Introduction

There are 4060 species and 372 genera in the Brassicaceae family of mustards, which are found around the world [1], The Mediterranean, American, and northern areas are habitat of this family, which is extensively distributed throughout the world in tropical, subtropical, and temperate climates [2] and [3] . The Brassicaceae family of mustards is a member of the Brassicales or caparales order [4]. It is no longer the same as it was before because it has been split up into multiple families. However, some of its genera have been elevated to the level of a family, such as Brassica to Brassicaceae, and some of its plants are used in traditional medicine. [5] and the Brassicaceae family is one of the families around which there is wide controversy and taxonomic issues.

Transcribed spacer (ITS) of nuclear rDNA is one of the main markers used to infer phylogenetic relationships in Brassica and the wider Brassicaceae, but it has strengths and clear limitations ,The phylogenetic of genusBrassica studied by using ribosomal DNA regions 5.8S,ITS1 and ITS2, [6] . Roles and performance of ITS in Brassica / Brassiceae

ITS resolves relationships among closely related Brassica species and their crop wild relatives (CWRs), effectively identifying clades such as B. rapa,B. juncea and B. nigra, B. carinata, and supporting classical “U‑triangle” relationships [7] .

This study focused on the most significant recent and earlier research, attempting to resolve the problems and disputes over the family's origins because it is one of the least researched families in Iraq and there aren't many studies like the study of [8] for the genus Brassica [9], This study sought to determine the genetic distance between the species under study using molecular markers like ITS to genetically distinguish the species because there were insufficient studies conducted on this genus specifically in Iraq and the Arabic world.

Material and Methods

Molecular Study

Isolation of Genomic DNA

The DNA was extracted from small fresh leaves of the Brassica species under study, obtained from trips in the center and north of Iraq as Erbil, Sulaimani, Kirkuk, Mousl, and Salahaldin Figure 1, using Cetyltrimethylammonium bromide (CTAB) and the method according to [10], based on the principles laid down by [11]. The protocol of genomic DNA was followed during the extraction procedure [12].

After the extraction process, the purity and concentration of DNA were determined using the NanoDrop instrument, where one drop of the previously extracted and stored DNA was taken and placed in the designated place for the sample in the device. Then, the device was given an order to measure via the computer program after calibrating the device on the (Elution Buffer (Tris-HCL, pH 8.5) solution. Then, the measurement was shown on the computer screen by the device, and the purity concentration was accurately measured in nanograms per milliliter. Then, the samples were diluted until the concentration became 50 ng/µL and stored in a freezer at -80° until they were used to study molecular typing by amplifying the nuclear ribosomal DNA (rDNA) genes ITS and making nucleotide sequences for these genes and matching them with the global gene bank to know the evolutionary line of the ranks of species in the Brassicaceae family. More than one specialized primer was used in this study, and its nucleotide sequences were designed by Bioneer-Korea Company, Table 1 & 2.

Gel Electrophoresis

To verify that DNA was present, DNA samples were tested on a 1% Agarose gel. This method replaced density gradient techniques, was found in the 1970s. It is a common and quick technique for sorting, recognizing, and purifying DNA fragments as they move through the gel. It is used to separate DNA fragments and determine the molecular weight of the molecules that are extracted from the gel by comparing them to the DNA ladder [13].

ITS interactions

DNA samples from the ten species of Brassica under investigation were subjected to ITS reactions in accordance with [14].

Polymerase Chain Reaction ( PCR) of the ITS marker

The following procedures were followed to apply the ITS molecular marker used in this investigation in the PCR technique:

1- All solutions were kept in ice, and All procedures were performed in a sterile laminar flow hood while wearing gloves.

2- The prepared Master Reaction tube was filled with 5 microliters of template DNA and 2 microliters of primer. Distilled deionized water was then added to the reaction to obtain a final volume of 20 microliters.

3- For every set of primers, a unique program was set up for the thermocycler.

Detection of PCR products

Following the conclusion of the reaction time, the tubes were taken out of the thermal polymerization apparatus, and 10 microliters were extracted and placed onto the agarose gel that had been previously prepared at a concentration of 1%, with the DNA ladder loaded on one side. The samples were then transferred by running the electrophoresis apparatus for an hour at 70 volts. Following this, the products of DNA were exposed to ultraviolet light using the Gel documentation system for the capture of a photo [15].

Sequence data analysis for comparison and genotype mapping of the studied species:

After performing the PCR test, the PCR product was sent to the Korean company Macrogen to perform DNA sequencing using BLAST. program and using the NCBI) gene database and the BioEdit v 7.2 program. After that, the bases were saved, i.e. each base was placed against it, leaving spaces for mutations, if any, to start the analysis by three maximum similarity programs that are sent to Cipres to analyze the results related to the nucleotide sequences of the plant species under study, while the analysis by the programs (Average (UPGAM) and Neibor Join is directly implemented by the PAUP program on the computer to draw the Phylogenetic tree.

Data analysis

DNA sequencing was performed to determine the phylogenetic tree of the studied species by determining the nucleotide sequence of the ITS gene as a result of the PCR and using primers designed by Macro Gene (South Korea). After completing the product test was shipped to Macrogene Company in Korea so that DNA sequencing could be done with a genetic analyzer. Also, the BLAST tool was utilized to compare the products of ten species on the NCBI database, in addition to the MEGA 6 program to draw the phylogenetic tree.

Dendrogram

The UPGMA program was adopted to obtain the genetic relationship tree (evolution and development) using the biostatistical program PAST version 62.1, [16]. The mentioned program, the genetic dimension was extracted, and the genetic relationship tree was drawn [17].

After the DNA extraction process, concentration and purity were measured. The results showed that purity ranged between 1.56-1.98 in B.deflexa and B.juncea, respectively, while the highest concentration ranged between 73-140 nanograms per milliliter in B.tournifortii and the lowest concentration were in B.oleraceae. See Tables 1 and 2.

To diagnose the gene, PCR reactions were carried out using the reaction mixture following the extraction procedure, DNA collection, and measurement of the concentration and purity in the species under study. Table 3. Then, to obtain the gene product in accordance with the reaction protocol, The reaction mixture was loaded into the thermocycler listed in Table 3. Table 4.

To verify that DNA was present, genomic samples were tested on a 1% of gel. Figure 2 shows the DNA bands found for the ten species belonging to the Brassica genus after extraction. This is evidence of the success of the extraction process from the plant leaves of the studied species.

The results of the current study showed that most of the samples gave bands after being subjected to electrophoresis. These bands were characterized by their clarity and density. These bands were 758 base pairs after comparing them with the DNA Ladder. Because it provides a clear image of the strength of the relationship between several clans of the same origin, the appearance of bands with almost identical molecular sizes shows that they have the same genetic basis, according to [18]. The use of these specialized genes contributed to clarifying the correlation, Figure 3.

PCR is the approved basis for molecular classification and knowing the sequence of plant species. It depends mainly on the purity of the DNA extracted from the smallest amount of plant tissue from plant samples. This is what the results of the current study reached: reaching good results and then reaching knowledge of the sequence for most of the studied species and for the ITS region. This was confirmed by. The process of reaching knowledge of DNA sequences in plastids, nuclei, or mitochondria was used to track and find molecular genetics evolutionary relationships in plant species belonging to the genus Brassica, and in particular, the use of chloroplast genes.

The DNA amplification products were sent to the Korean company Macrogene to perform the nitrogenous base sequences of the studied species. The results were compared with the BLAST nd BioEdit v 7.2 programs with the genes registered in the NCBI National Center for Biotechnology Information gene bank. The results proved the success of the studied species, and the sequencing results of the studied species were matched with the sequences in the NCBI gene bank.

It became clear through the relationships of evolution that genetic similarity is a similarity to clarify the genetic relationships between the studied species in a single tree with high-resolution data reliability, and in terms of the general shape of the tree and the distribution of species into groups that did not differ from their predecessors, when analyzing the data with the MEGA6 program, The genetic structures of the studied species were divided into two main groups, the two groups are Group A and Group B. Group A included eight species, namely B.deserti, B.nigra, B.deflexa, B.juncea, B.elongata, B.tournifortii, B.napus, B.rapa, while group B included two species,B.aucheriand B.oleraceae. The genetic distance ratio ranged from 0.1639 to 7.8948; the shortest genetic distance was 0.1639 between B.tournifortii and B.napus, while the longest genetic distance was 7.8948 between B.aucheri and B.tournifortii Figure 4.

Based on DNA fragments obtained from the amplification products of primers and by the ITS-PCR technique in regions 1 and 2, as well as the analysis of the results obtained from the studied species using the program MEGA 6. The genetic distance ratio ranged from 0.1639 to 7.8948; the shortest genetic distance was 0.1639 between B.tournifortii and B.napus, while the longest genetic distance was 7.8948 between B.aucheri and B.tournifortii Table 5. To ground this in current science, we can look at research regarding Wild Brassica Species.Recent studies, such as those by, emphasize that wild relatives like B. tournefortii and B. deserti are crucial for "climate-proofing" modern crops. A study published in regarding the genetic diversity of the Brassiceae tribe notes that the C-genome group which includes B. oleracea often shows higher divergence from the A-genome group B. rapa. This aligns perfectly with your data's separation of Group A and Group B. Furthermore, the high distance of B. aucheri is supported by recent barcoding studies that identify it as a distinct lineage with unique resistance to high-salinity environments. These results align with recent genomic surveys [19] that emphasize the role of wild Mediterranean Brassica species as distinct genetic reservoirs, essential for enhancing the climate resilience of modern cultivars.

Bootstrap Analysis

Bootstrap analysis was performed to assess the reliability of the phylogenetic tree constructed from ITS sequences. The results showed high support values for the main clades. Group A, which includes eight species (B. deserti, B. nigra, B. deflexa, B. juncea, B. elongata, B. tournefortii, B. napus, and B. rapa), showed strong support with a bootstrap value of approximately 92%. The closest relationship between B. tournefortii and B. napus showed the highest bootstrap support (98%), confirming their close genetic similarity. A sub-cluster including B. nigra, B. deflexa, and B. juncea was supported by a bootstrap value of approximately 85%. Group B (B. aucheri and B. oleracea) showed strong support (90%), and the separation between Group A and Group B showed very strong support (95%), reflecting significant genetic divergence between the two lineages figure 5.

Conclusion

The present study demonstrated that the ITS marker is an effective molecular tool for resolving phylogenetic relationships among Brassica species in Iraq. The phylogenetic tree clearly separated the studied species into two major genetic clusters corresponding to known evolutionary lineages within the genus.

The close relationship between B. tournefortii and B. napus suggests potential genetic exchange and breeding value, while the divergence of B. aucheri highlights its uniqueness as a genetic resource. These results contribute to understanding the genetic diversity of Iraqi Brassica species and provide a scientific basis for future conservation and breeding programs.

Figure 1. Figure 1: regions of collection of specimen for species under study.

Figure 2. Figure 2: Gel electrophoresis of genomic DNA extraction from the studied species

1. B.deserti 2. B.nigra 3. B.deflexa 4 B.juncea 5. B.elongata 6. B.tournifortii 7. B.napus 8. B.aucheri 9. B.rapa 10. B.oleraceae

Figure 3. Figure 3: ITS primer results on 1.5% agarose gel for 50 min with 758 bp size marker for species belonging to the genus Brassica .

1. B.deserti 2. B.nigra 3. B.deflexa 4 B.juncea 5. B.elongata 6. B.tournifortii 7. B.napus 8. B.aucheri 9. B.rapa 10. B.oleraceae

Figure 4. Figure 4: Phylogenetic tree for ten species of genus Brassica by MEGA 6

Figure 5. Figure 5: Bootstrap analysis for ten species of genus Brassica

1. B.deserti 2. B.nigra 3. B.deflexa 4 B.juncea 5. B.elongata 6. B.tournifortii 7. B.napus 8. B.aucheri 9. B.rapa 10. B.oleraceae

Figure 6. Table 2: Gene-specific primer sequence used on this study.

1. B.deserti 2. B.nigra 3. B.deflexa 4 B.juncea 5. B.elongata 6. B.tournifortii 7. B.napus 8.B.aucheri 9. B.rapa 10. B.oleraceae