Abstract: (6 Views)
Introduction and objective: The date palm (Phoenix dactylifera L.) is a monocotyledonous plant, from the Palmaceae, which is cultivated in many countries from tropical and subtropical regions including Iran. Today, thanks to the progress of technology, molecular identification methods have special importance in systematic studies. One of these methods is DNA barcoding which is species identification based on the sequences of conserved DNA regions that provides a valuable tool for assessing phylogenetic relationships. In this method, comparison of the sequences of special conserved DNA fragments is used to study the diversity of populations and determining the relationships between different species. Accordingly, different regions of nuclear DNA (ITS, ITS2) and chloroplast (rbcL, matK, trnH-psbA, atpF-atpH, nhdJ, psbK-psbI) have been suggested as important barcodes in plants. The matK chloroplast gene, which has a high evolutionary speed at the nucleotide and amino acid levels, is one of the most suitable chloroplast genes for analysis of phylogeny and evolutionary relationships in a range of taxonomic levels from species to genus, phylum and even super phylum, in Angiosperms in particular. This gene can be used individually or together with other genes to identify and introduce unknown species. Examining the genetic diversity of the available germplasm reserves is essential for the improvement and propagation of cultivars with desirable traits, and the first step in this process is to identify the existing cultivars and classify them. Due to the importance of production of dates in the country, browsing for new cultivars, breeding existing cultivars, and studying the existing diversity between different cultivars are of important strategies to improve quantity and quality of date palm production. However, there are not enough studies on the investigation of relationship or genetic distance of different date palm cultivars using suitable genetic markers. Therefore the main objective of this research was determining the capability of matK marker for analysis of genetic diversity and phylogenetic relations of the studied date palm cultivars.
Material and methods: In this research, the chloroplast gene matK was used to investigate the genetic diversity among 15 local date varieties from Saravan and Jalgh, Nahok and Sinokan regions of Sistan and Baluchistan province. DNA extraction was done using the Dellaporta (1993) method from young leaves of different cultivars, and then the quality and quantity of DNA was determined using 1.5% agarose gel electrophoresis and a spectrophotometer. PCR was performed using specific primers of matk gene, and the fragment of about 900 bp was amplified and sent for sequencing. The quality of the sequencing results was checked and then aligned using BioEdit software, and the regions with low sequencing quality were removed from the 3' and 5' ends. Next, the sequences were blasted for alignment with other sequences in the NCBI database and the degree of similarity with other registered sequences was examined. After sequence alignment, key genetic parameters including the total number of mutations, nucleotide diversity, the number of mutation sites, and the extent of their variation were comprehensively analyzed. Then, MEGA7 software was used to determine the relationships and genetic distance between the studied cultivars, and UPGMA method was also used to draw the phylogeny tree.
Findings: The comparison of the sequences showed that there is a high homology between these sequences and the sequences of Phoenix dactylifera species available in the gene bank. Sequencing results of investigated cultivars were registered in NCBI database. The results of the current research showed that a total of 1019 positions were identified for the matk marker used in this study, of which 672 positions were included deletions and additions, and 347 positions were without deletions or additions. In this population, 15 haplotypes and four conserved DNA regions were identified. The numerical value dN/dS was 0.169, and the genetic distance was ranged from 0.019 to 1.238. The highest genetic distance of 1.238 was observed between the samples of Halile20 from Saravan and Pimazo16 from Jalgh. After drawing the phylogeny tree, the studied cultivars were divided into three branches, which Halileh 20 from Saravan was located in the first branch. In the second branch, Pimazo 16 variety from Jalgh was placed. The third branch was divided into two sub-branches, in which there were a total of 6 numbers in the first sub-branch and a total of 7 numbers in the second sub-branch. According to this classification, Halileh 20 of Saravan is the most distant from other varieties.
Conclusion: Based on the obtained results, it can be concluded that the use of matk marker has been useful and suitable for studying and understanding the diversity and intraspecies relationships of date palms. Therefore, in future date palm studies to investigate genetic diversity, it is possible to suggest the use of this barcode in addition to other suitable molecular.
Material and methods: In this research, the chloroplast gene matK was used to investigate the genetic diversity among 15 local date varieties from Saravan and Jalgh, Nahok and Sinokan regions of Sistan and Baluchistan province. DNA extraction was done using the Dellaporta (1993) method from young leaves of different cultivars, and then the quality and quantity of DNA was determined using 1.5% agarose gel electrophoresis and a spectrophotometer. PCR was performed using specific primers of matk gene, and the fragment of about 900 bp was amplified and sent for sequencing. The quality of the sequencing results was checked and then aligned using BioEdit software, and the regions with low sequencing quality were removed from the 3' and 5' ends. Next, the sequences were blasted for alignment with other sequences in the NCBI database and the degree of similarity with other registered sequences was examined. After sequence alignment, key genetic parameters including the total number of mutations, nucleotide diversity, the number of mutation sites, and the extent of their variation were comprehensively analyzed. Then, MEGA7 software was used to determine the relationships and genetic distance between the studied cultivars, and UPGMA method was also used to draw the phylogeny tree.
Findings: The comparison of the sequences showed that there is a high homology between these sequences and the sequences of Phoenix dactylifera species available in the gene bank. Sequencing results of investigated cultivars were registered in NCBI database. The results of the current research showed that a total of 1019 positions were identified for the matk marker used in this study, of which 672 positions were included deletions and additions, and 347 positions were without deletions or additions. In this population, 15 haplotypes and four conserved DNA regions were identified. The numerical value dN/dS was 0.169, and the genetic distance was ranged from 0.019 to 1.238. The highest genetic distance of 1.238 was observed between the samples of Halile20 from Saravan and Pimazo16 from Jalgh. After drawing the phylogeny tree, the studied cultivars were divided into three branches, which Halileh 20 from Saravan was located in the first branch. In the second branch, Pimazo 16 variety from Jalgh was placed. The third branch was divided into two sub-branches, in which there were a total of 6 numbers in the first sub-branch and a total of 7 numbers in the second sub-branch. According to this classification, Halileh 20 of Saravan is the most distant from other varieties.
Conclusion: Based on the obtained results, it can be concluded that the use of matk marker has been useful and suitable for studying and understanding the diversity and intraspecies relationships of date palms. Therefore, in future date palm studies to investigate genetic diversity, it is possible to suggest the use of this barcode in addition to other suitable molecular.
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