Research Article

Evaluation of Genetic Diversity in Wild Rhynchosia capitata through Morphomatric and Biochemical Character Collected from District Dir Lower

Shahid Iqbal1, Arshad Khan, Ali Hazrat1*, Gul Rahim, Mohammad Ihsan1, Umar Zad Gul1, Maryam Bibi1, Khadija Bibi1 and Muhammad Mukhtiar2

1Department of Botany, University of Malakand, Chakdara, Dir (Lower)-18800, Khyber Pakhtunkhwa, Pakistan; 2Department of Pharmacy University of Poonch Rawalakot, Azad Kashmir, Pakistan.

Received | November 02, 2023; Accepted | January 10, 2024; Published | February 15, 2024

*Correspondence | Ali Hazrat, Department of Botany, University of Malakand, Chakdara, Dir (Lower)-18800, Khyber Pakhtunkhwa, Pakistan; Email: [email protected], [email protected]

Citation | Iqbal, S., A. Khan, A. Hazrat, G. Rahim, M. Ihsan, U.Z. Gul, M. Bibi, K. Bibi and M. Mukhtiar. 2024. Evaluation of genetic diversity in wild Rhynchosia capitata through morphomatric and biochemical character collected from district Dir Lower. Sarhad Journal of Agriculture, 40(1): 213-220.

DOI | https://dx.doi.org/10.17582/journal.sja/2024/40.1.213.220

Keywords | Morphology, Cluster analysis, PCA, Correlation

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The Rhynchosia capitata (Roth) DC, belong to Fabaceae which is widely distributed in tropical areas, In Pakistan, Rhynchosia capitata, is a perennial summer plant, It is indigenous to Sri Lanka, India and Pakistan Southern Punjab, is appropriate a more serious pest of agricultural (Dogra et al., 2009; ILDIS, 2010; Ali et al., 2013). After watering, the plant emerges from the seed in the field, it is an annual plant with numerous branches that surround and root at each node. A nearly month-old plant starts to bloom and produces oval-shaped pods with two seeds in each pod. Typically, the spherical seeds are dark in hue. The plant starts to dry out after three months, and the seeds mature. From May to October, when the typical low and high temperatures are 29/21 °C and 39/29 °C, respectively, the season for growth is in effect (Noor et al., 2018).Despite the fact that they are influenced by environmental changes and conflict with the study of genetic variability, morphological traits are important for the estimation of genetic diversity in plants (Nisar et al., 2016). On the other hand, there are several advantages to using DNA-based techniques and molecular methodologies for genetic diversity estimation as opposed to traditional morphology (Ndiaye et al., 2012). Comparatively to biochemical analysis at the protein level, investigating DNA characterization at the molecular level is too expensive (Win et al., 2011). The protein isolation technique is a straightforward, trustworthy, affordable, and ecologically consistent method for biochemical measurements (Wadood et al., 2016). SDS-PAGE method that is increasingly being used to determine the genetic makeup of agricultural species (Hameed et al., 2009). SDS-PAGE has received a lot of attention over the last 20 years for evaluating genetic variation, making reliable decisions, and cataloguing plant species. Cultivated variants of a number of crop plant species have been successfully identified and taxonomic relationships determined using seed storage protein characterization; Beans Chickpea (Ghafoor et al., 2003; Nisar et al., 2007), Pisum sativa (Nisar et al., 2009), Lens culin (Win et al., 2011). The latent uses of the wild relatives as genetic tools for crop breeding and enhancement are the focus of this assessment, and we concise the different genetic/genomic slants postulated for these drives. For tree improvement, programs can be developed on the results of the proper study using morphological and biochemical characterization. The aim of the present research is to find out the genetic diversity in food legumes. To evaluate the genetic diversity in Rhynchosia capitata through morphological characterization. To score out total seed storage proteins using SDS-PAGE methods also to score out the most promising genotypes future breeding program.

Materials and Methods

The present study was carried out in 2023 at the University of Malakand Department of Botany. A total of 36 genotypes of Rhynchosia capitata were used for agro morphological and biochemical characteristics. A total of 12 morphological traits in which nine quantitative traits: Petiole length, leaf length, leaf width, seed length, seed width, and seed weight, pod length, number of seed per pod, number of pods per plant, number of branches per plant, and plant height, and three qualitative traits were used in the current study.

SDS-PAGE analyses were performed on all the studied genotypes to determine total seed storage proteins. Five healthy and mature seeds were finely ground with a mortar and pestle, about 0.02g of the ground material was weighed and transferred into 1.5 ml centrifuge tubes with 400 protein extraction buffer. The material was than homogenized for 1 minute in a vortex before being centrifuged at 12,000 rpm for 10 minutes at room temperature. For total seed protein, 12.5% polyacrylamide gels were used, as per Laemmli’s 1970 procedure.

Data analysis

To determine genetic diversity, five plants were randomly selected and the mean value of each genotype was used for data analysis. Microsoft Excel 2016 was used to compute basic statistics (Mean, Maximum, Minimum, and Coefficient of variation). For cluster analysis and principal component analysis PC ORD software version 6, and correlation analysis SPSS version 22 were used. Similarly, for total seed storage protein the binary matrix data was calculated as the presence and absence of bands. Two way cluster Dendrograms were generated with the help of PC ORD version 6 using the unweighted pair group method with arithmetic average (UPGMA) technique.

Results and Discussion

Genetic diversity based on agro morphological characterization

The current research investigated at both qualitative (3) and quantitative (9) traits in Rhynchosia capitata genotypes from Dir (L). During the investigation 3 qualitative characters of Rhynchosia capitata were studied which are leaf colors, seed coat color, and seed shape. A single allele was found to be in charge of regulating leaf color; each was determined to be yellow-green. Every seed was circular and had a head on top. For all of the genotypes, the colour of the seeds was found to be brown 42%, grey 54%, and pale grey 4%. There were two types of seed texture: Rough 55% and smooth 45%.

Quantitative characters

During the present study, 9 quantitative traits were studied in Rhynchosia capitata collected from 2 Districts, and there was found a large number of variations for most of the traits. The maximum value for internodes length was recorded 4.1 cm while minimum was 1.7 cm with the mean value of 2.58 cm. Minimum value for biomass per plant of 2 gm and maximum value of 8 gm with the mean value of 4.14 gm, standard deviation 0.328, sample variance 3.773. .Minimum value 0.2 cm and maximum of 2 cm for the Petiole length with the mean of 1.01 cm, with a mean value of 18.9 cm, a standard deviation of 6.568, and a sample variance of 43.14 percent, the highest range for plant height was 35 cm, while the minimum was 10 cm. Leaf length had a mean value of 3.24 cm, a standard variation of 0.877, a sample variance of 0.770, and a range of 2 cm to 6 cm, respectively. The mean value for leaf width was 2.36 cm, standard deviation of 0.597, sample variance of 0.356, with the minimum value of 1 cm and maximum value of 3.2 cm. Similarly pod length has maximum range from 18 cm while minimum range from 1.1 cm with the mean value of 2.87 cm, standard deviation 2.738 and sample variance 7.495%. Furthermore, seed per pod with the mean value of 2.86, standard deviation 0.912, sample variance 0.832%, minimum value of 2 and maximum value of 4. The mean value for pod per plant was 14.2 standard deviation 5.759, sample variance 33.165, minimum value 8 and maximum value of 40 pods per plant. Similarly the association coefficients among 9 quantitative traits are, Internodes length (2.56), Branch per plant (3.96), petiole length (0.94). Total plant height (31.42), leaf length (3.28), leaf width (2.35), pod length (3.05), seed per pod (2.79) and pod per plant (14.54) (Table 1, Figure 1).

Correlation analysis

For nine morphological characteristics, correlation was calculated using MS Excel 2016. Internode length showed a negative correlation with biomass per plant (-0.07), petiole length (-0.15), pod length (-0.14), and pod per plant (-0.13), while a positive correlation was seen with plant height (0.03), leaf length (0.18), leaf width (0.10), and seed per plant (0.12). Biomass per plant show positive correlation with leaf length (0.11), leaf width (0.02) and seed per plant (0.22) whereas negative correlated with petiole length (-0.04), plant height (-0.02), pod length (-0.05) and pod per plant (-0.09). Petiole length show negative correlation to plant height (-0.15), leaf length (-0.08), leaf width (-0.27) and seed per plant (-0.03) while positive correlated to pod length (0.08) and pod per plant (0.04). Plant height show positive correlation to leaf length (0.16), leaf width (0.15), pod length (0.13), seed per pod (0.10) and pod per plant (0.18). Leaf length show positive correlation to all traits except seed per pod (-0.32). Leaf width show positive correlation to pod length (0.12) and seed per pod (0.24) while negative to pod per plant (-0.31).

 

Table 1: Descriptive statistics of Rhynchosia capitata collected from Dir Lower.

Traits	Mean	Standard error	Standard deviation	Sample variance	Minimum	Maximum	CV %
IN	2.58	0.125	0.739	0.546	1.7	4.1	0.29
B/P	4.14	0.328	1.942	3.773	2	8	0.47
PtL	1.01	0.084	0.495	0.245	0.2	2	0.49
PH	18.9	1.110	6.568	43.14	10	35	0.35
LL	3.24	0.148	0.877	0.770	2	6	0.27
LW	2.36	0.101	0.597	0.356	1	3.2	0.25
PL	2.87	0.463	2.738	7.495	1.1	18	0.95
S/P	2.86	0.154	0.912	0.832	2	4	0.32
P/P	14.2	0.973	5.759	33.165	8	40	0.41

IN, Internodes length; B/P, branch per plant; PtL, petiole length; PH, total plant height; LL, leaf length; LW, leaf width; PL, pod length; S/P, seed per pod; P/P, Pod per plant.

 

 

Pod length show negative correlation with seed per pod (-0.05) while positive to pod per plant (0.16). Seed per plant show negative correlation with pod per plant (-0.11) (Table 2).

 

Table 2: Correlation analysis in 36 R. capitata collected from Dir Lower.

Traits	IN	B/P	PtL	PH	LL	LW	PL	S/P	P/P
IN	1
B/P	-0.07	1
PtL	-0.15	-0.04	1
PH	0.03	-0.02	-0.15	1
LL	0.18	0.11	-0.08	0.16	1
LW	0.10	0.02	-0.27	0.15	0.01	1
PL	-0.14	-0.05	0.08	0.13	0.00	0.13	1
S/P	0.12	0.22	-0.03	0.10	-0.32	0.24	-0.06	1
P/P	-0.13	-0.09	0.04	0.18	0.01	-0.31	0.16	-0.11	1

IN, Internodes length; B/P, branch per plant; PtL, petiole length; PH, total plant height; LL, leaf length; LW, leaf width; PL, pod length; S/P, seed per pod; P/P, Pod per plant.

 

Table 3: Principal component analysis (PCA) based on 9 morphological traits.

AXIS	PC1	PC2	PC3	PC4
% of Variance	19	15.85	14.51	12.02
Cum.% of Var.	19	34.87	49.38	61.4
Eigenvalue	2.83	1.829	1.329	0.996
Traits	Eigenvector
IL	0.33	-0.17	0.37	0.235
B/P	0.22	0.119	-0.02	-0.85
PL	-0.4	0.317	-0.05	-0.08
PH	0.15	-0.51	-0.38	-0.12
LL	0.01	-0.57	0.395	-0.31
LW	0.54	-0.11	-0.22	0.227
PDL	-0.1	-0.25	-0.53	0.127
S/P	0.43	0.35	-0.37	-0.14
P/P	-0.4	-0.28	-0.3	-0.12

 

Principal component analysis (PCA)

Principal component analysis (PCA) based on 9 quantitative traits shown significant divergence among the 36 genotypes of R. capitata. It was determined that 4principal component with an Eigen value of above (0.99) accounted for 61.4% of the total variation (Table 3, Figure 2). In PC1 the total variation was 19% and the variation found with the highest values are associated with internode length (0.33), branches per plant (0.22), leaf width (0.54) were found positively weighted. The entire variance in PC2 was 34.87%. Plant height (-0.51) and leaf width (-0.57) were found to be negatively weighted, while the variation associated with pod length (0.31) and seed per pod (0.35) was found to be favorably weighted. Similarly, internode length (0.37) and leaf length (0.39) were favorably correlated with weight in PC3, while plant height (-0.38), seeds per pod (-0.37), and pod length (-0.53) were negatively correlated. The total variation in PC3 was 49.38%. Internode length (0.23) and leaf width (0.22) were found to have positive weight contributions in PC4, whereas branches per plant (-0.85), leaf length (-0.31), and seeds per pod (-0.14) were found to have negative weight contributions. The overall variation in PC4 was 61.4% (Table 3).

 

SDS-PAGE characterization and genetic association within genotypes

To determine the genetic variation in seed storage proteins, all R. capitata genotypes were examined using total seed storage proteins. In the current research, 12.5% acrylamide gel was employed. There were a total of 14 polymorphic bands observed; the highest degree of variation was observed in B1 (0.96%) and B2 (0.94%), B4 (0.85%), B6 (0.75%), and B7 (0.73%), in that order. Similar to B12 (0.2%), B14 (31%), B13 (0.34%), B11 (0.41%), B9 (0.55%), and B8 (0.65%) all showed modest levels. cluster dendogram was construct to show the level of genetic variation among the collected genotypes (Figure 3 & 4), and all the genotypes were divided into 2 main linkage and further divided in sub cluster. Linkage 1 consist of 4 cluster such as C-1 (RC01, RCO5, RC04, RC08, RC09, RC10, RC02, RC03) C-2 (RC06) C-3 ( RC18, RC23, RC19, CR20, RC24) C-4 (RC07, RC21) and linkage 2 consist of 2 sub cluster, C-5 (RC11, RC13, RC22, RC17) C-6 (RC12, RC14, RC15, RC16).

 

 

The validation of group analysis by clustering by scatter plot by Principal Components based on SDS-PAGE in 20 R. capitata genotypes. The entire population, which has been divided into 9 groups, exhibits a high degree of genomic divergence rather than genetic connection. Each group contains a striping pattern that denotes a particular landrace (Figure 2).

The importance of genetic diversity within genotypes for crop growth programmes cannot be overstated (Win et al., 2011; Simon et al., 2007). The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of seed storage protein is a common method for demonstrating genetic diversity and relationships between different taxa (Nisar et al., 2007). Researchers have studied genetic variation in different plant species using electrophoretic representations of total seed proteins (Sundin et al., 2004; Ayten et al., 2009; Ghafoor and Arshad, 2008). SDS-PAGE has been used in numerous Leguminosae studies (Hussain et al., 2005; Hussein and George, 2002). Although in Pakistan, the current research represents the first known attempt to use SDS-PAGE to identify intra-specific genetic polymorphism in R. capitata genotypes. In the current study, phenotypic characterization of the seeds and SDS-PAGE descriptions of 36 genotypes of R. capitata showed a high degree of intra-genotypic diversity. One allele was found to be responsible for regulating the specific foliage color. For all genotypes, the colour of the seeds was discovered to be one of brown, grey, or pale grey. There were two kinds of seed texture: Rough and smooth. Roughness was 55%, while smoothness was 45%.

Because environmental changes have no impact on storage proteins, SDS-PAGE protein profiling is regarded as a reliable technique for economically describing germplasm (Javid et al., 2004; Iqbal et al., 2005). The protein profiles of 20 cowpea landraces were examined using 10% slab gel electrophoresis, and the findings demonstrate how similar the genotypes are. To eliminate duplicates and create a core collection of R. capitata, improved gene bank management requires an accurate and thorough knowledge of agricultural and biochemical data (protein and DNA). Variations in SDS-PAGE can be used to understand the degree of genetic variation and the connections among Pakistani R. capitata.

Conclusions and Recommendations

A significant genetic diversity was found in qualitative characters on the basis of frequency distribution shown variation. On behalf of these traits descriptive statistics, correlation and cluster analysis was made the quantitative traits was observed with significant variation for all the traits. Their genetic diversity studied through the biochemical analysis by SDS-PAGE. There was significant diversity among the genotypes. A total 14 bands were found, all the protein bands were polymorphic, there were no monomorphic bands found.

Novelty Statement

This is the first study of evaluating the Genetic Diversity in wild Rhynchosia capitate in Dir Lower (KP), Pakistan.

Author’s Contribution

Shahid Iqbal: Helped in collection of data fromthe field.

Arshad Khan: Result collection and field work,Objective and title configuration.

Mohammad Ihsan: Resultcalibration with software’s.

Ali Hazrat: Discussion calibrationwith result.

Gul Rahim: Help in experimental work.

Maryam Bibi: References designing according to the journal standard.

Umar Zad Gul: Review of literature.

Khadija Bibi and Muhammad Mukhtiar: Overall compilation of the paper.

Conflict of interest

The authors have declared no conflict of interest.

References

Achuba, F.I., 2006. The effect of sublethal concentrations of crude oil on the growth and metabolism of cowpea (Vigna unguiculata) seedlings. Environmentalist, 26: 17-20. https://doi.org/10.1007/s10669-006-5354-2

Albert, P.S., Z. Gao, T.V. Danilova and J.A. Birchler. 2010. Diversity of chromosomal karyotypes in maize and its relatives. Cytogenet. Genome Res., 129(1-3): 6-16. https://doi.org/10.1159/000314342

Ali, H.H., A. Tanveer, M.A. Nadeem, H.N. Asghar and M.M. Javaid. 2013. Germination ecology of Rhynchosia capitata: An emerging summer weed in Asia. Planta Daninha, 31: 249-257. https://doi.org/10.1590/S0100-83582013000200002

Beckstrom-Sternberg, S.M., 1989. Two-dimensional gel electrophoresis as a taxonomic tool: Evidence from the Centrospermae. Biochem. Syst. Ecol., 17(7-8): 573-582. https://doi.org/10.1016/0305-1978(89)90102-6

Bravo, R., 1984. Two-dimensional gel electrophoresis of proteins: Methods and applications. Academic Press.

Celebi, A., L. Acik and Z. Aytac. 2009. Biosystematics studies among Ebenus L. species based on morphological, RAPD-PCR and seed protein analyses in Turkey. Pak. J. Bot., 41(5): 2477-2486.

Chen, F., H. Liu, Q. Yao, P. Fang and F. Lv. 2015. Genetic variations and evolutionary relationships among radishes (Raphanus sativus L.) with different flesh colors based on red pigment content, karyotype and simple sequence repeat analysis. Afr. J. Biotechnol., 14(50): 3270-3281. https://doi.org/10.5897/AJB2015.14911

Cholastova, T. and D. Knotova. 2012. Using morphological and microsatellite (SSR) markers to assess the genetic diversity in Alfalfa (Medicago sativa L.). Int. J. Agric. Biosyst. Eng., 6: 781-787.

Das, A.B., I.C. Mohanty, D. Mahapatra, S. Mohanty and A. Ray. 2010. Genetic variation of Indian potato (Solanum tuberosum L.) genotypes using chromosomal and RAPD markers. Crop Breed. Appl. Biotechnol., 10: 238-246. https://doi.org/10.1590/S1984-70332010000300009

Dogra, K.S., S.K. Sood, P.K. Dobhal and S. Kumar. 2009. Comparison of understorey vegetation in exotic and indigenous tree plantations in Shivalik Hills of NW Indian Himalayas (Himachal Pradesh). J. Ecol. Natl. Environ., 1(5): 130-136.

Egbadzor, K.F., K. Ofori, M. Yeboah, L.M. Aboagye, M.O. Opoku-Agyeman, E.Y. Danquah and S.K. Offei. 2014. Diversity in 113 cowpea (Vigna unguiculata (L) Walp) accessions assessed with 458 SNP markers. Springer Plus, 3(1): 1-15. https://doi.org/10.1186/2193-1801-3-541

Ghafoor, A. and M. Arshad. 2008. Seed protein profiling of Pisum sativum L., germplasm using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) for investigation of biodiversity. Pak. J. Bot., 40(6): 2315-2321.

Ghafoor, A., F.N. Gulbaaz, M. Afzal, M. Ashraf and M. Arshad. 2003. Inter-relationship between SDS-PAGE markers and agronomic traits in chickpea (Cicer arietinum L.). Pak. J. Bot., 35(4): 613-624.

Hameed, A., T.M. Shah, B.M. Atta, N. Iqbal, M.A. Haq and H. Ali. 2009. Comparative seed storage protein profiling of Kabuli chickpea genotypes. Pak. J. Bot., 41(2): 703-710.

Hussein, H. and N.M. George. 2002. Taxonomic importance of floral morphology, chromosome number and seed protein electrophoretic patterns in some species of tribe Vicieae (subfamily: Papilionoideae-Leguminosae). Egypt. J. Biotechnol., 11: 106-123.

Hussein, H., N.M. George and M.M. El-Dimerdash. 2005. Taxonomic importance of seed protein electrophoretic patterns in some taxa of the subfamily Mimosoideae-Leguminosae. Assiut. Univ. J. Bot., 34(2): 101-130.

ILDIS, 2010. online. International Legume Database and Information Service. 2010. (Available from: http://www.ildis.org/LegumeWeb, 2010).

Iqbal, S.H., A. Ghafoor and N. Ayub. 2005. Relationship between SDS-PAGE markers and Ascochyta blight in chickpea. Pak. J. Bot., 37(1): 87-96.

Javid, A., A. Ghafoor and R. Anwar. 2004. Seed storage protein electrophoresis in groundnut for evaluating genetic diversity. Pak. J. Bot., 36(1): 25-30.

Kaga, A., N. Tomooka, Y. Egawa, K. Hosaka and O. Kamijima. 1996. Species relationships in the subgenus Ceratotropis (genus Vigna) as revealed by RAPD analysis. Euphytica, 88: 17-24. https://doi.org/10.1007/BF00029261

Khan, M.K.U., N. Muhammad, N.A. Nisar-Uddin, I. Khan and R. Ullah. 2019. Genetic polymorphism within the wild population of Rhynchosia himalensis Benth. ex Baker. Int. J. Biosci., 14(3): 54-64. https://doi.org/10.12692/ijb/14.3.54-64

Ladizinsky, G. and T. Hymowitz. 1979. Seed protein electrophoresis in taxonomic and evolutionary studies. Theor. Appl. Genet., 54: 145-151. https://doi.org/10.1007/BF00263044

Lioi, L., F. Sparvoli and R. Bollini. 1999. Variation and genomic polymorphism of lectin-related proteins in Lima bean (Phaseolus lunatus L.) seeds. Genet. Resour. Crop Evol., 46: 175-182. https://doi.org/10.1023/A:1008630330008

Manifesto, M.M., A.R. Schlatter, H.E. Hopp, E.Y. Suárez and J. Dubcovsky. 2001. Quantitative evaluation of genetic diversity in wheat germplasm using molecular markers. Crop Sci., 41(3): 682-690. https://doi.org/10.2135/cropsci2001.413682x

Ndiaye, M., B. Faye, R. Tine, J.L. Ndiaye, A. Lo, A. Abiola and O. Gaye. 2012. Assessment of the molecular marker of Plasmodium falciparum chloroquine resistance (Pfcrt) in Senegal after several years of chloroquine withdrawal. Am. J. Trop. Med. Hyg., 87(4): 640. https://doi.org/10.4269/ajtmh.2012.11-0709

Ndoutoume-Ndong, A. and D. Rojas-Rousse. 2007. Y at-il\’elimination d’Eupelmus orientalis Crawford par Eupelmus vuilleti Crawford (Hymenoptera: Eupelmidae) des syst\emes de stockage du ni\’eb\’e (Vigna unguiculata Walp)? arXiv preprint arXiv:0705.4630. https://doi.org/10.1080/00379271.2007.10697503

Ng, N.Q. and L.M. Monti. 1990. Cowpea genetic resources.

Nisar, M., A. Ghafoor, M.R. Khan and Asmatullah. 2009. First proteomic assay of Pakistani Pisum sativum L. germplasm relation to geographic pattern. Russ. J. Genet., 45: 805-810. https://doi.org/10.1134/S1022795409070072

Nisar, M., A. Ghafoor, M.R. Khan, H. Ahmad, A.S. Qureshi and H. Ali. 2007. Genetic diversity and geographic relationship among local and exotic chickpea germplasm. Pak. J. Bot., 39(5): 1575-1581.

Nisar, M., S.F. Wadood, A. Iqbal, A. Nausheen and A. Ghafoor. 2016. Intra and inter specific profiling of Pakistani Quercus species growing in the hilly areas of District Dir Khyber Pakhtunkhwa. Pak. J. Bot., 48(1): 263-270.

Noor, M., N. Ali, M. Nisar, E.F. Abd-Allah and A. Hashem. 2018. Genetic diversity within natural populations of the medicinal plant Rhynchosia minima (L.) Dc. Appl. Ecol. Environ. Res., 16(5): 5633-5651. https://doi.org/10.15666/aeer/1605_56335651

Oppong-Konadu, E.Y.R., H.K. Akromah, A. Dapaah and E. Okai. 2005. Genetic diversity within Ghanaian cowpea germplasm based on SDS-PAGE of seed proteins. Afr. Crop Sci. J., 13(2): 117-123. https://doi.org/10.4314/acsj.v13i2.27852

Pal, T., S. Ghosh, A. Mondal and K.K. De. 2016. Evaluation of genetic diversity in some promising varieties of lentil using karyological characters and protein profiling. J. Genet. Eng. Biotechnol., 14(1): 39-48. https://doi.org/10.1016/j.jgeb.2016.03.003

Pasquet, R.S. and J.P. Baudoin. 2001. Cowpea. In: Charrier A. Jacquot M., Hamon S. andNicolas D. (eds), Tropical plant, breeding. Science publishers, Enfield, pp. 177–198.

Potokina, E., D.A. Vaughan, E.E. Eggi and N. Tomooka. 2000. Population diversity of the Vicia sativa agg.(Fabaceae) in the flora of the former USSR deduced from RAPD and seed protein analyses. Genet. Resour. Crop Evol., 47: 171-183. https://doi.org/10.1023/A:1008756420011

Simon, L., S. Shyamalamma and P. Narayanaswamy. 2007. Morphological and molecular analysis of genetic diversity in jackfruit. J. Hortic. Sci. Biotechnol., 82(5): 764-768. https://doi.org/10.1080/14620316.2007.11512302

Singh, B.B., H.A. Ajeigbe, S.A. Tarawali, S. Fernandez-Rivera and M. Abubakar. 2003. Improving the production and utilization of cowpea as food and fodder. Field Crops Res., 84(1-2): 169-177. https://doi.org/10.1016/S0378-4290(03)00148-5

Sundin, B.A., C.H. Chiu, M. Riffle, T.N. Davis and E.G. Muller. 2004. Localization of proteins that are coordinately expressed with Cln2 during the cell cycle. Yeast, 21(9): 793-800. https://doi.org/10.1002/yea.1133

Varshney, R.K., U. Beier, E.K. Khlestkina, R. Kota, V. Korzun, A. Graner and A. Börner. 2007. Single nucleotide polymorphisms in rye (Secale cereale L.): discovery, frequency, and applications for genome mapping and diversity studies. Theor. Appl. Genet., 114: 1105-1116. https://doi.org/10.1007/s00122-007-0504-6

Wadood, S.F., N. Hassan, A. Khaliq, T. Nausheen, T. Jan, A. Ghafoor and M. Nisar. 2016. Genetic polymorphism in Lens culinaris collected from Malakand division Khayber Pakhtunkhwa, Pakistan. J. Biodiv. Environ. Sci., 8(2): 53-60.

Win, K.T., A. Zaw, K.L. New, M.S. Thein and H. Yutaka. 2011. Diversity of Myanmar cowpea accessions through seed storage polypeptides and its cross compatibility with the subgenus Ceratotropis. J. Plant Breed. Crop Sci., 3(5): 87-95.

Xu, Y., 2009. Molecular plant breeding. Cabi. https://doi.org/10.1079/9781845933920.0000