Phylogenetic study and evaluation of genetically diverse

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Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973

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RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2021 18(1): 62-76. OPEN ACCESS

Phylogenetic study and evaluation of genetically diverse germplasms in selected Cucumis L. Species provide a base for future breeding programs

Noor Muhammad*1&2, Muhammad Khalil Ullah Khan1, Nisar Uddin1, Syed Fazal Wadood2 and Niaz Ali*2 1Department of Botany Hazara University, Mansehra 21300, PK, Pakistan 2Department of Pomology, College of Horticulture, Agricultural University of Hebei, Baoding 071001, Hebei, China 3College of Life Sciences, Sun Yat˗ Sen University, Guangzhou, Guangdong, 510275, China *Correspondence: [email protected], [email protected] Received 31-12-2020, Revised: 20-01-2021, Accepted: 22-01-2021 e-Published: 024-01-2021

The genetic diversity of plants has been reduced by breeding and domestication, this reduction in genetic diversity constrains our need to expand a crop's range. This study aimed to study the phylogenetic relationship and to evaluate genetically diverse germplasms in selected Cucumis L. species In this study, genetic variation and Phylogenetic relationship in 90 genotypes of Cucumis (C. sativus, C. melo, and C. melo var agrestis. (30 genotypes each) were assessed, collected from various localities in swat Khyber Pakhtunkhwa Pakistan using morphometric and seed storage protein profiling. A total of 23 morphometric characters were noted for evaluation of the phylogenetic relationship and genetic polymorphism through traits similarity index and cluster analysis. Protein profiling was accessed on 12% slab gel electrophoresis; nine reproducible bands were detected in C. melo eight bands in C. melo var. agrestis and seven bands/loci in C. sativus. With a molecular weight ranging from 10KDa to 180KDa. Intra and inter genetic disagreement was 22.222% in C. melo and 33.333% in C. melo var. agrestis and C. sativus. Interspecific locus contribution toward genetic disagreement was found 55.55%. Among the nine loci, L-4 to L-9 were monomorphic in C. melo while L-4 to L-8 were monomorphic in C. melo var. agrestis. L-4 to L-7 was monomorphic in C.sativus. Interestingly, Locus 4(L-4), 5 (L-5), 6(L-6), and locus 7 (L-7) were monomorphic in all genotypes of the three taxa and marked as generic specific loci for Cucumis species. The present data demonstrated that Cucumis species show intra and interspecific genetic polymorphism, but upheld species-specific distinctiveness in the area regardless of ecological changes. Genetic differentiation through SDS-PAGE characterization indicated the important and diverse genetic makeup of genotypes for breeding programs.

Keywords: Cucumis; Genetic Association; Genetic variability Morphological and biochemical Characterization

INTRODUCTION

It is important to know the natural composition and closest relatives of the genus Cucumis L., as plant breeders worldwide struggle to improve melon and cucumber with traits through wild relatives (Chung et al. 2006). After tomatoes and onion, melon and Cucumber are considered as some of the most frequently growing crops in the

world (Pitrat et al. 1997). From an evolutionary point of view, Cucumis organellar genomes are unusually labile and main chromosome rearrangements are thought to have taken place, at the time of the evolution of Cucumis (Cummings et al. 2003; Lilly, et al. 2001). In the genus Cucumis, C. sativus is one of the species with a chromosome number of n = 7, which shows

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Muhammad et al. Study of genetically diverse germplasms in selected Cucumis L. species

Bioscience Research, 2020 volume 18(1): 62-76 63

that it has been evolved from a supposed familial karyotype (n = 12), but the main information regarding reduction of chromosome number is unclear (Pitrat et al. 1997; Yang, 2014). Therefore the genus Cucumis holds striking interest to study the Phylogenetic relationship among Cucumis species (Ritschel et al. 2004). Cucumis is a genus of the family Cucurbitaceae; order Cucurbitales (Whitaker & Davis, 1962). Cucurbitaceae family comprising of about 825 species and 119 genera, including the genus Cucumis (Szabo et al. 2008). The genus Cucumis consisting of increasing numbers of species i.e. nine species were identified by Kirkbride, (1993), thirteen by Naudin, (1859), thirty-two by Kirkbride, (1993), and (thirty-four) were identified by Chen et al. (1997). Out of them melon and cucumber are the two economically important commercial vegetable crops (Whitaker & Davis, 1962; Dane & Tsuchiya., 1976). Understanding the nearest relatives and natural arrangement of Cucumis is very important because of ongoing struggles by breeders all over the world to enhance melon and Cucumber with characters from wild relatives (Kocyan et al. 2007; Ghebretinsae et al. 2007; Renner et al. 2007).

The genetic diversity and Phylogenetic relationship are significantly important for conservation, maintenance, and improvement in crop productivity (Muhammad et al. 2018). Plant genetic diversity can be predicted and kept in the form of plant genetic resources, like gene banks which may be in turn be exploited for crop improvement to offer security against abiotic and biotic stresses (Muhammad et al. 2018; Garzón-Martínez et al.2015; Govindaraj et al.2015). Reduction in genetic diversity and emerging pests are the main causes of reduced yields for Cucumis species. Wild relatives of crops frequently have valuable characters, such as disease resistance, for crop and yield enhancement. Such characters can possibly be introgressed into crops by crossing with the wild species for the development of introgression lines (Muhammad et al. 2018; Zamir, 2001).

Nowadays, many tools are available to examine the genetic diversity and phylogenetic relationship among the germplasms of plant species. At the start and beginning of agricultural improvement programs, morphological characterization was used but in the majority of cases, morphological characterization is unstable due to the environmental fluctuations (Muhammad et al. 2018; Muhammad et al.2019). Germplasm evaluation and genetic variation through DNA

based molecular marker is a very prominent approach but is more expensive. Compared to DNA marker characterization, seed storage protein characterization is free of environmental fluctuations and is easy to carry out in developing countries like Pakistan (Muhammad et al. 2018; Khan et al. 2020).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been successfully used to evaluate taxonomic relationships in many plant species and to identify cultivated varieties like Cleome viscosa L. (Muhammad et al. 2018a), Rhynchosia minima (L.) DC. (Muhammad N et al.2018), Punica L (Muhammad et al.2019), Rhynchosia L. species (Muhammad N et al. 2019a)

There is a scarcity of knowledge of the phylogenetic relationship and genetic variation among Cucumis species growing in the hilly areas of Swat. A broad genetic base is key to adaptation and will assess the potential extent of the impacts of climate change (Muhammad N et al. 2018). Some 10,000 years ago, after domestication, the genetic diversity of the crops has been narrowed due to intense plant breeding and selection (Ali, 2012). Nowadays, mankind relies on less than a dozen plant species for 80% of all caloric intake (McCouch et al. 2013). Unless effective contingency measures, this will not be sufficient to meet the 2050 feeding requirements in the face of climate change, habitat degradation, and dwindling water and land resources (Muhammad N et al. 2018; Garzón-Martínez et al., 2015; Kazi et al.2017). Therefore, there is an intense need to conduct research work that identifies the genetically diverse genotypes for breeding programs and it is necessary to understand the degree of genetic variation and phylogenetic relationship in Cucumis genotypes. The present study was aimed to examine genetically diverse germplasms and phylogenetic relationship among three species of Cucumis (C. melo L., C. melo var. agrestis Naudin, C. sativus L.) growing in hilly areas of District Swat and adjoining areas of Swat, Khyber Pakhtunkhwa Pakistan. MATERIALS AND METHODS

Plant materials Various study tours were organized to

different areas of Swat in 2017–2018 for seed collection and morphological characterization to study genetic variation and phylogenetic



Table 1:Samples collection Sites for the current study

Note: Cm= C. melo, Cma= C. melo var. agestris, Cs= C. sativus

Sites Genotypes Sites Genotypes Sites Genotypes Sites Genotypes

Kabal Cm1 Jabagi Cm16 Soray Cma 31 Ziarat Cma46

Kotlai Cm2 Swegalai Cm17 Tangai Chena Cma32 Rangila Cma47

Melaga Cm3 Jawand Cm18 Qalagy Cma33 Garai Cma48

Akhunkalay Cm4 Kohay Cm19 Sarkhanai Cma34 Dadahara Cma49

Shaoor Cm5 Kodbaoro Cm20 Yakhtangay Cma35 Sara Shah Cma50

Barikot Cm6 Kan Sar Cm21 Dokat Cma36 Daam Cma51

Jandrai Cm7 Banda Cm22 Sarkhazano Cma37 Gumbatay Cma52

Islam-Gat Cm8 Garam Chashma

Cm23 Sarbala Cma38 Serai Cma53

Haji Shai Cm9 Ziarat Cm24 Qambo Cma39 Jawand Cma54

Kharkani Cm10 Boni Cm25 Landai Shah Cma40 Chinar Dara Cma55

Thal Cm11 Daam Cm26 Kasai Cma41 Zawra Cma56

Kalkot Cm12 Bar Daam Cm27 Banjo Banda Cma42 Sharif Abad Cma57

Lamotai Cm13 Parai Dara Cm28 Gadi Cma43 Nasafai Cma58

Jagram Cm14 Nagoha Cm29 Mula Hassan Baba Cma44 Gwar Dand Cma59

Bandagai Cm15 Gumbatona Cm30 Malak Abad Cma45 Qudraman Cma60

Cucumis sativus

Sites Genotypes Sites Genotypes

Ziarat Cs61 Qabar shah Cs81

Swegalai Cs62 Landakay Cs82

Dadahara Cs63 Kota Cs83

Kohay Cs64 Aboha Cs84

Gadi Cs65 Manyar Cs85

Sharif Abad Cs66 Terang Cs86

Zarkhela Cs67 Dool Cs87

Jalala Cs68 Chargo Tangay Cs88

Gat Koto Cs69 Alook Garai Cs89

Gora gat Cs70 Shamra Cs90

Chongai Cs71 Aboha Cs75

Qabar Shah Cs72 Ziarat Cs76

Ziarat Cs73 Shamra Cs77

Swegalai Cs74 Kota Cs78

Chongai Cs80 Gora gat Cs79



Figure 1: Representation of a phylogenetic relationship through morphometric traits in 90 various samples of Cucumis L. species collected from Swat. Cs indicates genotypes of C. sativus, Cma indicate genotypes of C. melo var agrestis, and Cm represents C. melo



The seeds of each genotype were deposited in the seed repository at the molecular laboratory, Department of Botany Hazara University, Mansehra, KP, Pakistan. These sites are shown below in table 1.

Morphological characterization Morphological characterization of 90

genotypes (total 23 characters i.e. 14 quantitative and 9 qualitative) both qualitative and quantitative characters were studied following descriptor for melon IPGRI, (Kerje, 2003). Quantitative characters include petiole length (PL), leaf length (LL), leaf width (LW), flower length (FL), fruit length (FtL), No. of seed per fruit (S/Ft), 100 seed weight, No. of branches/ plant (B/P), plant height (PH), Biomass, Yield/plant (Y/P) and qualitative characters include leaf shape (LS), leaf color (LC), Leaf pubescent (LP), fruit color (Ft), seed texture (St), seed coat color (SCc), seed shape (SS), flower color (FC), presence of spots on fruit (SpFt). These morphological characters of 90 genotypes (total 23 characters) were recorded and the morphological data were subjected for the analysis, to computer software’s PC-ord. v.5 McCune (McCune B & Mefford M J, 1997) and SPSS (Renner et al 2007; IBM Corp, 2017) (figure 1).

Protein Profiling For seed storage protein characterization, two

to three seeds of each genotype were ground into powder. To explore the level of phylogenetic relationship SDS-PAGE was performed using the method of Laemmli, 1970 modified by Muhammad et al. (2018). The 0, 1 data was noted from the gel on the excel sheet and, cluster analysis was performed using software PC-ord. v 5 McCune & Mefford, (1997).

RESULTS AND DISCUSSION

A total of 90 genotypes of genus Cucumis; Cucumis sativus (30 genotypes), Cucumis melo (30 genotypes), and Cucumis melo var. agrestis (30 genotypes) were assessed, collected from various localities of swat Khyber Pakhtunkhwa Pakistan. However, up to date, the present investigations for the assessment of inter-species phylogeny among the selected species through agro morphological and seed storage protein profiling is one of the huge and updated reports from Swat Khyber Pakhtunkhwa, Pakistan. The present data demonstrated that Cucumis L. species shows intra and interspecific genetic

polymorphism, but upheld species-specific distinctiveness in the area regardless of ecological changes.

Morphological Characterization Pearson correlation coefficient was performed

to study the correlation amongst the 23 characters for the three species of Cucumis (Table 2, 3 & 4). In the correlation study, the petiole length (PL) in the genotypes of C. melo, C. melo var agrestis, and C. sativus leaf length was positively correlated with the leaf length of C. melo, C. sativus, and C.melo var. agrestis. The fruit length was positively significant with the number of the seeds per fruit at (0.05) in the genotypes of C. melo var. agrestis and C. sativus whereas negatively correlated in the genotypes of C. melo with fruit width. The number of seeds/ fruit was positively correlated with the number of fruit in the genotypes of C. melo var agrestis while negatively correlated with the genotypes of C. sativus and so on as shown in detail in tables 2, 3, 4. One of the studies carried out in watermelon genotypes, by Guner and Wehner, (2004), represented that a long and highly branching system in cultivars will lead to high yields. The weight of 100 seeds was negatively correlated with the number of branches in genotypes of C. melo while significantly positively correlated with the genotypes of C. melo var. agrestis and C. sativus Number of the branches were negatively correlated with Biomass in the genotypes of C. melo whereas significantly positively correlated with genotypes of C. melo var agrestis and C. sativus. Biomass (BM) was negatively correlated with yield per plant in the genotypes of C. sativus and C. melo; also another study in the Cucurbitaceae species was carried out by Gichimu et al. (2009) they studied morphological characterization among three watermelon cultivars in Kenya and found significantly high fruit number, branch number, high fruit weight and lower seed number in watermelon cultivars and found high morphological diversity (53-54% diversity). The morphological data were examined for the making phylogenetic tree to denote the similarity of the three taxa of Cucumis (Fig 1). The dendrogram divided all three species into three groups (Fig: 1). Group I consisted of 30 genotypes of C. melo var agrestis. Group II comprised of all genotypes of C. sativus. Group I and Group II have 63% similarity whereas Group III was comprised of 30 genotypes C. melo. Group II and Group III had a 68.75% similarity.



Table 2: Identified Correlation among seventeen quantitative traits of C. melo

Table 3: Identified Correlation among seventeen quantitative traits of C. melo var agrest

Traits PL LL LW FL FW SL Ft L S/Ft Ft/P 100 SWT BP PH BM Y/P

PL 1.00

LL 0.11 1.00

LW 0.11 .749** 1.00

FL -0.24 0.03 -0.12 1.00

FW -0.29 0.04 -0.14 0.789** 1.00

SL 0.15 0.06 0.14 -0.22 -.362* 1.00

Ft L 0.35 0.06 0.13 -0.369* -.446* 0.14 1.00

S/Ft 0.29 0.01 -0.06 -0.08 -0.10 0.22 .384* 1.00

Ft/P .412* -0.07 -0.04 -0.04 -0.34 0.28 -0.07 0.23 1.00

100SWt 0.05 -0.15 -0.24 -0.01 -0.02 0.19 0.28 0.399* -0.12 1.00

BP 0.08 0.18 -0.02 0.17 0.25 -0.25 -0.06 0.00 -0.19 -0.13 1.00

PH 0.09 0.02 -0.02 0.06 0.18 -0.09 -0.08 0.13 -0.09 -0.02 -0.04 1.00

BM -0.22 -0.14 0.03 -0.01 0.05 -0.18 -0.05 -0.01 -0.26 0.366* 0.25 0.33 1.00

Y/P -0.23 -0.17 -0.14 -0.14 -0.02 -0.05 -0.19 0.03 0.04 0.29 0.10 0.04 .536** 1.00

*. Correlation is significant at the 0.05 level

**. Correlation is significant at the 0.01 level

Note: The abbreviations are listed in table 10

Traits PL LL LW FL FW SL Ft L S/Ft Ft/P 100SWT BP PH BM Y/P

PL 1.00

LL 0.15 1.00

LW 0.07 .858** 1.00

FL -0.17 -0.16 -0.07 1.00

FW -0.11 -.526** -0.32 0.23 1.00

SL -0.31 0.24 0.23 0.20 -0.09 1.00

Ft L 0.08 0.01 0.13 -0.16 -0.23 0.02 1.00

S/Ft -0.16 -0.13 0.03 .682** 0.35 0.13 -0.06 1.00

Ft/P 0.10 -0.30 -.511** 0.05 0.01 -0.21 -0.11 0.04 1.00

100SWt .639** 0.32 0.32 -0.14 -0.15 -0.06 -0.30 -0.20 -0.15 1.00

BP -.496** -0.36 -0.36 0.20 0.26 0.09 0.26 0.22 0.19 -.864** 1.00

PH .533** 0.32 0.28 -0.33 -0.31 -0.19 -0.17 -0.36 -0.08 .846** -.841** 1.00

BM 0.00 -0.28 -0.35 -0.30 0.13 -0.29 0.06 -.427* 0.03 -0.08 -0.04 0.12 1.00

Y/P -0.24 -.402* -0.31 0.16 0.23 0.02 0.11 -0.08 0.13 -0.571** 0.463** -.507** 0.20 1.00






Table 4. Identified correlation among seventeen quantitative traits of C. sativus

Traits PL LL LW FL FW SL Ft L S/FtL FtL/P 100 SWT

BP PH BM Y/P

PL 1.00

LL 0.790** 1.00

LW 0.793** .985** 1.00

FL 0.25 0.21 0.23 1.00

FW 0.416* 0.31 0.33 0.886** 1.00

SL 0.692** .834** .833** 0.20 0.33 1.00

Ft L 0.764** .786** .804** 0.30 0.35 .661** 1.00

S/FtL -0.01 0.04 0.08 .501** .469** 0.09 0.30 1.00

FtL/P 0.02 -0.11 -0.14 0.09 -0.21 -0.12 -0.06 0.15 1.00

100SWT -.504** -.736** -.735** -0.30 -.383* -.752** -.400* -0.20 0.15 1.00

BP -0.18 -0.22 -0.22 -0.20 -0.14 -0.21 -0.20 -0.04 -0.03 0.26 1.00

PH -.426* -.661** -.680** -0.17 -0.24 -.609** -.524** -0.17 0.16 .622** 0.05 1.00

BM -0.13 -0.04 -0.05 -.591** -.533** -0.08 0.09 0.05 -0.13 0.18 .382* -0.09

1.00

Y/P -0.09 -0.03 -0.02 -.452* -.381* -0.01 0.21 0.24 -0.07 0.20 0.28 -0.23

.839** 1.00




Table 5. Genetic diversity between and within spps. C. melo, C. melo var agrestis, C. sativus identified by 22 morphological characters

Trait C. melo C. melo var. Agestris C. sativus Trait similarity index

Cm & Cma Cs & Cma Cs & Cm

PL 6.59656 2.793111 4.5882 NA NA NA LL 13.8948 8.48 39.125 NA NA NA LW 12.4727 6.8 29.765 NA NA NA FL 7.12358 4.766667 3.1956 NA NA NA FW 6.19201 5.3 2.1237 NA NA NA SL 2.51683 2.633333 5.0893 2.5745* NA NA Ft L 45.3747 57.27111 60.902 NA NA NA

S/FtL 131.2 38.9 80.067 NA NA NA FtL/P 14.6667 7.2 26 NA NA NA

100SWT 6.2 14.858 27.62 NA NA NA BP 6 12.44444 12.967 NA 12.707* NA PH 258.767 295.0444 337.24 NA NA NA BM 285.39 109.3493 328.57 NA NA NA



Y/P 266.287 23.98667 315.45 NA NA NA

LS Cordate Ovate* Ovate* NA Ovate* Ovate* LC Green** Green** Green** Green** Green** Green** LP Present** Present** Present** Present** Present** Present** FtS Ellipsoid* Ellipsoid* Oblong NA Ellipsoid* Ellipsoid* FC Yellow** Yellow** Yellow** Yellow** Yellow** Yellow** St Smooth** Smooth** Smooth** Smooth** Smooth** Smooth**

SCc White yellow** White yellow** White yellow** White yellow** White yellow** White yellow** SS elliptic** elliptic** elliptic** elliptic** elliptic** elliptic**

SpFt Absent Present* Present* NA Present* Present*

Total TSI = (homologous trait/total traits)*100) 30.4348 43.47825 39.1304

*- Traits similarity within two species

**- Traits similarity within three species


Table 6: Inter-locus variations among C. melo L., C. melo var agrestis Naud. and C. sativus L.

Locus Present (%) Absent (%) Variation (%) status GD

L-1 19(21.11) 71(78.88) 78.88 polymorphic 0.2111

L-2 30(33.33) 60(66.666) 66.66 polymorphic 0.3333

L-3 76(84.44) 14(15.555) 15.555 polymorphic 0.8444

L-4*(generic specific locus) 90(100) 19(16.37) Nil monomorphic 1.0000

L-5 *(generic specific locus) 90.0(100) 0.000 Nil

monomorphic 1.0000

L-6*(generic specific locus) 90.0(100) 0.000 Nil monomorphic 1.0000

L-7 *(generic specific locus) 90.0(100) 0.000 Nil monomorphic 1.0000

L-8 57(63.33) 33(36.666) 36.666 polymorphic 0.6333

L-9 30(33.333) 60(66.66) 66.666 polymorphic 0.3333

GD =55.556% (Polymorphic loci/total loci*100)

. * represents generic specific loci.



Table 7: Genetic diversity at each locus within 30 genotypes of C. melo L

Table 8: Genetic diversity at each locus within 30 genotypes of C. melo var agrestis Naud.

Table 9: Genetic diversity at each locus within 30 genotypes of C. sativus L.

Locus Present (%) Absent (%) Variation

(%) status GD

L-1 13(43.333) 17(56.666) 56.666 polymorphic 0.43333

L-2 13(43.333) 17(56.666) 56.666 polymorphic 0.43333

L-3 25(83.333) 5(16.666) 16.666 polymorphic 0.8333


L-5* (generic specific locus) 30.0(100) 0.000 Nil monomorphic 1.0000


L-7* (generic specific locus) 30.0(100) 0.000 Nil monomorphic 1.0000

L-8 0.0000 30(100) Nil monomorphic 0.0000

L-9 0.0000 30(100) Nil monomorphic 0.0000


Note: * represents generic specific loci.

Locus Present (%) Absent (%) variation status GD

L-1 2.0(6.666) 28(93.333) 93.333 polymorphic 0.6666

L-2 18.0(60.0) 12(40) 40 monomorphic 0.600

L-3 30.0(100) 0.000 Nil monomorphic 1.000



L-6*generic specific 30.0(100) 0.000 Nil monomorphic 1.000




GD =22.222 %( Polymorphic loci/total loci*100).

* represents generic specific loci.

Locus Present (%) Absent (%) Variation (%) status GD

L-1 4.0(13.333) 26(86.666) 86.666 polymorphic 0.13333

L-2 2.0(6.666%) 28(93.333) 93.33% polymorphic 0.6666

L-3 21.0(70) 9(30) 30.00% polymorphic 0.7000

L-4*(generic specific locus) 30.0(100%) 0.000 Nil monomorphic 1.0000




L-8 30.0(100%) 0.000 Nil monomorphic 1.0000

L-9 0.0(0.0%) 30(100) Nil monomorphic 0.0000


Note: * represents generic specific loci.



Table 10: List of abbreviations used for descriptors in current study Traits Symbols Traits Symbols

Petiole length PL Fruit shape FtS

Leaf length LL Fruit Color FC

Leaf width LW Seed texture St

Flower length FL Seed coat color SCc

Flower width FW Seed Shape SS

Seed length SL Spots on fruit SpFt

Fruit length FtL trait similarity index TSI

No. of seeds per fruit S/ Ft C. melo var agrestis Cma

No. of fruit/plant Ft/P C. sativus Cs

Genetic Disagreement GD C. melo Cm

No. of fruit/plant Ft/P Biomass BM

Seed weight SWt Yield/ Plant Y/P

No. of branches/ Plant BP leaf Shape LS

Plant height PH leaf color LC

Locus LC leaf pubescent LP

Figure 2: Phylogenetic relationship identified through SDS-PAGE in 90 samples of Cucumis L. species

picked from Swat. Note: Cs= C. sativus L., Cma= C. melo var agrestis (Naudin), Cm= C. melo L. Tree A represents the relationship of the genotypes in groups (Group I, II, III) with one another, Tree B represents

Bands (B-1 to B-7) and C represents mark coding; presence or absence of the band



Similarity indexes were performed for all genotypes of three species that was 30.4348 for C. melo and C. melo var agrestis, 43.47825% for C. sativus, and C. melo var. agestris whereas C. sativus and C. melo were 39.1304% similar morphologically (Table 5). The study is in agreement with the findings of Szamosi et al. (2010) who studied the comparison in 58 Cucumis melo accessions of Hungarian and Turkish melon (Cucumis melo) germplasm using morphometric characterization and observed that both Turkish and Hungarian germplasm showed a wide range of diversity.

Genetic diversity in Cucumis Species through SDS- PAGE profiling

Seed storage protein profiling through SDS- PAGE has been considered as a valuable tool for the assessment of genetic diversity and phylogenetic relationships in angiosperm plant species (Renner et al. 2007; Gichimu et al. 2009; Szamosi et al. 2010; Crawford, 1983) and maintain species-specific identity (Zamir, 2001; Muhammad et al. 2019), because seed storage protein profiling shows genetic similarities within taxon and even between different biological taxa (Ladizinsky, 1979; Crawford, 1983; Muhammad et al. 2019c; Muhammad et al. 2019d). Nine 9 reproducible protein bands were detected in C. melo L., 8 in C. var agrestis, and 7 bands in C. sativus. The phylogenetic association among all the three species through dendrogram has been shown in (Fig 2). The tree divided all the genotypes of three species into three groups. Group I comprises 30 genotypes of C. sativus (Cs61 (Ziarat), Cs62(Swegalai), Cs63( Dadahara), Cs64(Kohay), Cs65 (Gadi), Cs66 (Sharif Abad), Cs67 (Zarkhela), Cs68 (Jalala), Cs69 (Gat koto), Cs70 (Gora gat), Cs71 (Chongai), Cs72 (Qabar Shah), Cs73 (Ziarat), Cs74 (Swegalai),Cs80 (Chongai), Cs81 (Qabar shah), Cs82 (Landakay), Cs83 (Kota), Cs84 (Aboha), Cs85 (Manyar), Cs86 (Terang), Cs87 (Dool), Cs88 (Chargo Tangay), Cs89 (Alook Garai) and Cs90 (Shamra), Cs75 (Aboha), Cs76 (Ziarat), Cs77 (Shamra), Cs78 (Kota), Cs79 (Gora gat) whereas the Group II comprised of C. melo var. agrestis (Naudin) (Cma 31 (Soray), Cma32 (Tangai Chena), Cma33 (Qalagy), Cma34 (Sarkhanai), Cma35 (Yakhtangay), Cma36 (Dokat), Cma37 (Sarkhazano), Cma38 (Sarbala), Cma39 (Qambo), Cma40 (Landai Shah),Cma41 (Kasai), Cma42 (Banjo Banda), Cma43 (Gadi), Cma44 (Mula Hassan Baba), Cma45 (Malak Abad), Cma46 (Ziarat), Cma47 (Rangila), Cma48 (Garai),

Cma49 (Dadahara), Cma50 (Sara Shah), Cma51 (Daam), Cma52 (Gumbatay), Cma53 (Serai), Cma54 (Jawand), Cma55 (Chinar Dara), Cma56 (Zawra), Cma57 (Sharif Abad), Cma58 (Nasafai), Cma59 (Gwar Dand), Cma60 Qudraman). Group I and Group II were 24% similar genetically. Group III; Cm1 (Kabal), Cm2 (Kotlai), Cm3 (Melaga), Cm4 (Akhunkalay), Cm5 (Shaoor), Cm6(Barikot), Cm7 (Jandrai), Cm8 (Islam-Gat), Cm9 (Haji Shai), Cm10 (Kharkani), Cm11 (Thal), Cm12 (Kalkot), Cm13 (Lamotai), Cm14 (Jagram), Cm15 (Bandagai), Cm16 (Jabagi), Cm17 (Swegalai), Cm18 (Jawand), Cm19 (Kohay), Cm20 (Kodbaoro), Cm21 (, Kan sar), Cm22 (Banda), Cm23 (Garam Chashma), Cm24 (Ziarat), Cm25 (Boni), Cm26 (Daam), Cm27 ( Bar Daam), Cm28 (Parai Dara), Cm29 (Nagoha), Cm30 (Gumbatona). The genotypes of Group II clustered adjacent to Group III was 62.5% similar genetically.

Locus variation Nine loci (L1-L9) were noted, out of which L-4

to L-7 were monomorphic and were marked as generic specific. These common bands propose the common ancestry of the studied taxa of genus Cucumis Similarly, the earlier studies (Muhammad et al. 2019; Muhammad et al. 2019d) reported that the existence of monomorphic loci in Rhynchosia species and Ipomoea species described their common evolutionary origin. Moreover, the loci L-1, L-2, L-3, L-8, and L-9 were marked as polymorphic with 78.88, 66.66, 15.555, 36.666, and 66.666 percent genetic diversity, respectively. The genetic disagreement among the species was 55.555% in the collected 90 genotypes of the selected three species of the genus Cucumis L. (Table 6).

Intraspecific diversity among 30 genotypes of C. melo was described in Table 7. Notably, L-3 to L-9 were monomorphic in C. melo L. L-1 and L-2 were polymorphic which showed 93.33% and 40% variation respectively and the total genetic diversity of C. melo L. was 22.222% (Table 7).

Intraspecific variation among 30 genotypes of C. melo var. agrestis, demonstrated high intra-specific diversity. Among nine loci L-4 to L-8, were monomorphic, while L-2 and L-3 were polymorphic. The L-9 was missing in 30 genotypes of C. melo var. agrestis. Hence this absent band can be helpful to categorize this species. L-1, L-2 and L-3 represent 86.666%, 93.333% and 30% diversity respectively. The total genetic disagreement within the C.melo var. agrestis (Naudin) genotypes was 33.333% (Table



8). Among 30 genotypes of C. sativus, nine loci

were detected, out of which L-1, L-2, and L-3 were polymorphic, while L-4 to L-7 were monomorphic. The L-8 and L-9 were missing in 30 genotypes of C. sativus L. These missing bands distinguished this species from the other. L-1, L-2 and L-3 represent 56.666%, 56.666 and 16.666% variation. The genetic disagreement within C. sativus genotypes was 33.333% (Table 9). In one of the study, carried out by Esmmailnia et al. (2015) who found the least distance among the species of Cucurbits with a genetic similarity value of 68.34%, while the highest genetic distance was found among species of Cucurbita and Cucumis whereas a high degree of polymorphism in Cucumis melo L. was reported by Levi et al. (2001; 2004); In different studies such as in Citrullus lanatus by Stepansky et al. (1999); Cucumis sativus by Djè et al. (2010); C. lanatus by Dey et al. (2006) and Cupresses sempervirens L. genotypes by Manohar and Murthy, (2012) and Hassan et al. (2016) five loci (bands) were detected, out of total only one locus (Locus-1) was monomorphic while the remaining were polymorphic. Out of polymorphic loci, locus-5 displayed a high degree of intra-specific locus variation (ISLV) of 54.54% followed by Locus-3 (36.36%) ISLV. Intra specie locus contribution was 80% and found that the genotypes of each zone occupied a separate cluster (Hassan et al. 2016).

Phylogenetic relationship based on phenotypic and SDS-PAGE characterizations

To determine the degree of genetic diversity and phylogenetic relationship among the Cucumis species, morphological and seed storage protein analysis (SDS-PAGE) was performed, as germplasms were collected from different zones. Seed storage protein profiling is one of the most widely used procedures to separate and characterize protein and to know the phylogenetic relationship of plant species (Crawford, 1983; Muhammad et al. 2019). The protein in SDS-PAGE is separated according to the change in their molecular weight (Muhammad et al. 2018). SDS-PAGE analysis of 90 genotypes of three species was carried out. In our present investigation, dendrogram based on seed storage protein analyses of selected species of Cucumis L. disclosed that the three species of Cucumis had a close resemblance to one another. The result showed that the C. sativus was clustered adjacent to C. melo var agestris showed 24%

genetic similarity, Whereas C. melo var. agestris variety had clustered near to C. melo has revealed genetic relatedness of 62.5% to each other.

CONCLUSION The results obtained after morphometric and

seed storage protein profiling disclosed that the techniques provided a powerful tool for consistent germplasm differentiation based on genetic dissimilarities in seed storage and the identified high genetically diverse germplasms like in C. sativus L. (Cs70 to Cs79), in C.melo var agestris Naudin. (Csa54, 55, 56 and 60) whereas in C. melo (Cm3, Cm5) of the genus Cucumis L. are suggested for future breeding programs. This examined diversity 55.556% and (diverse germplasms) should be kept in the form of plant genetic resources, which may, in turn, be exploited for Cucumis species improvement in order to provide security against abiotic and biotic stresses. The genetic diversity and phylogenetic relationship among the species of the genus Cucumis may demonstrate significance in improving the economically important Cucumis L. crops by manipulating their wild relatives. The Cucumis L. taxa designated for the analysis by SDS-PAGE exposed an extensive genetic disagreement in the studies of total genotypes and hence the results obtained by this study could be helpful in the identification and selection of desired genotypes of Cucumis for breeding programs in the future. Intra and inter genetic diversity was 22.222% in C. melo L., 33.333% in both C. melo var. agrestis and C. sativus. Interspecific locus contribution toward genetic disagreement was found to be 55.55%. Among the nine loci, L-4 to L-9 were monomorphic in C. melo L. while L-4 to L-8 were monomorphic in C. melo var. agestris (Naudin). L-4 to L-7 were monomorphic in C. melo L. Interestingly, locus 4, 5, 6, and 7 were monomorphic in all genotypes and marked as generic specific loci for Cucumis L. species. The present data demonstrated that Cucumis L. species shows intra and interspecific genetic polymorphism, but upheld species-specific distinctiveness in the area regardless of ecological changes. Genetic differentiation through molecular characterization indicated the importance of the study area regarding genetic estimation and species conservation CONFLICT OF INTEREST

The authors declared that the present study was performed in absence of any conflict of



interest. ACKNOWLEDGMENT

The authors are thankful to Department Hazara University and Hebei Agricultural University for the provision of the chemicals for experimental work. AUTHOR CONTRIBUTIONS

NM did the experimental work and wrote the Manuscript, MKUK help in analysis. NU, SFW, and NA helped in experimental work and reviewed the article.

Copyrights: © 2021@ author (s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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