Chemical and molecular examinations of some cowpea genotypes using simple sequence repeat and intersimple sequence repeat DNA markers in relation to their cooking quality

Abstract Cowpea is a leguminous crop that has received widespread attention due to its high nutritional value. However, it is prone to losing some of its nutritional content due to the long cooking process. In this study, fourteen cowpea genotypes were evaluated for their chemical properties before and after cooking, along with the effect of different cooking treatments on the cooking time, considered as the main indicator of the cooking quality. Moreover, the correlation between molecular markers (simple sequence repeat (SSR) and intersimple sequence repeat (ISSR)) and the cooking time of cowpea genotypes was determined. The obtained results showed significant differences between all the investigated genotypes before and after the cooking procedure, reflecting significant genotypic and heritability estimates. Kareem 7 and Greenish Black Balady genotypes showed the shortest cooking time. Microwave's treatment manifested the shortest cooking time compared with the other treatments, which appeared as a new approach to improve the cooking quality of cowpea seeds. Spearman's rank correlation showed that the calculated values were smaller than the tabulated value at 0.05, reflecting the existence of a rank correlation between SSR/ISSR‐PCR banding products and the cooking time of cowpea genotypes. Such a study appeared to be a new approach, particularly in Egypt for the proper selection of the optimal cowpea seed on the basis of its cooking quality.


| INTRODUC TI ON
is a legume crop cultivated in tropical and sub-tropical countries. This crop exhibits a wide range of differences in its seed characteristics, either morphological or chemical properties (Boukar et al., 2019;Langyintuo et al., 2003). The homeland of cowpea was Southern Africa (Beshir et al., 2019), and later it moved to East and West Africa, and recently India is considered the modern center of diversity of this crop. Cowpea is one of the nutrition staple foods in many parts of the globe (Odedeji & Oyeleke, 2011). Indeed, it is an important source of proteins and carbohydrates already used for the human diet, in addition to minerals, vitamins, and cereals (Gomes et al., 2019;Horn & Shimelis, 2020).
Thus, it is a wealthy plant protein source for people who cannot get proteins from animal sources (Akpapunam & Sefa-Dedeh, 1997), and also for young children as the main component of their diet.
This plant is widely distributed and exhibiting a wide range of variations in its characteristics. Understanding such variabilities seems to be the key to establish some improvement programs. Simple sequence repeat (SSR) and intersimple sequence repeat (ISSR) primers have been widely used for studying genetic varieties among different genotypes.
Both markers are advantageous over the others owing to their easier use, lower cost, faster activity, and absence of radioactive substances.
SSR and ISSR markers have the potential to identify polymorphisms and determine genetic diversity in intermicrosatellite loci, using primers designed from repetitions of di-or tri-nucleotide simple sequences (Araújo et al., 2019;Badiane et al., 2012;Chen et al., 2017). The polymerase chain reaction (PCR) technique is developed to introduce several assays based on selective DNA amplification, such as SSR and ISSR primers to detect specific variations of nucleotide sequences of polymorphic DNA (Araújo et al., 2019). Moreover, the cowpea seeds are very susceptible to the process treatment and, mainly, to the cooking time, being thermolabile. Therefore, the cooking time is considered the main point of the cowpea seeds cooking quality. Longer cooking time is associated with a decrease in the nutritional value of the cooked seeds.
Shorter cooking time is more acceptable and desirable as it would allow reducing the process duration and the energy use, and also saving the associated costs (Hamid et al., 2015;Ngoma et al., 2018).
In this framework, the current study aimed to evaluate fourteen cowpea genotypes for their chemical properties before and after cooking and to investigate the effect of various cooking treatments on the cooking time. Furthermore, the association between SSR or ISSR DNA markers and the cooking time of cowpea genotypes was evaluated since it may play an important role in the selection of the best genotypes for their cooking quality.

| Cowpea seeds, cultivation conditions, and chemicals
The plant materials used in this study comprised 14 cowpea genotypes, including 9 Egyptian (5 Cultivar and 4 Balady) and 5 introduced ones (1 Brazilian and 4 Chinese) (external morphological properties, Figure 1). The seed of these entries was planted in the summer season of 2018. The plants received equal normal practices of cowpea cultivation to obtain more new seeds. The chemicals and buffer solutions used for molecular analysis such as dNTPs, Tris HCl buffer, Taq DNA polymerase, and Tris-Borate EDTA buffer were obtained from Sigma-Aldrich Chemical Co., Ltd. All other reagents and chemicals were of analytical grade, from local suppliers (Egypt).

| Chemical examination of cowpea seeds
Five grams of dry cowpea seeds were randomly taken from each genotype and grounded manually. The powdered samples were divided into three replicates. Moisture and protein contents were determined according to Chemists and Horwitz (1975). The ash content was detected by burning the samples in a muffle furnace for 2 hr at 550°C (Hamid et al., 2016). The fibers amount was determined according to the method described by Umar (2014). The nonprotein substances, that is, carbohydrates, lipids, and vitamins were calculated by subtracting protein, ash, and fiber contents from 100.

| Cooking technique and treatments
Five grams of each cowpea genotype seed were taken, and three replicates were considered for each kind. The seeds of each genotype/ replicate were subjected to different treatments before the cooking procedure. These treatments included airily drying unsoaked seeds, toasting unsoaked seeds, dry heat pretreatment at 60°C for 1 hr, soaking seeds in distilled water for 4 hr, soaking seeds in tap water for 4 hr, soaking seeds in 0.5% of sodium bicarbonate (purity, 99.8%) solution for 4 hr, soaking seeds in 0.5% of baking powder solution for 4 hr, and exposing the soaked seeds in tap water to microwaves for 4 min. A cooking technique was carried out according to Yeung et al. (2009) with major modifications in the cooking procedure. The samples were placed in 250 ml flasks filled with 100 ml of cooking solution or water (tap or distilled) according to the treatments.
The flasks were covered with aluminium foils and placed in a boiling water bath. Standard laboratory hotplates were used to maintain uniform and constant temperature during cooking.
Cooking time represents the time from the beginning of the cooking liquid boiling to the complete cooking. During boiling, aliquots of 3-4 seeds were drawn at 5 min intervals up to 20 min, and after 2 min the seeds' softness was tested by pressing between the index finger and the thumb. When the cooked seeds reached their desirable tenderness, the cooking time was recorded. The optimal time was that corresponded to the softness of 85% of the seeds. At the end of the cooking procedure, each flask's content was transferred to a puncher funnel to separate the cooked seeds from the cooking liquid; the cooked seeds were weighed, put in plastic bags, and kept frozen till examination.

| Genomic DNA extraction
Five seeds from each cowpea entry were germinated in a Petri dish.
DNA was extracted from 0.1 g of fresh and young leaves of each entry, according to Dellaporta et al. (1983). The extracted DNA was measured using a UV spectrophotometer (Jenway, 6305) at 280 nm, and its purity was adjusted according to Karp et al. (2012). DNA samples were stored at −20°C till examination by PCR.

| SSR and ISSR analysis
SSR and ISSR-DNA markers technique was used to genetically characterize the studied cowpea genotypes at the molecular level. This technique is based on the amplification of short segments of target genomic DNA using SSR and ISSR primers (Williams et al., 1990). The names and sequences of SSR and ISSR primers used in this study are shown in Table 1

| Agarose gel electrophoresis of PCR products
The agarose gel electrophoresis to visualize the PCR products were prepared according to Armstrong and Schulz (2008) with some modifications. Briefly, placing 1.5 mg agarose in 100 ml Tris-Borate EDTA (TBE) buffer and then boiling it in a water bath. Ethidium bromide (5 µl) was added to the melted gel after the temperature decreased to 55°C. The melted gel was poured into a mini-gel apparatus and the comb was immediately inserted, then the comb was removed when the gel hardened, and the gel was covered by TBE buffer. Eight μL of PCR products were loaded in each well and run at 80 V. PCR products were visualized through UV light using a UV spectrophotometer (Jenway, 6305) at 280 nm. The bands of SSR and ISSR-PCR products were counted, scored, and ranked. The obtained bands were compared with the bands of the DNA ladder (Non-Liner Dynamic Lth, USA).
All bright and visible fragments were scored as (1) if present or (0) if absent (Table S-1,2). The banding patterns were studied taking into F I G U R E 1 External morphological properties of studied cowpea genotypes; (a) Cultivars genotypes, (b) Balady genotypes, (c) Introduced genotypes consideration the relative migration of their different sizes. To study the association between either SSR or ISSR-PCR banding products and the cooking time of the studied cowpea genotypes, Spearman's rank correlation coefficient (Rs) was calculated according to Sokal and Rohlf (1995) and was determined using the following equation Whereas d, the difference in ranks of the genotypes; n, the number of genotypes. The results were always between 1 (a perfect positive correlation) and −1 (a perfect negative correlation). The highest performance genotype presented the highest ranking value, and vice-versa, for either cooking time or PCR banding products categories.

| S TATIS TIC AL ANALYS IS
An analysis of variance (ANOVA) test for the chemical composition and cooking treatment of cowpea genotypes was performed using CoStat software (version 6.4, Monterey, CA, USA). The obtained data were expressed as mean ± SD, and the differences were considered to be statistically significant for p ˂ .05.

| Chemical composition of cowpea seeds
The chemical composition of cowpea seeds seemed to be of great importance in their cooking quality. Table 2 shows the chemical composition of cowpea genotypes seeds before and after cooking.
The data exhibited significant differences (p < .05) among cowpea genotypes for all the investigated chemical properties before and after cooking, except for the fiber content. Cowpea genotypes seeds presented different moisture content on the basis of their weights and/or sizes. The moisture contents ranged from 6.40% to 9.50% and 23.54% to 32.80% before and after cooking, respectively. The lowest values before and after cooking were recorded in Greenish Black Balady (6.40 ± 0.36 and 23.54 ± 0.62) while the highest values in Variegated Chinese (9.50 ± 0.28 and 32.80 ± 0.17) followed by Azmerlli (8.60 ± 0.16 and 29.34 ± 0.11), respectively. Cooked cowpea genotypes seeds showed an increase in moisture contents when compared to cowpea genotypes seeds before cooking. This increase can be explained by the imbibition of the cowpea seeds during cooking (Melo et al., 2017). Concerning the protein content, it ranged from 21.22% to 30.26% and 14.49% to 21.37% before and after cooking, respectively. Previous literature reported that cowpea seeds were rich in protein content, containing about 21%-31% Values with the different superscript letter (uppercase, same genotype before and after, within a row) are significantly different (p < .05).
*Cooking process conditions; the seeds are soaked in tap water for 4 hr, then placed in 100 ml of tap water and cooked in a boiling water bath until the end of cooking.
proteins per 100 mg (Anam 2016;El-Jasser, 2011;Henshaw & F.O., 2014 El-Jasser, 2011; Kamara et al., 2018;Silva et al., 2018). Cowpea genotypes seeds after cooking showed a reduction in ash when compared to uncooked cowpea genotypes seeds. This reduction in ash content can be attributed to the loss of minerals through diffusion in the water used in the thermal treatment (Melo et al., 2017). However, the cowpea genotypes seeds presented nearly similar fiber contents before and after the cooking process.

| Cooking time
Cooking time is considered the main indicator of cowpea seeds cooking quality and the pivotal factor in the choice of a particular variety by the consumers. Taking into account that the nutritional value of cowpea seeds strongly depends on their nutrients amounts and that the nutrients could be lost during the cooking process, any cooking treatment that efficiently reduces the cowpea seeds cooking time is highly beneficial (Hamid et al., 2015). The cooking times of cowpea seed genotypes estimated under different cooking treatments are compared in Table 3. The obtained results exhibited significant differences among all genotypes and cooking treatments, reflecting different reactions, and cooking time influence, correlated to their different genetic backgrounds. Similar results were previously reported about rapid methods to evaluate the cowpea cooking properties by soaking dry seeds for 12 hr, followed by cooking for 27 min (Mashi, 2006;Yeung et al., 2009). The authors found significant differences in cooking quality among the studied 52 cowpea cultivars (Mashi, 2006;Yeung et al., 2009).
Azmerlli and Variegated Chinese seeds, both unsoaked, air-dried, and toasted seeds, presented a longer time to cook, probably due to their larger and rounder shape, followed by the white Brazilian. On in fact, it contains further ingredients, such as sodium pyrophosphate and corn meal, in addition to sodium bicarbonate which could lead to improving the cooking time and quality of seeds (Bhokre & Joshi, 2015). Adding baking powder to soaking and cooking solutions of cowpea seeds seemed to be a new approach for improving the cowpea cooking quality. Regarding the exposure of soaked cowpea seed to microwave, Variegated Chinese presented the longest

Tap water and microwave
Azmerlli 64.00 ± 0. Thus, it is possible to conclude that the exposure of cowpea seeds to microwaves before cooking appeared to be a promising approach in cowpea cooking to improve their quality.
Generally, cowpea seeds consumption might be limited owing to the presence of several anti-nutrient factors such as tannins, phytate, and trypsin inhibitors, which reduced their availability (Khalid & Elhardallou, 2016). Soaking process in water before cooking might remove such factors: during soaking, cowpea seeds undergo F I G U R E 3 Electrophoretic banding patterns of ISSR primers produced from the studied cowpea genotypes physiochemical changes leading to softening the seed tissues and hence promoting a shorter cooking time (Hamid et al., 2016;Kouam et al., 2018;Yeung et al., 2009). Moreover, the soaking of cowpea seeds prior to cooking would remove the seed coat pigmentation and polyphenols; also, the content of proteins, sugars, and ash would be affected, which diffuse in the soaking solution. Finally, the soaking of cowpea seeds before cooking would reduce the risk of flatulence due to the oligosaccharides, which can be fermented by bacteriaproducing gas. For these reasons, Hawaiian cooks usually add few amounts of ginger to the cooking solutions in order to decrease the potential development of gases (Animasaun et al., 2015;Ngalamu et al., 2015;Yeung et al., 2009).
Finally, the ranking pattern of the studied cowpea genotypes according to their estimated cooking under different cooking treatments for genotypic evaluation is presented in Table 4. The highest score (14) was revealed in the case of the genotype with the shortest cooking time, whereas the lowest score (1) in the case of longest cooking time as expected. Then, the mean rank of each genotype was estimated.
The data showed that Kareem 7 ranked first with the shortest cook-

| Molecular studies
Molecular markers appeared to be useful tools for assessing genetic variations at the molecular level (Araújo et al., 2019). The electrophoretic banding patterns of SSR and ISSR primers produced from cowpea genotypes are illustrated in Figures 2 and 3, respectively. The distribution of SSR and ISSR bands among cowpea genotypes is given in

| CON CLUS ION
The cooking time is considered the main indicator of the cooking quality of cowpea seeds and an important factor in the selection of a particular variety by the consumers. The reduction in cooking time seems to be advantageous, requiring less energy and fuel, and mainly reducing the possible loss of cowpea seeds nutrients, thus improving their nutritional value. Among the fourteen investigated cowpea seed genotypes, the Egyptian cultivar Kareem 7 achieved the shortest cooking time, and the Black Balady genotypes manifested the greatest protein amount in their cooked seeds with respect to the other ones. Concerning the investigation of the genetic variations at the molecular level, the twelve SSR primers amplified 447 bands and were widely and differently distributed among the cowpea genotypes. In comparison, the ten ISSR primers amplified a total number of 892 bands, widely distributed among the studied genotypes. The association between the mean cooking times and SSR or ISSR-PCR banding products of the studied cowpea genotypes was estimated in underserved trials. Such a study appeared to be a new approach and, for the first time, was performed in Egypt. Ultimately, the use of SSR and ISSR primers gave a clearer picture as effective tools in assessing and detecting the molecular polymorphisms existing among the studied cowpea genotypes and assuring their phylogenetic relationships.

ACK N OWLED G M ENTS
The authors are grateful to the Faculty of Agriculture, Zagazig University, Zagazig, Egypt for their support.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The dataset supporting the conclusions of this article is included within the manuscript.