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Middle East Journal of Agriculture Research Volume: 10 | Issue: 01| Jan Middle East Journal of Agriculture Research Volume: 10 | Issue: 01| Jan. - March| 2021 EISSN: 2706-7955 ISSN: 2077-4605 DOI: 10.36632/mejar/2021.10.1.13 Journal homepage: www.curresweb.com Pages: 227-237 Plant genetic barcoding of some Legume tree species grown in Egypt Houssam El-Din M.F. El-Wakil1, Aly Z. Abdelsalam2, Hesham M. Aly3, Asma Aboshady2, Samar M.A. Rabie1, Mohamed E. Hasan4 and Nader R. Abdelsalam1 1 Agricultural Botany Department Faculty of Agriculture, Saba-Basha, Alexandria University, Egypt. 2 Genetics Department, Faculty of Agriculture, Ain-Shams University, Egypt. 3 Department of Forestry and Wood Technology, Horticulture Institute, Agriculture Research Center, Antoniadis Botanical Garden, Alexandria 21554, Egypt. 4Bioinformatic Department, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Egypt Received: 20 December 2020 Accepted: 10 February 2021 Published: 25 February 2021 ABSTRACT DNA barcoding as tools for rapid species documentation based on DNA sequences. During the current study fifteen species were collected, belonged to family fabacea from Antoniades Garden Alexandria between July 2019 and January 2020 RbcL and matK of the plastid genomes were used to study the sequence of the nucleotides bases of these genetic materials, alignment the current genetic sequence which obtained from NCBI and CBOL and matched the observed sequence with other in the GeneBank, calculating the differential between the different species of trees using precise genetic coding instead of the phenotypic distinction and finding the evolutionary relationship between the types of these trees. The results showed that the quality of the extracted DNA which detected by using 1% agarose gel electrophoresis there were no fragmentation was observed in extracted DNA. Also, the extracted DNA was directly used in PCR amplification for the two genes; RbcL (600 bp) and MatK gene (900 bp). The average of nucleotides composition for all the plant species are A=27.23%, G=20.86%, G=22.43% and T=29.45%, while the G+C= 40.30% and A+T= 56.69% in a total number 585.26 nucleotides. Also, the data of MatK gene, showed highly similiters percentage ranged from 95.63 to 99.77 for all the studied species with others in gene bank. For instance, Cassia fistula was identical with the same species on GenBank (99.77%), while Delonix regia showed high similarities with Delonix regia, Delonix boiviniana voucher Bruneau (99.54). The average of nucleotides composition for all the plant species are A=37.30%, G=15.11%, C=16.84% and T=30.75%, while the G+C= 31.95% and A+T= 68.05% in a total number 833 nucleotides. Regarding to RbcL gene, there are high significant homogeneity between the tested species as follow: Tipuana tipu with Schotia brachypetala, Parkinsonia aculeata, Leucaena leucocephala, Delonix regia and Dalbergia sissoo with P-values were 0.010, 0.014, 0.030, 0.000 and 0.024. While other significant was observed between Schotia brachypetala Parkinsonia aculeata with Erythrina humeana by 0.004; Erythrina_humeana with Delonix regia and Cassia javanica by 0.004 and 0.030 and finally, Delonix regia with Cassia fistula by P-values 0.032. On the other hand, for MatK, there are significant homogeneity between Erythrina_humeana and the fowling species were Sophora secundiflora, Tipuana tipu, Senna surattensis, Schotia brachypetala, Parkinsonia aculeata, Leucaena leucocephala and Acacia saligna (0.000, 0.000, 0.004, 0.000, 0.000, 0.000 and 0.000). while Tipuana tipu with Enterolobium_contortisiliquum by (0.036). Also, Erythrina_humeana showed significant homogeneity with the species Enterolobium contortisiliquum, Dichrostachys cinerea, Delonix regia, Dalbergia sissoo, Cassia javanica, Albizia lebbek and Cassia fistula. Keywords: DNA sequences, rbcL and matK genome, Legume, fabacea, gene Bank Corresponding Author: Houssam El-Din M.F. El-Wakeel, Agricultural Botany Department Faculty of Agriculture, Saba-Basha, Alexandria University, Egypt. 227 Middle East J. Agric. Res., 10(1): 227-237, 2021 EISSN: 2706-7955 ISSN: 2077-4605 DOI: 10.36632/mejar/2021.10.1.13 1. Introduction DNA barcoding as tools for rapid species documentation based on DNA sequences, share an emphasis on huge scale genetic data which give the accurate difference among species. DNA barcode consists of a standarized short sequence of DNA (400 to 800 bp) should be easily created for all species on the planet has been stablished to stimulate the creations of data base of documented reference DNA sequences to serve as a library for undefined taxa. DNA barcode is one of the advanced molecular markers-based method that classify target plant species in short period. The purpose of DNA barcodes is nucleotide sequence-based documentation of multiple plant species with accuracy and is one of the widely accepted technology (Group et al., 2009) DNA barcodes help to discover new species and to identify cryptic species (Ward et al., 2008) and might facilitate biodiversity valuations and minor the cost and time requirement of traditional taxonomic biodiversity research (Gaston and O’Neill 2004; Hebert et al., 2004; Pauls et al., 2010). Becker et al. (2011) reported that DNA barcodes are a perfect established technique and has been showed to bring substantial benefits to different application such as species interactions, biodiversity valuation and plant conservation (Valentini et al., 2009 and Francis et al., 2010), in addition its supplement to traditional morphology-based taxonomy (Hebert and Gregory 2005; Packer et al., 2009 and Wang et al., 2016). Also, DNA barcodes can show the distribution of intra and intra- specific variations based on the distance and similarity (Hebert et al., 2003; Meyer and Paulay 2005). Sarvananda (2018) reviewed that DNA barcoding is the identical system for fast and accurate to identify the species especially for the ecological system that using short DNA sequence. They renamed the short DNA sequence from the standard region of genome as marker. Also, they classified the genes to, CO1 cytochrome c oxidase 1 used for animals; matK and rbcL used for plants and (ITS) Internal Transcribed Spacer using for fungus. Tahir et al. (2018) tested six DNA barcodes, notably ITS2, matK,rbcL a,ITS2+matK,ITS2+rbcL a, matK+rbcL a and ITS2+matK+rbcL a, for their ability to identify frequently consumed but geographically isolated medicinal species of Fabaceae and Poaceae native to the Cholistan Desert. The results showed that the comparison of six barcode regions showed that ITS2 had the highest number of variables. Marizzi et al. (2018) reviewed the fact that DNA barcoding is an essential tool for research and science education. They said that this technology enables rapid and accurate genetic diversity using only a minimal amount of tissue samples taken from any organism at any stage of development. They discovered that DNA barcoding allows citizens scientists to make preliminary taxonomic identifications and to make a contribution. da Silva et al. (2018) reported that Fabaceae is among the most diverse family in the world, recognized for its ecological and economic potential; divided into the following sub-families (Caesalpinioideae, Cercidoideae, Detarioideae, Dialioideae, Duparquetioideae). They discussed various species, divided into 14 genera and four subfamilies. Macêdo et al. (2018) reported that Fabaceae is one of the major groups of ethnopharmacological significance. The most versatile Fabaceae species and the agreement of use between informants were evaluated in the Chapada do Araripe Savanna. Semi-structured interviews were conducted with 126 informants, adopting a snowball method and using a structured protocol. Therefore, this proposed research is scrutinized to barcode some legume trees plants distributed in Egypt based on morphological and DNA barcode studies using rbcL and matK. 2. Materials and methods The current work was carried out in The Faculty of Agriculture, Agricultural Botany Department, Saba Basha, Alexandria University, Alexandria, Egypt during the period of 2019 to 2021 to study accurate taxonomical identification of some Legume tree species grown in Egypt by applying plant genetic barcoding. During the current study fifteen species were collected (Table 1), belonged to family fabacea from Antoniades Garden's between July 2019 and January 2020. The morphological characters were detected based on previous publications, besides photographed all the plant species by the own authors. 228 Middle East J. Agric. Res., 10(1): 227-237, 2021 EISSN: 2706-7955 ISSN: 2077-4605 DOI: 10.36632/mejar/2021.10.1.13 Table 1: Scientific name for studied species No. Scientific name 1 Cassia fistula 2 Cassia javinca 3 Albizia lebbek 4 Delonix regia 5 Senna surattensis 6 Parkinsonia aculeata 7 Schotia brachypetala 8 Tipuana tipu 9 Erythrina humeana 10 Sophora secundiflora 11 Leucaena leucocephala 12 Enterolobium contortisiliquum 13 Dichrostachys cinerea 14 Acacia saligna 15 Dalbergia sissoo 2.1. Molecular Genetic analysis (plant barcoding): 2.1.1. DNA extraction Genomic DNA was extracted from fresh leaves tissue using i-genomic plant DNA extraction Mini kit @ iNtron Biotechnology according to the protocol associated with the Plant Genomic DNA Kit (iNtRON Bio Co., South Korea) according to the manufacturer descriptions as following: 2.1.2. PCR amplification PCR amplification was conducted by using Applied Biosystems® ProFlex™ PCR System (Figure 3). The PCR amplification conditions were optimized for the all reactions by using extracted DNA in a total volume
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