Characterization, Amplication, and Phylogenetic Analysis of Gossypium herbaceum using rbcL Molecular Marker

the PCR products was done for DNA sequencing. After that, all the DNA sequences were compared with the available DNA sequences in public databases. The relationship between Gossypium herbaceum and other related species was evaluated using the neighbour-joining method for phylogenetic analysis. Results: The results showed a high percentage of pairwise nucleotide sequence identity with Gossypium richmond and Gossypium hirsutum . The study demonstrated the potential of DNA barcoding using the rbcL gene as a reliable method for identifying and con�rming Gossypium herbaceum . Also, this study provides valuable insight into the phylogenetic relationships of this medicinal plant species. Conclusions: The �ndings support the conservation and appropriate use of medicinal plants and highlight the importance of ensuring the authenticity and quality of herbal products.

Historically, this particular plant has been included in medicine and food preparation. Vitamin E can be found in cottonseed, which is used as a pain reliever, antioxidant and laxative. The root bark of the plant acts as an aphrodisiac, and a decoction made from it is used to treat amenorrhea [5]. In Unani medicine, leaves of G. herbaceum are useful in ishal al-atfal (childhood diarrhoea), and seeds are used to cure in qillatul laban [6]. The morphological characteristics of Gossypium herbaceum are given in Table  1. Unfortunately, for the last few decades, people's trust in the herbal mode of treatment is going decreased due to the unhygienic and unethical approach, i.e., adulteration (mixing or replacement of a particular medicinal plant with a morphologically similar plant that has no or least therapeutic values) [7]. Adulterating herbal medicines is becoming increasingly common, harming individuals and signi cantly negatively impacting the herbal industry. Medicinal plants are commonly traded in traditional herbal shops and markets as dried leaves, roots, bark, processed mixtures, extracts, and powdered parts. These plant materials must possess the necessary morphological characteristics to ensure accurate identi cation for retailers and consumers [8]. A more recent term used in the scienti c literature [10] is "DNA barcoding", which involves using standardised DNA regions to accurately identify species [9]. The method follows globally recognised protocols and DNA regions to establish a comprehensive database of organisms [11]. The importance of DNA barcoding in plants lies in the need to identify speci c species for their conservation and use, although in some regions, this may require more taxonomic expertise [12]. When macroscopic or microscopic identi cation methods present a challenge, DNA barcoding offers a reliable alternative for identifying biological materials [13]. It can detect and differentiate species at any stage of growth or processing, allowing DNA extraction [14]. DNA barcoding has already found practical applications in various elds, such as authenticating plant products, including medicinal plants [13]  Gossypium herbaceum is not a preferred choice for cotton production among cotton farmers in Pakistan, but it is occasionally cultivated in arid areas of Balochistan.
It is considered as native from Soviet Central Asia, and of adjacent Iran and Afghanistan.

Shrubby or herbaceous
Perennial Early July and blooms through August The plant can be a shrub or herb or annual, or perennial. Branches, petioles and pedicels may have stellate tomentose with or without common spreading hairs. The leaves are rounded in outline, measure 2-5 cm in length and width, and are glabrous above with a hairy margin. The lower nerves have stellate pubescence, which can sometimes be mixed with simple hairs. The Leaves are mainly deeply cordate and palmately (3-)5(-7)-lobed, with lobes that are oblong, elliptic, ovate, or broadly ovate. The lobes are entire and obtuse or obtuse at the apex. Stipules linearlanceolate or obliquely ovate, 6-12 mm long and 1.5-5 mm wide, while the petiole is 2-3.5 cm long. Pedicel 7-15 mm long. Epicalx segments are deeply cordate at the base, measuring 1-2 cm long and wide, and 6-8 toothed or lobed at the apex. The central part is the largest, with lanceolate teeth. Calyx cup-shaped, densely blackdotted, 7-10 mm long and wide, and 5-lobed or shorter. The corolla is yellow, 4-6 cm long, and the petals are ovate, measuring 2.5-3.5 cm long and 3-4 cm wide. The staminal column is 1 cm long and contains anthrafurans at the top. The capsule is

Ethnobotanical uses
Cotton has various uses, such as relieving symptoms of nausea, fever, headache, diarrhoea, dysentery, neuralgia and bleeding. Women have been known to use cotton to control menstrual disorders and menopausal symptoms. It has also been used to induce labour and childbirth and expel the afterbirth. For some women, cotton is used to increase breast milk production.
o b l o n g o r r o u n d , 2 . 5 -3 c m l o n g , comprehensive, and beaked at the apex. They are 3-5 celled, pitted and glabrous, each containing 5-7 seeds. The seeds are ovoid, measuring 6-9 mm long and 3-7 mm wide, covered with long white hairs ( and images of herbarium specimens. BOLD provides a means of managing projects and allows trace les, scans of herbarium specimens, and photographs to be stored alongside DNA sequences [12]. The purpose of this current study was to provide awareness among people about the importance of using authentic medicinal plants, adulteration in them, the effect of quality of particular medicinal plants and their harmful impacts on human health. We can improve our economy by promoting herbal plant treatment and their export. This is the only way to keep the quality of a particular herbal drug. The prime focus of this study is to provide facilities to learn more about the techniques used to indicate the adulteration persisting in plants, i.e., DNA Barcoding. nanodrop spectrophotometer (Thermo Scienti c, Wilmington, USA) was used to con rm DNA samples' concentration, purity, and quantity by recording the absorbance at 260 nm. DNA samples were typically diluted 20-fold, and a double-beam spectrophotometer model Halo DB20 was used to quantify DNA [24]. The gel electrophoresis technique was used to con rm plant DNA. A 1% agarose gel was formed in 1X TAE buffer, and the gel was allowed to solidify. The gel tray was immersed in a gel electrophoresis tank containing TAE buffer (1X), and the comb was carefully removed. To load the samples into the gel, 10 µl DNA sample and 4 µl loading dye were mixed well and loaded with a 100 bp ladder (Thermo Fisher Scienti c, USA). The gel was allowed to run at 100 volts for 30 minutes, and the appearance of bands in the gel con rmed DNA in the samples.   [21,22]. The plant was placed in a zip-lock bag after collection and labelled with the location and date of collection, common name, and plant identi er. The plant was transferred to the Plant Molecular Lab, Department of Botany, Govt-Graduate College of Science, Wahdat Road Lahore, at Ice Box. The collected plant leaves were stored in a -20 °C freezer before further processing. DNA analysis was used to detect adulteration in plant samples, and a protocol was developed to detect possible adulteration. The Doyle and Doyle method [23] was used for DNA extraction. Genomic DNA extraction involved crushing a 1 g plant sample in a sterile ice-cold pestle and mortar with liquid nitrogen, and the ground material was transferred to an Eppendorf. 750 µl of Cetyltrimethylammonium Bromide (CTAB) was added and incubated in a water bath at 65 °C for 30-45 min. Eppendorf added equal volumes of chloroform and isoamyl alcohol (24:1) solution and centrifuged at 10,000 rpm for 15 min. The supernatant was removed to another Eppendorf, and 750 µl of chloroform and isoamyl alcohol solution was added. This was centrifuged at 10,000 rpm for 15 min, and this step was repeated three times. The clear supernatant was collected in another Eppendorf, and 2/3 volume of ice-cold isopropanol was added to each Eppendorf containing the supernatant. DNA strands appeared as minute threads, and these Eppendorf were placed in a -20°C freezer overnight to allow the DNA strands to thicken and set. The following day, these Eppendorf tubes were centrifuged at 10,000 rpm until the DNA strands resolved as a pellet, and the pellet was washed three times with 200 µl washing buffer. The pellet was allowed to dry, and the DNA was dissolved in 50-70 µl of distilled water.

Primer sequence
[27] [28] [29] [30] A volume of 25 µl was prepared in PCR tubes containing the reaction mixture. PCR pro le was adjusted on the machine at the initial denaturation temperature of 94°C for 3 minutes, the annealing temperature of 48°C for 1 minute, and the extension temperature of 72°C for 10 minutes. All the temperature sequences were maintained to operate for 40 cycles. The PCR product (5 µl) was mixed with 3 µl of loading dye (5X) and allowed to run on 1% agarose gel to con rm DNA ampli cation (Figure 2).

R E S U L T S
After DNA ampli cation, the next step involves purifying the plant DNA sample. To achieve this, a technique involved adding distilled water to Eppendorf containing DNA, resulting in a total volume of 100 μl. An equal volume of a mixture of phenol and chloroform (100 µl) was added to the same Eppendorf, and the entire mixture was mixed by inversion. The Eppendorf was then centrifuged at 14000 rpm for 6 min, and the top aqueous phase of the solution was transferred to a new tube. Then, 9 µl of a 3-molar solution of sodium acetate (1/10th volume, pH 5.2) and 250 µl of absolute ethanol (2.5% volume) were added to the Eppendorf and then incubated at -20 °C for 30 min. The tubes were centrifuged at 14000 rpm for 10 min to precipitate the DNA and remove the ethanol. The DNA pellet was washed with 100 µl 70% ethanol, centrifuged at 14000 rpm for 2 min, and then the ethanol was removed. The DNA pellet was kept dry, and 20 µl sterile distilled water (SDW) was added to dissolve it. To validate DNA puri cation, 4 µl of DNA solution was loaded onto a 1% agarose gel. Puri ed PCR products were sent to "CELEMICS BTSeq™, Seoul, Korea" for sequencing, and the results were analysed by comparative study. Nextgeneration sequencing (NGS) was used to perform sequencing instead of Sanger sequencing because it is a more reliable and accurate method. NGS is an impressive sequencing technique that provides extraordinary levels of throughput, scalability, and speed, determining the nucleotide sequence of entire genomes or speci c regions of DNA or RNA. With NGS, the possibilities are endless, and the future looks bright for advances in genomics research [31]. The sequences were blasted on NCBI to nd the most closely related sequences. Twelve closely related sequences for plant samples were downloaded and used for the DNA analysis. The neighbour-joining method [32] was conducted using MEGAX software [33] for the phylogenetic analysis. The Bootstrap test with 1000 replications was included during the phylogenetic tree  Agarose gel electrophoresis was performed by allowing the gel to run for 30 minutes at a voltage of 100 volts. The observance was done under a UV illuminator in the Gel Documentation system (Bio-Rad, USA), and the appearance of bands in the gel con rmed the DNA in the samples (Figure 3). The concentration of ds DNAs of plants was determined using a spectrophotometer at 260 nm wavelength using distilled water as blank. The following formula calculated the DNA concentration [24]. DNA concentration (µg/ml) = E × OD260 × dilution factor. (E is extinction coe cient = 50 for dsDNA) (Table 3). DNA to pure is about 1.8. However, if the ratio falls below 1.6, it may indicate the presence of contaminants such as proteins or phenols, which absorb strongly at or near 280 nm [34]. Therefore, the C-1 and H-1 used for further working [35] (Table 4). Gossypium herbaceum samples were collected from different province of Punjab, Pakistan, stored in labelled zip lock bags and transported to the Plant Molecular Biology Laboratory at Government Graduate College of Science, Wahdat Road, Lahore. The plant was characterised morphologically, including its common name, genus, stem, leaves, owers and seeds. DNA was extracted using s t a n d a r d p r o t o c o l s a n d c o n r m e d b y 1 % g e l electrophoresis before ampli cation by polymerase chain reaction (PCR) with the various primers matK, rbcL, psbA-trnH, and ITS. was In 2009, CBOL (The Consortium for the Barcode of Life) recommended the use of the two loci rbcL and matK as a universal plant DNA barcode. The psbA-trnH spacer and nuclear internal transcribed spacer 2 (ITS2) can also be widely used [37]. Selected plants were successfully a m p l i e d w i t h p r i m e r r b c L , a n d t h e r e s u l t i n g ampli cations were puri ed. Nucleotides obtained from the rbcL gene were sequenced using next-generation sequencing technology at "CELEMICS BTSeq™, Seoul, Korea". The rbcL gene is an e cient DNA barcoding tool for G. herbaceum belonging to the Malvaceae family. It can identify processed plant products used in food, medicine or cosmetics. The results were consistent with previously reported rbcL gene sequences [38], and NCBI BLAST searches revealed the closest matches with the same species at 99%. The ampli ed rbcL gene sequence from Gossypium herbaceum showed a maximum percentage pairwise nucleotide sequence identity of 99.2% with rbcL gene sequences of G. richmondii and G. hirsutum and 99.0% with Gossypium barbadense. The identity of the studied plant (G. herbaceum) was con rmed by NCBI Blast and by studying many plants for their morphology and crossreferencing with different articles. We examined the nucleotide distances of different plants by comparing intraspeci c and interspeci c pairwise sequences.
Interspeci c homology was more signi cant than intraspeci c homology, indicating species having a higher percentage of homology with its closest relative. A  Figure 5).

Polymerase Chain Reaction (PCR)
The PCR of the plant DNA sample was carried out using DNA barcoding primer pairs (matK, rbcL, nrITS and trnH-psbA) [26] in Bio Rad Thermocycler. 10 µl of PCR product was used in the 1% agarose gel electrophoresis. The required size band was obtained and captured with the help of available gel documentation. Only the rbcL primer pair gave positive results for the plant sample ( Figure 4). successful DNA barcode must show the difference between intraspeci c and interspeci c distances. Animals typically show a high degree of variation between species in mitochondrial COI, resulting in a "barcode gap" that allows species differentiation with reasonable certainty. However, this "barcode gap" in the plastid DNA regions mark, and rbcL is absent in closely related land plant species. In our study, when we performed intraspeci c versus intraspeci c sequence divergence [39] analysis, many sequences overlapped, requiring no further testing to determine the barcode gap. Some researchers have used the barcode gap and distance method to distinguish plant groups beyond the species or generic levels, many plant species' mathematics at the species level and the absence of barcode gaps [40] in rbcL have been used and well documented. To assess the effectiveness of barcodes in separating species, we performed cluster analysis and constructed NJ trees with bootstrap analysis [42]. Gossypium herbaceum (MS05) was found to be in the same clade as Gossypium richmondii and Gossypium hirsutum with a 99.2% bootstrap value based on the phylogenetic tree using the DNA sequence of the rbcL gene. Our results demonstrated that a barcoding gene (rbcL) can identify G. herbaceum. The rbcL gene sequence has a high discrimination e ciency for G. herbaceum, making it a valuable barcode for species identi cation. The short sequence of the single gene rbcL is an informative and potentially powerful molecular tag for identifying grass and cultivated plant species.

C O N C L U S I O N S
In this study, it has been found that herbal products are widely used in different aspects of life, but they need to be identi ed and checked consciously, and they may also lead to disastrous results for human health. Therefore, it is compulsory to con rm whether the herbal product is the same as required or whether it has been adopted to obtain their personal goals. In future, Molecular identi cation (DNA Barcoding) will be a bene cial tool to identify plant or plant products for their e cient use in various elds of life.