims to investigate the populations of this species in Isfahan province using the CDDP molecular marker. Materials and Methods To investigate the diversity in the populations of this species, 55 populations from 15 cities in Isfahan province were identified, with one individual sampled from each population. After extracting their genomes, 10 CDDP marker primers were utilized to examine the genetic diversity of these plant populations. For data analysis, NTSYS-pc, GenAlex, and PowerMarker software were employed. Genomic DNA was extracted from plant leaves. The quantity and quality of the extracted DNA were evaluated using a spectrophotometer at a 260 nm wavelength and through 0.8% agarose gel electrophoresis. The initial annealing step for isolating and denaturing the template DNA was set for 4 minutes at 94°C. This was followed by 35 cycles, each including an annealing step performed at 94°C for 1 minute. The PCR reaction products were visualized using electrophoresis on 1.5% agarose gel, followed by documentation with a gel documentation machine. Research Findings The results indicated that using 10 primers, a total of 124 bands were produced, of which 102 bands (80.168%) were polymorphic. The WRKY-F1 primer produced 16 bands, identifying more gene loci than other primers, while the WRKY-R3 primer produced the fewest, with 8 bands. The lowest and highest levels of polymorphism were associated with the primers WRKY-R3, MADS-4 (with 5 bands, 62.50%, 45.45%), and ABP1-1 (with 15 bands, 100%), respectively. On average, each primer generated 85.7% polymorphic bands. The average polymorphic information content (PIC) was calculated to be 0.308. The highest PIC value was associated with the MADS-2 primer at 0.455, while the lowest was for the KNOX-2 primer at 0.0975. The average marker index for each primer was 3.865, with the highest and lowest values being for ABP1-1 (6.664) and KNOX-2 (1.56), respectively. Molecular analysis of variance revealed that intra-population variation was higher than inter-population variation. Cluster analysis and PCoA demonstrated the existence of two distinct populations and three subpopulations among them. Mantel's test results showed the amount of genetic difference, genetic distance, and geographic distance between the populations of saltcedar, indicating no significant relationship between genetic distance and geographic distance at the 5% level. Discussion of Results and Conclusion In the present study, a very high level of genetic diversity was observed within the populations at each location. Considering the ecological conditions prevalent in the Isfahan region, this degree of diversity is not unexpected. Indeed, unfavorable environmental conditions and geographical isolation are factors that can lead individuals of a species to accumulate genetic changes during the process of adaptation to their environment. The efficacy of the CDDP marker in differentiating the populations of T. ramosissima, as well as the high genetic diversity within these populations, which is a valuable trait for breeding purposes, was confirmed. Additionally, the analysis of population similarity, as demonstrated through dendrogram construction and PCoA coordinate axis plotting, revealed that the studied populations are divided into two main groups and three subpopulations. The genotypes found in Isfahan exhibited the highest genetic diversity among the populations.