Morphological, Biochemical and Barcoding Analysis of Different Egyptian Jew’s mallow ( Corchorus olitorius L. ) Landraces

: Jew's mallow is used as a food source, medicinal plant and natural antioxidant agent as well as biofuel source. Its numerous beneficial properties made it a valuable crop worldwide. This study was carried out to investigate the morphology, biochemical genetic analysis and DNA barcoding for three different Jew’s mallow ( Corchorus olitorius ) Egyptian landraces. They were collected from different location within Egypt namely, Fallahy (North), Seady (South) and Swain (West). Results revealed that, Siwan landrace had higher values for plant height, leaves number/plant, branches number/plant and fresh weight followed by Seady, then Fallahy. Biochemical values generally showed no significant differences between most of the measured elements. Mineral composition data showed numerical increase in the Siwan landrace in some minerals than others including Boron, Sodium, Magnesium, Aluminum, Calcium, Titanium, Chromium and Zinc, while Seady landrace recorded the highest values for Cobalt, Nickel, Copper, Indium and Bismuth. PCR was performed for the landraces along with two universal primers ( RbcL-MatK ). RbcL gene was amplified at 800-900 bp, while MatK gene was at 900 bp. Data was uploaded on DNAman program and partial sequences of RbcL and MatK genes were submitted to DDBJ Gene Bank with accession numbers of LC732565, LC732566 and LC732567 for RbcL gene and LC732049, LC732050 and LC732051 for MatK gene. This study showed that, the Siwan landraces was more unique from the other two landraces; furthermore, DNA barcoding using RbcL and MatK genes is an effective and sufficient tool in identifying different C. olitorius landraces in Egypt.

genetic barcoding using two plastid genes RbcL and MatK can differentiate them from one another according to the different cultivation locations (Hollingsworth et al., 2009). The matK gene sequence is more challenging to be amplified and sequenced than RbcL as; it is longer than RbcL by 300bp, and more delicate to be degraded, that's why, sequence reaction quality which has mononucleotide repeats is easily affected (Cohen, 2011). Therefore, this investigation was carried out to study the morphological description along with biochemical genetic analysis and the molecular barcoding of three different Egyptian landraces of Jew's mallow, (C. olitorius) cultivated in Egypt.

MATERIALS AND METHODS
The current work took place at the Agriculture Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, with collaboration with the Medicinal and Aromatic Plants Research Department, Horticultural Research Institute, Agricultural Research Center, Alexandria, Egypt. Three landraces of Jew's Mallow (C. olitorius L.) namely: Fallahy, Seady and Siwan were used during the current study. The plant material (seeds) were collected and selected from different locations at Northern, Southern and Western Egypt, respectively. Seeds were cultivated at Faculty of Agriculture Farm, Alexandria University, during the cultivation seasons 2021 and 2022.

Morphological Studies:
Random sample of 20 plants were selected for the morphological measurements of the three landraces under study. Morphological studies included; plant height (cm), number of leaves/plant, number of branches/plant as well as fresh weight (g), dry weight (g) and moisture content. The field experiment design was a randomized complete blocks with three replicates for each landrace. Seeds of each Jew's Mallow landrace were sown in rows approximately 40cm apart. Each experimental unit consisted of 10 rows, 3.5m along and 0.20m a part. The sowing date was the 24 th June on 2021 summer season.

Biochemical Assay:
Chlorophyll contents (A and B) were measured according to Barros et al. (2011) with some modifications. Also, total phenolic compounds, total flavonoids were extracted using methanol as described by the previous author. Proline content was determined following Bates et al. (1973) procedure, using Aqueous Sulfosalicylic Acid (3%), Acid Ninhydrin; Ninhydrin (1.25 g), glacial acetic acid (30 ml) and 6M phosphoric acid (20 ml). Total dietary fiber was estimated by nonenzymatic gravimetric technique of Li and Cardozo (1994). The soluble and insoluble dietary fibers were determined according to Asp et al. (1983). Total soluble solids were directly measured using a hand refractometer (Model: ATAGO. Tokyo, Japan). Vitamin C content was estimated using the 2, 6-dichlorophenol indophenol method as illustrated by AOAC (1975). Quantification of total protein content, Bovine serum albumin (BSA) was used as standard reagent for the standard curve preparation which helped in estimating the total protein content (Sarkar et al., 2020). Plant leaves content of actual nitrogen (N) was calculated using the following equation.
Where ν the sample consumed volume, νb is the control treatment consumed volume, NH2SO4 is the normality of sulphuric acid at 0.014, meq is the dry weight sample. Mineral composition and determination were done using the atomic absorption spectrophotometer with the dry burning method (Kacar and Inal, 2008).

Molecular Studies
Isolation of the genomic DNA was done from seeds of the three landraces under study according to Irfan et al. (2013), with some modifications. DNA extraction as well as purification was achieved using CTAB (Cetyltetramethyl ammonium bromide) method (Muray and Thompson, 1980). PCR amplification was performed using biosystems® proflex™ PCR system. The PCR amplification conditions were optimized for all reactions by using extracted DNA in a total volume reaction of 25 µl as; Master Mix (12.50 µl) and primers (1.25 µl of each) ( Table 1) (forward and reverse), DNA template (2 µl) and de-ionized water (up to 25 µl) (Erlich, 1989).

RESULTS AND DISCUSSION
Three landraces of Jew's Mallow (Fallahy, Seady and Siwan) seeds were collected and selected from Egyptian different locations to study their morphological, biochemical and molecular barcoding variations.

Morphological studies:
No variations were observed in seed germination among the three Jute Mallow landraces under study. Results indicated that the germination percentage ranged from 98 to 100%. Morphological measurements are shown in Table  (2). Siwan landrace highest mean value was recorded for plant height (50.00 cm) compared with the lowest value in Fallahy (38.67 cm), while Seady recorded (46.00 cm). As for no. of leaves/plant, no. of branches/plants as well as fresh weight, Siwan landrace showed the highest values followed by Seady, then Fallahy with no significant difference among them as presented in Table (2). Those results were in agreement with previous studies which stated that, morphological differences among the landraces very important and of useful significance.

Biochemical Assay:
Biochemical values of the tested Jew's mallow landraces were recorded in Table (3) as mean of three replicates ± standard error. Generally there were no significant differences between most of the measured elements; however there were slit increase among some of them. Chlorophyll A content was high in Seady (8.326), followed by Siwan landrace (8.026) and finally, Fallahy (7.885), while Chlorophyll B and Chlorophyll A+B, values were similar and ranged from 3.182 to 3.268 and 11.153 to 11.526, respectively. Fiber content in each gram was 2.187, 2.054 and 1.642 for Siwan, Seady, and Fallahy respectively. For protein, Siwan recorded the highest value (13.586) and was superior on Fallahy (12.489) and Seady (12.241) landraces. Biochemical assay values of C. olitorius followed the same trend as mentioned by Ahmed and Nizam (2008) who reported that, Corchorus spp. contain a considerable amount of vitamins, calcium and carotene. This was also supported by Nemb et al. (2011) who stated that, C. olitorius leaves contain protein, carbohydrates, fiber, Ca, Fe, vitamin A and ascorbic acid. Mahmood et al., (2021) reported that, Jew's mallow contains a variety of flavonoids with the highest concentration found in the leaves of the plant; and that, flavonoids concentration and distribution were influenced by factors such as light intensity, temperature, and plant maturity. Ismail et al. (2020) added that Jew's mallow was found to have beneficial properties like high antioxidant activity, which is attributed to phytochemicals existence like flavonoids, phenolic acids, and carotenoids, also, can be used in food preservation due to its elevated level of antioxidants. Mineral composition results of the C. olitorius landraces under study were expressed in ppm (milligrams/kg) as illustrated in Table (4). Data showed numerical increase in the Siwan landrace for some minerals than others including Boron, Sodium, Magnesium, Aluminum, Calcium, Titanium, Chromium and Zinc which recorded; 9.2, 6918. 6, 808.1, 194.9, 232.9, 40.9, 5.9

Molecular studies:
Extracted DNA concentration was measured (400 to 600 ug/ml) and determined using a Nano drop spectrophotometer (Thermo Scientific™ NanoDrop™ One/OneC Microvolume UV-Vis). PCR was performed for the two universal primers (RbcL-MatK), using PCR program as mentioned before.
Electrophoresis of the RbcL amplified gene took place as illustrated in Figure (1-A). Gene amplified fragments were located between 800-900 bp. This was performed using the retrieved DNA directly. MatK amplified gene is presented in Figure (1-B) for the three landraces of Jew's mallow genomic DNA samples. Gene was located at 900 bp.

Single Nucleotide Polymorphism (SNP) Identification and Frequencies:
Nucleotides were identified using DNAMAN® software (Lyon BioSoft, Quebec, Canada). Sequences were added in DDBJ and the accession number has been obtained as demonstrated in Table (5). Fasta format files were uploaded on DNAman program and partial sequences of RbcL and MatK genes obtained from the three landraces of C. olitorius were compared with each other. The three sequences were submitted in DDBJ Gene Bank with accession numbers of LC732565, LC732566 and LC732567 for RbcL gene and LC732049, LC732050 and LC732051 for MatK gene.

Barcoding analysis of RbcL:
Sequences were compared to the three landraces of Corchorus olitorius, the highest SNP's frequency was found in LC732567. Data in Table ( 28.43%, C: 20.02%, G: 22.82%, T: 28.72% while GC content was 42.84% and AT content was 57.16% in the total number of nucleotides (1034). RbcL phylogenetic tree was divided into two main clusters with C. olitorius Fallahy and C. olitorius Siwan at the same branch, while C. olitorius Seady was in a separate branch, as shown in Figure (2).   The phylogenetic analysis of the three C. olitorius landraces using Rbcl genes sequencing were recorded in Figure (3), dendrogram was divided into two main branches, one of them had the three landraces under study, C. olitorius Fallahy, C. olitorius Siwan and C. olitorius Seady in the same cluster. Barcoding analysis of MatK gene compared with the three landraces of C. olitorius revealed that, the highest SNP's frequency was found in LC732051. There were no base substitutions between all sequences aligned according to data in Table (5). There were three deletions found in nucleotide 257, 568, 569 and 577 in C. olitorius Siwan landrace. Results in Table (9) represented amino acid composition of MatK sequences of the three landraces of C. olitorius under study as compared with genes in NCBI by BioEdit sequence Alignment Editor Version 7.2.5(12/11/2013). Cysteine and Glycerin usually ranged between 33.66 and 34.64, meanwhile Alanine and Threonine ranged between 65.36 and 66.34. Data in Table (10) showed similarity percentage calculated by BLAST and the phylogenetic similarity based on MatK gene plant sequencing. There was high similarity percentages ranged from 99.62 to 100 for the landraces under study compared with others in the gene bank. C. olitorius Seady collected sample was identical to C. olitorius of the database with 100%, while C. olitorius Fallahy showed high similarity of 99.62%, as shown in Table (10).
Data in Table (11) presented the nucleotides content of the landraces under study and the total number of nucleotides. Nucleotides average number was 775 nucleotides using MatK gene. The nucleotide composition percentages of the landrace C. olitorius Fallahy were A= 29.81%, C= 16.90%, G= 16.39% and T= 36.90%, while the G+C= 33.29% and the A+T= 66.71%. It's clear that the G+C content and A+T content in Seady was identical with Siwan as G+C= 33.81% and A+T= 66.19% in a total number of 775 nucleotides, as shown in Table (11). The phylogenetic tree was divided into two main clusters; one of them contains Seady and Siwan as shown in Figure (4). This phylogenetic differentiation might be due to the difference in the environmental conditions which lead to natural selection which agreed with previous studies supporting that, DNA polymorphisms is a powerful approach for understanding the evolutionary of different landraces and determine how genomic regions is functional (Begun et al.,  2007). Amino acids amount of change is nowadays easily to be determined to be used in adaptation studies, the locations of these changes in the protein's three-dimensional structure and mechanisms by which substitutions influence stability and kinetic properties (Somero, 2010). It was also reported that DNA barcoding projects begin with a tissue sample from which all subsequent genetic processing takes place. "Barcode" in a Gene bank is achieved by DNA barcode records submission with specific metadata elements associated with the specimen source (Kress and Erickson, 2012)   Phylogenic tree (Figure 4) was designed with a scale, at which a branch lengths was measured with number of substitutions /site. According to the previous data of MatK genes and using the three landraces of C. olitorius, the cluster analysis was divided into two branches; Fallahy was in separated branch and both Siwan and Seady in a separated branch. The phylogenetic analysis of the three C. olitorius landraces using MatK genes sequencing are presented in Figure (5). Dendrogram in Figure (5) was divided into two main branches, one of them had the three landraces under study, C. olitorius Falahy and C. olitorius Siwan were in the same cluster, while Corchorus olitorius Seady followed them.

Figure (5) Phylogenetic tree of C. olitorius landraces under study with genes in NCBI using MatK gene
Current study declared that RbcL and MatK genes are effective tools to be used in barcoding to relate plants, as well as the difference between species and individuals as supported by Hebert et al., (2004), who reported that, Taxonomists are highly interested in DNA barcoding, it is widely used to support ownership or intellectual property rights. The same authors defined DNA barcode, as one or more short genetic sequences obtained from a genomic uniformed portion (standard) to identify species. Similarly, Cowan et al. (2006) mentioned that barcoding allows scientists to identify and recover information about known species quickly and accurately. Cohen (2011) mentioned that RbcL and MatK, were identified as core barcode loci. However, RbcL gene is much easier to amplify and sequence than MatK for several reasons. In addition, studying the biochemical differences among different landraces can lead to identifying the most valuable one, containing the beneficial active ingredient and the most delicious as an edible one (Islam, 2013). He also added, barcoding is a characterizing method for species involving a small sequence from a specific and agreed-upon position at the organism genome. Kress et al. (2005) stated that RbcL is commonly used in phylogenetic investigations with wide range on the Gene bank (≤50000). This gene is easy to be amplified, sequenced, and aligned at most plants, as it has a barcoding region specified for the species, genus and the families they belong to. However, RbcL genes loci are hard to change and have the lowest plastid genes divergence in flowering plants. Mildenhall (2006); reported that using DNA barcoding to maintain food hygiene and labeling licensing for identity confirmation The MatK gene, also known as Megakaryocyte-associated tyrosine kinase, is a member of the non-receptor tyrosine kinase family. This gene is found on chromosome 10 in humans and is expressed in various tissues, including the bone marrow, spleen, and thymus. (El-Rokiek et al., 2016). The RbcL gene, known as ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, is a significant gene involved in photosynthesis in plants (Alqurashi and Awad, 2011). Previous investigations found that Jew's mallow contains a significant amount of RbcL and MatK genes expression in both leaves and stems. It was also found that, those genes expression were high in the early stages of growth and decreased as the plant matured. Furthermore, researchers observed that their expression was influenced by environmental conditions. It was found also that; gene expression increases under low-temperature conditions and decrease under high-temperature conditions.
That's to say, Jew's mallow is a valuable base for RbcL and MatK genes, as they are influenced by environmental factors. (Alqurashi and Awad, 2011 and El-Rokiek et al., 2016).

CONCLUSION
Based on the morphological and molecular studies it was concluded that Siwian landraces was more unique from the other two landraces, also DNA barcoding using Matk and Rbcl genes is an effective and sufficient tool in identifying different Corchorus olitorius landraces in Egypt.