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

INTRODUCTION

Jew's mallow, (Corchorus olitorius L.) commonly recognized as Melokhya in Arabic, belongs to Malvaceae family. It is a tropical green leafy summer vegetable, traditionally cooked in Middle East, Asia and different parts of Africa (El-Haddad et al., 2020). Culinary leaves taste very delicious among Egyptians, juicy leaves soften quickly when cooked, resulting in a sticky, slimy broth that can be eaten with starchy foods (Adediran et al., 2015). 100 g of edible leaves of C. olitorius contains, approximately 85g water, 5g protein, 0.7g oil, 5g carbohydrates, 4g dietary fiber, 258g Mg and Ca, 6mg iron, 3000IU vitamin A, 1.5mg nicotinamide, 75mg ascorbic acid (Aluko et al., 2014). It is well-thought-out to be the major source of natural fibers worldwide (El-Haddad et al. 2020). C. olitorius L. was described as a medicinal plant. Leaves can be used as laxatives either in a juice form, or fried for skin care in creams and as a therapeutic agent for a wide range of health disorders. Its fresh leaves are known to be high in beta-carotene for sharp eyesight, iron for rich red blood cells, calcium for healthy bones and teeth, and vitamin C for smooth skin, strong immunity system and rapid wound healing (Islam, 2013). Moreover, it was found that, plant extraction has antitumor-promoting activity using an immune- blotting analysis (Furumoto et al., 2002). Its high nutritional value and potential health benefits make it a valuable addition to the Egyptian diet (El-Haddad et al., 2020).

C. olitorius has high morphological diversity based on different measurable traits on wide range of cultivated areas including Nigeria, Senegal, Egypt, Pakistan, beside other countries around the world (Adebo et al., 2015). Over the years, crop improvement programs were directed to select high-quality and fine-fiber varieties. However, growing Jew’s mallow, as a vegetable, is rare in breeding programs (Adjatin et al., 2019). In Egypt, Seady and Fallahy Jute Mallow (Molekhya) landraces are the most famous one, while Siwan landrace, is not well known according to its geographical isolated habitat in Siwa Oasis although it’s the most valuable among them. Morphological description, molecular analysis and 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.

2. 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.

2.1. 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.

2.2. 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 non-enzymatic 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.

= x  x 0.014  x 100

Where  the sample consumed volume,  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).

2.3. 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 (Cetyl-tetramethyl 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).

Table (1): Primer sequence, Tm value and GC (%).

PCR amplification conditions for RbcL gene were 30 cycles as follows: 94 °C / 5 minutes (1 cycle); 94 °C / 45 sec, 50 °C / 45 sec, 72 °C / 45 sec (30 cycles); 72 °C / 7 minutes (1 cycle); 4 °C (24 hrs) as described by Fay et al. (1997). WhilePCR amplification conditions for MatK gene were 45 cycles as follows: 94 °C / 5 minutes (1 cycle); 94 °C / 30 sec, 45 °C / 30sec, 72 °C / 30 sec; 72 °C / 7 minutes (1 cycle); 4 °C (24 hrs). Bands of the previously mentioned genes were eluted using an elution kit (@iNtRON Biotechnology, Inc. Korea) as described by Bjourson et al. (1992). Sequence analysisincludes all Malvaceae family plant species were available on GenBank to find interspecific and intergeneric variation. C. olitorius species sequences for RbcL and MatK were obtained in Fasta format from NCBI. Several sequence alignments of the RbcL and MatK genes were performed using the PROMALS server (Pei and Grishin, 2007), Clustal Omega server (Sievers et al., 2011), the BIOEDIT software (Hall, 2001), Clustal X and MEGA - x (Stecher et al., 2020). Those areoffline-software which performed optimum alignment for sequence to extract the conserved regions. Evolutionary history was derived following the neighbor-joining method (Tamura et al., 2004). Mafft server (Katoh et al., 2015) Clustal Omega server and MEGA-x software (www.megasoftware.net) were used for the phylogenetic tree construction.

3. 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.

3.1. 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. Helaly et al. (2017) reported that, wild landrace can be significantly superior in most morphological parameters compared to the domesticated variety. Also, current results agreed with morphological studies on Jew’s mallow by Sajib et al. (2008) whodescribedC. Olitorius and C. Capsularis morphologically. Adebo et al. (2015) whoreported that, C. Olitorius has high morphological diversity based on the different weather conditions in Egypt. Furthermore, current findings agreed with Abd-Allah et al. (2006) who reported that, six genotypes of Jew’s mallow were obviously different in their morphological traits as they were cultivated at different regions of Egypt.

Table (2): Morphological characters of the three tested Jew’s mellow.

* Mean of 20 randomly selected plants ± Standard error (SE)

2. 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 assayvalues ofC. olitorius followed the same trend as mentioned by Ahmed and Nizam (2008) whoreported that,Corchorus spp. contain a considerable amount of vitamins, calcium and carotene. This was also supported by Nemb et al. (2011) whostated 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.

Table (3):  Biochemical means of the three tested Jew’s mallow.

*Values are presented as means of three replicates ± (Standard Error)

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 and 18.6 ppm, respectively. While Fallahy landrace recorded the lowest values for Sodium, Aluminum, Calcium, Titanium, Iron and Zinc which recorded; 2630.2, 157.2, 181.6, 27.5, 10.7 and 12.4 ppm respectively, whereas Seady landrace recorded the highest values for Cobalt, Nickel, Copper, Indium and Bismuth, were 1.4, 4.4, 12.3, 1.2 and 2.3 ppm and the lowest values were detected in Seady for Magnesium, Potassium, and Barium (631.5, 5677.9 and 2.5 ppm, respectively). Current findings were in agreement with Helaly et al. (2017) who described Jute Mallow plantsas a medicinal plant in the Middle East, as they contains phenolic compounds, polysaccharides, carotenoids, minerals, sugars, proteins and multi-vitamins.

Table (4): Minerals composition of the three tested Jew’s mallow.

3.3.1. 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.

Table (5): Authors and Accession numbers for RbcL and MatK gene recorded for Corchorus olitorius landraces under study.

3.3.1.1. 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 (6) illustrated the amino acid composition of RbcL gene sequence for the three landraces under study grown in Egypt by bioedit sequence alignment editor version 7.2.5 (12/11/2013). The highest Alanine value was recorded in Seady landrace (30.02%), followed by both the other landraces 26.96 and 26.89 as presented in Table (6). A+T as well as C+G value was the highest at Fallahy and Siwan landraces. Phylogenetic analysis data for chloroplast RbcL gene of the three landraces for Jew’s Mallow grown in Egypt is shown in Table (7). Data showed high similarity percentage and phylogenetic similarity based on RbcL gene plant sequencing. Similarity percentage ranged from 99.51 to 99.88 for all the studies landraces with others in gene bank. C. olitorius Seady collected sample showed the highest similarity with C. olitorius (99.88%) with the other matching as presented in Table (7). Data in Table (8) showed the nucleotides content in the studied landraces. Average nucleotides number was 1034 nucleotides using RbcL gene. GC content and AT content in C. olitorius Fallahywas identical with C. olitorius Seady. GC content represent 42.46% while AT content represent 57.54% in the total number of nucleotides (Table 8). C. olitorius Siwan, nucleotides composition was A: 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 Fallahyand C. olitorius Siwanat the same branch, while C. olitorius Seadywas in a separate branch, as shown in Figure (2).

Table (6): Amino acid composition of RbcL sequences of C. olitorius landraces grown in Egypt and it’s similar in NCBI.

Figure (2): Phylogenetic tree of the three C. olitorius landraces under study using RbcL gene.

Table (8): Nucleotides content of RbcL sequences of C. olitorius landraces grown in Egypt.

Figure (3): Phylogenetic tree of the three C. olitorius landraces grown in Egypt using RbcL gene.

Barcoding analysis of MatK genecompared 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 Siwanlandrace.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)

Table (9) Amino acid composition of MatK sequences of three landraces of Corchorus olitorius grown in Egypt.

Table (10): MatK gene sequences similarities for C. olitorius landraces under study.