Comparison of genetic characteristics among species of Suaeda spp. (Amaranthaceae) in the western Anbar plateau - Iraq

Background: Comparison of genetic characteristics among species of Suaeda spp. Objective: identifying the genetic characteristics among species Suaeda (Amaranthaceae) in Anbar Governorate. (From August 2020 to September 2021 fresh plant samples were collected in the flowering stage for the studied species). Materials and methods: The genetic diversity of the Suaeda species was studied after DNA extraction and using the Inter Simple Sequence Repeats Reaction method to recorded in the genetic aspects. DNA was extracted from young leaves of the studied species. The concentration and purity of the DNA were determined, and the detection of genetic relationships between the studied plant species as well as finding this genetic fingerprint by ISSR Reaction. Results: The results showed that the plant samples collected during study belong to species of the genus Suaeda , and these species were: S. aegyptiaca, S. altissima, S. carnosissma, S. fruticosa, S. monoica, S. vera and S. vermiculata symbolized by (S1, S2, S3, S4, S5, S6, S7) respectively for the purpose of brevity. The results showed that there was the highest close between S. aegyptiaca and S. vera which amounted to 0.6319, and the results showed that there was the least closeness between S. aegyptiaca and S. carnosissma which amounted to 0.4231. Conclusion: Genetic traits are among the stable traits that can be adopted in separating the studied species. The genetic study, especially at the DNA level, is one of the most important modern taxonomic studies that rely on PCR technology for the accuracy of its results and the speed of obtaining results.There are at least seven species of the genus Suaeda in the western province of Anbar Governorate - Iraq.


Introduction
The Suaeda genus of plants is also known as seaweeds as well as seablites (1).Most species are confined to saline or alkaline soil habitats, such as coastal salt flats and tidal wetlands.They contain a trait seen in different plant genera that thrive in saline habitats (halophiles).There are about 110 species of the genus Suaeda (2).
The genus Suaeda includes plants that use the carbon-fixing pathway (C3 or C4), and the C4 pathway evolved independently in the same genus, and there are now about 40 species of the genus Suaeda that use the C4 pathway.Suaeda aralocaspica, classified in its Borszczowia section, uses a specific type of C4 photosynthesis without the typical Kranz anatomy leaf anatomy (3)(4) (5).
Suaeda can synthesize natural substances of strong antioxidant activity.It is considered a renewable source of energy, food and edible oil for a large number of people who live in a harsh environment with high salinity and drought conditions.This is due to its relatively large amounts of fixed oils, minerals and vitamins, which make it a potential renewable source for foods.These plants are also of great benefit because they are used as alternative medicines.In addition, Suaeda is used to treat various diseases due to its high content of polyphenols and flavonoids (6) (7).
Despite the multiplicity of the importance of the sage family, it has not been studied well in the world from a taxonomic point of view, due to the limited available taxonomic characteristics and the succulent nature of several types of them, and the delay in flowering and fruiting time as well as the unattractiveness of many of its types (8).
The Inter Simple Sequence Repeats reaction is used to find the contrast between different organisms (9) (10).It did not receive the required attention in Iraq and the world.However, there are recent contributions to cover the lack of information on the classification of family members, especially in Africa and Asia (11).
Based on what has been mentioned and the scarcity of local studies on this genus and the absence of information about it and its relationship to different photosynthesis pathways on the one hand, and its relationship to the taxonomic and evolutionary aspect on the other hand, the current study aims to record as much information as possible about several species of the genus Suaeda spp.In Anbar Governorate, western Iraq.

Sample collection
Plants were collected from the western plateau of Anbar Governorate (Ramadi, Fallujah, Khalidiya, Habbaniyah, Al-Muhammadi, Hit, Al-Baghdadi, Haditha), where several tours were conducted in the region and the tours were between periods of time ranging between 15/8/2020 and 15/9/2021 and through it the study samples were collected, which represented seven species belonging to the genus Suaeda.

Sample preservation
The samples were preserved after collection for the purpose of preparing them for the study.Parts of the sample were pressed with a wooden piston to be dried and to study the morphological and taxonomic characteristics.Parts of the samples were also placed in plastic bags, they were kept at a temperature of 4 o C until conducting the genetic study.

Method of crushing plant leaves of the studied species
Weigh (1 g) of the leaves of the studied samples and cut them into several small pieces using sterile scissors and put them in the pre-cooled ceramic mortar after which liquid nitrogen is added.The temperature is -20 o until the start of the DNA extraction process.

Isolation of Genomic DNA
DNA was isolated from the young leaves of the above study species using the Genomic DNA Kit Plant supplied by SCIENTIFIC (USA) IBI.Electrophoresis was carried out using a 1% agarose gel, where the samples were carried over with a voltage difference of 100 millivolts for an hour, and the DNA was investigated by exposing it to a UV transilluminator with a wavelength of 256 nanometers.

Determination of the extracted DNA concentration and purity
The DNA concentration was estimated by measuring the absorbance of the UV spectrum using a spectrophotometer and at a wavelength of (260) nm.A DNA sample was added to 1980 μl of the solute solution and then placed in a Spectrophotometer at a wavelength of (260) nanometers, and after reading the screen of the device, the following equation was applied to calculate the concentration of DNA: DNA concentration µg/µL = absorbance reading per 1 ml of sample at wavelength (260) x inverse of dilution (100) x (50/1000) As for the purity of each sample of the study, it was estimated by dividing the absorbance reading at the wavelength (260) nm by the reading of the absorbance at the wavelength (280) nm (12).

ISSR reactions
ISSR reactions were performed on 7 samples from the studied family, based on the (13).
 The Substances and solutions required for ISSR reactions:

3.Results: 3.1. genomic DNA extraction
The agarose gel electrophoresis at a concentration of (1%) showed the emergence of a single bund representing the chromosomal DNA of the different species of Suaeda (Fig. 1).

Results of ISSR reactions
Table (3) and Figure (2) shows the results of the genetic study.In this study, 10 primers were used, as all the primers showed doubling.The results of doubling were appropriate to reveal the genetic relationships between the studied plant species as well as finding their genetic fingerprint.The results data were recorded on the basis of the presence or absence of DNA doubling sites, association sites, total bundles, and the percentage of total bundles, as well as the variation in the sizes and numbers of loci, the highest and lowest value.For link sites in every type and in every primer, the presence of unique sites and absent sites.https://doi.org/10.24126/jobrc.2023.17

•Inferring the ISSR reactions for the prefixes used
Table (3) and Figure (2) shows the results of ISSR reactions.The highest primer in terms of the number of binding sites was (SSK7) with (55) bundles, and the lowest primer in terms of the number of binding sites was the primer (SSK2) with (27) bundles, the highest molecular size in binding sites were (1200bp) in the primer (SSK4) and the lowest molecular size was (100bp) in the primer (SSK6).

Results of ISSR reactions for the studied species
Table (4) and Figure (2) shows the use of the ISSR index to analyze genetic variation among eight types of cruciferous plants, determine the genetic dimension and find genetic fingerprints for some species.Primarily, as well as the lowest and highest molecular size, the number of unique and absent sites for each species.

• Inferring the ISSR reactions for the studied species
Table (4) and Figure (2) shows that the species (S7) had the highest number of binding sites (70) sites and a percentage (15.837%)and the lowest number of binding sites (52) sites and a percentage (11.765%)appeared in the type (S4), which had the highest molecular size It appeared in the type (S5) (1200bp) and the lowest molecular size (100bp) appeared in the type (S6), the highest number of binding sites for unique packages (10) sites appeared in the type (S3), and the lowest number of binding sites for unique packages (4) sites In the type (S6), and the type (S3) was distinguished by one link site for an absent band, while the results of the other studied types did not show any absent bands.

Estimation of genetic affinity based on the results of the ISSR .
The genetic affinity between the studied species was estimated using the genetic program (NTSYSpc 2.10e) based on the presence of bundles common to each of the studied species.The results were analyzed by the Nei equation (14).Table (5) and Figure (3) shows the genetic affinity between the studied species, which ranged between (0.4231 -0.6319).When the genetic material (DNA) between the two species matches, it indicates that the value of the genetic distance between them should be zero, and the percentage of genetic similarity between the two species was (100%) (15).

Discussion:
Previous studies of molecular and morphological evolution on the genus Suaeda by (3) contributed to clarifying the taxonomic system of the sub-genus and the order of divisions in the genus Suaeda, while recent molecular genetic studies have found unclear relationships between Suaeda spp.At the species level (16) (17).

Conclusion:
Genetic traits are among the stable traits that can be adopted in separating the studied species.The genetic study, especially at the DNA level, is one of the most important modern taxonomic studies that rely on PCR technology for the accuracy of its results and the speed of obtaining results.There are at least seven species of the genus Suaeda in the western province of Anbar Governorate -Iraq.
of PCR reaction products of Primers SSK1, SSK2, SSK3, SSK4, SSK5, SSK6, SSK7, SSK8, SSK9 and SSK10 by ISSR indicators of Suaeda plant species on agarose gel at a concentration of 1% and at a voltage difference of 100 mA for 45 minutes

Figure ( 3 )
Figure (3): Genetic relationship between the studied species according to ISSR indicators.Symbols represent the studied samples