# Evaluation of Morphological and Molecular Diversity among South Asian Germplasms ofCucumis sativus and Cucumis melo

Authors: Chi Zhang; Arun S. Pratap; S. Natarajan; L. Pugalendhi; Shinji Kikuchi; Hidenori Sassa; Natesan Senthil; Takato Koba
Journal: ISRN Agronomy
 (2012)

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## Abstract

Cucumber,Cucumis sativus (2n=2x=14), and melon, Cucumis melo (2n=2x=24), are two common vegetable crops worldwide. The present study evaluated eighteen Cucumis accessions (nine C. sativus and nine C. melo) that were collected from three South Asian countries that have the most diversity of Cucumis. Nine quantitative and twenty-three qualitative characteristics were measured. The values of fruit weight displayed the biggest divergence among the nine quantitative traits and much variation was displayed in twenty-three qualitative traits among eighteen accessions. For eight morphological quantitative traits other than fruit weight, eighteen accessions were divided into three groups by using Principle Component Analysis and K-means cluster analysis. Also, two chloroplast genes rbcL and matK of eighteen accessions were sequenced. Combined sequences were subjected to construct phylogenetic trees by Neighbor-Joining and Maximum Likelihood methods. Topologies of nine melon accessions were same in these two methods and nine cucumber accessions showed difference. The genetic distances among each of C. sativus and C. melo accessions were not high. We conclude that the genetic relationship among the eighteen accessions used in this study is not distant although they display significant morphological variations. The information on novel Cucumis germplasms provided here would contribute to breeding program as well as evolutional study in Cucumis.


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## Body

## 1. Introduction

Cucumber,Cucumis sativus (2n=2x=14), and melon, Cucumis melo(2n=2x=24), are two common vegetable crops worldwide, which are both within the 20 most important vegetables [1]. Within the genus Cucumis, two groups (African group and Asian Group) are accepted. They are defined by geographic origin and chromosome number. Geographically, chromosome number of the African group is 2n=2x=24. Africa, which contains the most numerous wild species, was assumed to be the place of origin of C. melo [2].C. sativusis indigenous to South Asia and estimated to be domesticated about 300 years ago and was soon cultivated in the south and east of the Himalayas. Diversity in C. sativus, the Asian group, is not abundant as that in C. melo, the African group. Cucumber, C. sativus, is thought to be the representative of Asian group with the chromosome number 2n=14 [3, 4]. On the other hand, a recent study proposed a different opinion about the origin of C. meloandC. sativus. Sebastian et al. proposed that melon and cucumber were both of Asian origin and had numerous relatives in Australia and around the Indian Ocean which were overlooked previously [5].It was known that there are thirty subspecies inC. melo, but only two varieties in C. sativus (C. sativus var. sativusand C. sativusvar.hardwickii). C. sativusvar. hardwickii (2n=14) is a wild, sympatric botanical variety of C. sativus var. sativus, which grows in the foothills of Himalayas mountains. This botanical variety has the multiple fruiting and branching habits which are not present in C. sativus var. sativus.C. sativusvar.hardwickii is cross-compatible withC. sativus var. sativus.Therefore,C. sativus var. hardwickiihas a potential for increasing the genetic diversity available for breeding commercial cucumber [6].C. melo is thought to contain the most diverse varieties in the genus Cucumis [7]. Significant morphological variations exist in fruit traits such as color, size, shape, texture, and taste. In 2005, 67 Japanese melon cultivars were evaluated by RAPD and SSR markers. These cultivars were clustered to three horticulture variety groups: Group Cantalupensis, Group Inodorus, and Group Conomon [8]. In China, the important secondary center of diversity, the genetic diversity of C. melowas firstly assessed by Luan et al. [9]. Sixty-eight ChineseC. melo cultivars were evaluated by RAPD markers, and the results indicated that the Chinese accessions were seem to be rich resources of genetic diversity for plant improvement. Furthermore, to show the genetic diversity of Chinese Hami melon, 120 melon accessions, including 24 accessions of Hami melon, were analyzed by RAPD markers and the accessions of Hami melon were classified into the large-seed type [10]. In C. sativus, 16 selected cucumber cultivars were divided into four groups by evaluating the traits related to fruit flavor such as taste, aroma, and dry matter, and the correlations among these traits were confirmed [11]. Recently, a high density (97.5% of chromosomes were covered) genetic map of cucumber was constructed, which was composed of 1,369 markers, including 1,152 SSR, 192 SRAP, 21 SCAR, and one STS locus [12]. Genetic diversity assessments and linkage map construction can increase the effectiveness of breeding programs [13].Elucidating the phylogenetic relationships in the genusCucumis is of great importance, because the closest relatives and natural composition can provide valuable information to improve melon and cucumber breeding [14]. By analyzing combined chloroplast sequences for 123 of the 130 genera of Cucurbitaceae, it was concluded that C. sativus evolved within an Australian/Asian clade and was phylogenetically far from C. melo than its relationship considered based on the current morphological classification [15]. Recently, by using DNA sequences from plastid and nuclear markers for 100 accessions from Africa, Australia, and Asia, it was shown that melon and cucumber are both of Asian origin and have some species-level relatives in Australia which were overlooked previously and further investigation of wild species should be concentrated in Asia and Australia [5].Here, we evaluate genetic diversity in eighteen accessions ofC. sativus and C. melo collected from South Asia. Also, the phylogenetic relationships are proposed based on the molecular data. Importance of genetic diversity for the purpose of crop improvement and evolutionary relationships are discussed.


## 2. Materials and Methods

### 2.1. Plant Materials

The materials used in this study comprised of eighteen genotypes of cucumber and melon collected from south Asian countries, most of which are from India (Table1). The field work was conducted at College Orchard, Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, India. All of the eighteen accessions were planted with two replications inside the shade net house and each replication had eight plants. All the recommended crop production and protection measures were adopted for raising a healthy crop.Table 1
Cucumber and melon accessions analyzed in this study.
Acc. No.
Germplasm
Spp.
Source
Country
1
AAUC 2
C. sativus
IIVR
India
2
Andaman
C. melo
TNAU
India
3
CM 1
C. melo
TNAU
India
4
Cus 125/71
C. sativus
CGN
China
5
IC 4079
C. sativus
CGN
India
6
JL 3 Dhillon
C. sativus
CGN
India
7
Long Green
C. sativus
CGN
India
8
Mayilam
C. melo
TNAU
India
9
Muskmelon Orange
C. melo
TNAU
India
10
Mysore
C. melo
TNAU
India
11
N 78
C. melo
NBPGR
India
12
Nanhai 2 Aouri
C. sativus
CGN
China
13
Nepal Local
C. sativus
CGN
Nepal
14
Olakkur
C. melo
TNAU
India
15
Perundurai
C. sativus
TNAU
India
16
Pudukkottai
C. melo
TNAU
India
17
Shantung Thorny
C. sativus
CGN
China
18
Theni
C. melo
TNAU
India
IIVR: India Institute of Vegetable Research
TNAU: Tamil Nadu Agriculture University
CGN: The Centre for Genetic Resources, the Netherlands
NBPGR: National Bureau of Plant Genetic Resources.


### 2.2. Evaluation of Quantitative and Qualitative Characters

Morphological characters were recorded using five plants in each replication according to combined standards as described by National Bureau of Plant Genetic Resources (NBPGR). In total, 32 traits including nine quantitative and 23 qualitative characters were recorded, which are shown in Table2 with their abbreviations.Table 2
Characters observed and their abbreviations and standards.
Character
Abb.
Standards
Fruit weight (g)
FW
The weight of fruits at fully ripe stage
Peduncle length (cm)
PL
The length from the base to fruit intersects peduncle
Fruit length (cm)
FL
The length from stalk end to the tip of the fruit
Fruit girth (cm)
FG
The girth at the middle portion
Leaf length (cm)
LL
The length from the tip to the leaf intersects petiole
No. of primary branches
NPB
The number of branches borne on the main stem
Leaf width
LW
The width at the broadest place of the leaf
Internodal length (cm)
IL
The length between two subsequent nodes
Leaf Petiole length (cm)
LPL
The length from the base of to the intersects petiole
Plant growth
PG
Determinate, semideterminate, and indeterminate
Stem shape
SS
Round, angular, and others
Stem color
SC
Yellow, light green, green, dark green, and others
Stem pubescence density
SPD
Sparse, medium, and dense
Stem pubescence type
SPT
Soft, intermediate, and hard
Leaf shape
LS
Orbicular, ovate, elliptical, reniform, cordate, triangular, entire, trilobite, pentalobate, and others
Leaf margin
LM
Crenate, denticulate, entire, lobate, serrate, serrulate, spiny, undulate, and others
Leaf color
LC
Light green, green, dark green, and others
Leaf glossiness
LG
Dull, intermediate, and glossy
Leaf pubescence density
LPD
Sparse, medium, and dense
Leaf pubescence type
LPT
Soft, intermediate, and hard
Flower color
FC
White, cream, yellow, orange, and others
Fruit shape
FS
Globular, flattened, oblate, elliptical, pyriform, ovate, acorn, elongated, and others
Stem end fruit shape
SFS
Depressed, flattened, rounded, and pointed
Blossom end fruit shape
BEFS
Depressed, flattened, rounded, and pointed
Fruit skin luster
FSL
Matt, intermediate, glossy, and others
Fruit skin texture
FST
Smooth, rough, spiny, and others
Fruit skin hardness
FSH
Soft, intermediate, hard, and others
Predominant fruit skin color
PFSC
White, yellow, cream, pale green, green, dark green, orange, brown, grey, and others
Secondary fruit skin color
SFSC
Absent, white, yellow, cream, pale green, green, dark green, orange, brown, grey, and others
Secondary skin color pattern
SSCP
Absent, spotted, striped, netted, short streaked, long Streaked, and others
Fruit mesocarp color
FMC
White, cream, green, orange, and others
Seed color
SC
White, cream white, yellow, light brown, and others


### 2.3. Extraction of DNA

Genomic DNA was extracted from fresh leaves. Seeds were sown on filter paper in petri dish and grown in room temperature. A week-old seedlings were ground by using mortar and pestle in liquid nitrogen to fine powder and genomic DNA was extracted by using the CTAB method [16]. DNA samples were diluted to 5 ng/μL for polymerase chain reaction (PCR) amplification.


### 2.4.rbcL and matK Gene Sequencing

PCR amplification was performed in a 10μL mixture containing 5 ng genomic DNA, 0.01 μmol primer, 0.4 mmol dNTP, 0.05 U Ex Taq (Takara, Japan), 1x PCR Buffer 2 mM MgCl2 (Takara, Japan). PCR reaction was performed in Takara PCR thermal cycler (Takara, Japan) and the cycle can be described as an initial denaturing step at 95°C for 5 min, then 30 cycles of at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 25 sec. The final extension step was at 72°C for 10 min. The PCR products were subjected to sequencing by using ABI 3130 Genetic Analyzer. Primers used in PCR and sequencing are shown in Table 3. Consensus sequences were assembled and edited by using the program GENETYX-MAC version 14 (Genetyx, Japan) followed by manual adjustments.Table 3
Primers used in PCR and sequencing.
Primer
Sequence
matK (Forward)
5′-CTATATCCACTTATCTTTCAGGAGT-3′
matK (Reverse)
5′-AAAGTTCTAGCACAAGAAAGTCGA-3′
rbcL (Forward)
5′-ATGTCACCACAAACAGAAACTAAAGC-3′
rbcL (Reverse)
5′-CTACGTCTGGAAGACCTGCGAATCC-3′


### 2.5. Data Analysis

Principal Component Analysis (PCA) and k-means cluster analysis was performed by using R version 2.15.0 with bpca package for statistical computing. Phylogenetic analysis was performed under neighbor-joining (NJ) and maximum likelihood (ML) methods. Sequences ofrbcL and matK were aligned with “clustalw2” on the website: http://www.ebi.ac.uk/Tools/msa/clustalw2/. The resulting aligned data matrix was prepared in MEGA 5.05. Bootstrap method is taken for the test of phylogeny. The number of bootstrap replications was 1000. The model of NJ tree was maximum composite likelihood, and the model of ML tree was Tamura-Nei model.


## 2.1. Plant Materials

The materials used in this study comprised of eighteen genotypes of cucumber and melon collected from south Asian countries, most of which are from India (Table1). The field work was conducted at College Orchard, Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, India. All of the eighteen accessions were planted with two replications inside the shade net house and each replication had eight plants. All the recommended crop production and protection measures were adopted for raising a healthy crop.Table 1
Cucumber and melon accessions analyzed in this study.
Acc. No.
Germplasm
Spp.
Source
Country
1
AAUC 2
C. sativus
IIVR
India
2
Andaman
C. melo
TNAU
India
3
CM 1
C. melo
TNAU
India
4
Cus 125/71
C. sativus
CGN
China
5
IC 4079
C. sativus
CGN
India
6
JL 3 Dhillon
C. sativus
CGN
India
7
Long Green
C. sativus
CGN
India
8
Mayilam
C. melo
TNAU
India
9
Muskmelon Orange
C. melo
TNAU
India
10
Mysore
C. melo
TNAU
India
11
N 78
C. melo
NBPGR
India
12
Nanhai 2 Aouri
C. sativus
CGN
China
13
Nepal Local
C. sativus
CGN
Nepal
14
Olakkur
C. melo
TNAU
India
15
Perundurai
C. sativus
TNAU
India
16
Pudukkottai
C. melo
TNAU
India
17
Shantung Thorny
C. sativus
CGN
China
18
Theni
C. melo
TNAU
India
IIVR: India Institute of Vegetable Research
TNAU: Tamil Nadu Agriculture University
CGN: The Centre for Genetic Resources, the Netherlands
NBPGR: National Bureau of Plant Genetic Resources.


## 2.2. Evaluation of Quantitative and Qualitative Characters

Morphological characters were recorded using five plants in each replication according to combined standards as described by National Bureau of Plant Genetic Resources (NBPGR). In total, 32 traits including nine quantitative and 23 qualitative characters were recorded, which are shown in Table2 with their abbreviations.Table 2
Characters observed and their abbreviations and standards.
Character
Abb.
Standards
Fruit weight (g)
FW
The weight of fruits at fully ripe stage
Peduncle length (cm)
PL
The length from the base to fruit intersects peduncle
Fruit length (cm)
FL
The length from stalk end to the tip of the fruit
Fruit girth (cm)
FG
The girth at the middle portion
Leaf length (cm)
LL
The length from the tip to the leaf intersects petiole
No. of primary branches
NPB
The number of branches borne on the main stem
Leaf width
LW
The width at the broadest place of the leaf
Internodal length (cm)
IL
The length between two subsequent nodes
Leaf Petiole length (cm)
LPL
The length from the base of to the intersects petiole
Plant growth
PG
Determinate, semideterminate, and indeterminate
Stem shape
SS
Round, angular, and others
Stem color
SC
Yellow, light green, green, dark green, and others
Stem pubescence density
SPD
Sparse, medium, and dense
Stem pubescence type
SPT
Soft, intermediate, and hard
Leaf shape
LS
Orbicular, ovate, elliptical, reniform, cordate, triangular, entire, trilobite, pentalobate, and others
Leaf margin
LM
Crenate, denticulate, entire, lobate, serrate, serrulate, spiny, undulate, and others
Leaf color
LC
Light green, green, dark green, and others
Leaf glossiness
LG
Dull, intermediate, and glossy
Leaf pubescence density
LPD
Sparse, medium, and dense
Leaf pubescence type
LPT
Soft, intermediate, and hard
Flower color
FC
White, cream, yellow, orange, and others
Fruit shape
FS
Globular, flattened, oblate, elliptical, pyriform, ovate, acorn, elongated, and others
Stem end fruit shape
SFS
Depressed, flattened, rounded, and pointed
Blossom end fruit shape
BEFS
Depressed, flattened, rounded, and pointed
Fruit skin luster
FSL
Matt, intermediate, glossy, and others
Fruit skin texture
FST
Smooth, rough, spiny, and others
Fruit skin hardness
FSH
Soft, intermediate, hard, and others
Predominant fruit skin color
PFSC
White, yellow, cream, pale green, green, dark green, orange, brown, grey, and others
Secondary fruit skin color
SFSC
Absent, white, yellow, cream, pale green, green, dark green, orange, brown, grey, and others
Secondary skin color pattern
SSCP
Absent, spotted, striped, netted, short streaked, long Streaked, and others
Fruit mesocarp color
FMC
White, cream, green, orange, and others
Seed color
SC
White, cream white, yellow, light brown, and others


## 2.3. Extraction of DNA

Genomic DNA was extracted from fresh leaves. Seeds were sown on filter paper in petri dish and grown in room temperature. A week-old seedlings were ground by using mortar and pestle in liquid nitrogen to fine powder and genomic DNA was extracted by using the CTAB method [16]. DNA samples were diluted to 5 ng/μL for polymerase chain reaction (PCR) amplification.


## 2.4.rbcL and matK Gene Sequencing

PCR amplification was performed in a 10μL mixture containing 5 ng genomic DNA, 0.01 μmol primer, 0.4 mmol dNTP, 0.05 U Ex Taq (Takara, Japan), 1x PCR Buffer 2 mM MgCl2 (Takara, Japan). PCR reaction was performed in Takara PCR thermal cycler (Takara, Japan) and the cycle can be described as an initial denaturing step at 95°C for 5 min, then 30 cycles of at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 25 sec. The final extension step was at 72°C for 10 min. The PCR products were subjected to sequencing by using ABI 3130 Genetic Analyzer. Primers used in PCR and sequencing are shown in Table 3. Consensus sequences were assembled and edited by using the program GENETYX-MAC version 14 (Genetyx, Japan) followed by manual adjustments.Table 3
Primers used in PCR and sequencing.
Primer
Sequence
matK (Forward)
5′-CTATATCCACTTATCTTTCAGGAGT-3′
matK (Reverse)
5′-AAAGTTCTAGCACAAGAAAGTCGA-3′
rbcL (Forward)
5′-ATGTCACCACAAACAGAAACTAAAGC-3′
rbcL (Reverse)
5′-CTACGTCTGGAAGACCTGCGAATCC-3′


## 2.5. Data Analysis

Principal Component Analysis (PCA) and k-means cluster analysis was performed by using R version 2.15.0 with bpca package for statistical computing. Phylogenetic analysis was performed under neighbor-joining (NJ) and maximum likelihood (ML) methods. Sequences ofrbcL and matK were aligned with “clustalw2” on the website: http://www.ebi.ac.uk/Tools/msa/clustalw2/. The resulting aligned data matrix was prepared in MEGA 5.05. Bootstrap method is taken for the test of phylogeny. The number of bootstrap replications was 1000. The model of NJ tree was maximum composite likelihood, and the model of ML tree was Tamura-Nei model.


## 3. Results and Discussion

### 3.1. Quantitative and Qualitative Characterization

Fruit appearances of eighteen accessions are shown in Figures1 and 2, and their morphological performances are shown in Tables 4 and 5. For the nine quantitative characters, there was a large divergence among the values of fruit weight.   C. melo “N78” and C. melo “Andaman” had the biggest fruit weights, 1,390 g and 1,350 g, respectively. These two values exceeded all the other accessions ranging from 210 g of C. sativus “Long Green” to 850 gC. melo “Theni.” C. melo cultivars had bigger fruit weights than those of C. sativus cultivars. C. meloaccessions were almost above 500 g and the mean was 825 g. On the other hand, C. sativusaccessions were 420 g on average and 400 g less than C. melo. The exceptions were C. melo “Mysore” and C. sativus “AAUC-2”, being the lightest C. melo and the heaviest C. sativus, with the weights of 290 g and 790 g, respectively (Table 4). López-Sesé et al. reported that the mean fruit weight for a collection of 125 Spanish accessions was 1.5 kg, while the mean fruit weight of Villaconejos landraces also from Spain was 2.08 kg [17]. It was also reported that the variability in fruit weight among Spanish varieties was 0.8 to 4.95 kg [18]. The range of fruit weight in the present study was 0.29 to 1.39 kg, not as heavy as the Spanish group. The difference might be due to the different collection region, as well as the different breeding process in the long history of cultivation between the Mediterranean region and south Asian region.Table 4
The values on 9 quantitative characters of 18 accessions.
Accession
Species
FW
PL
FL
FG
LL
LW
NPB
IL
LPL
(g)
(cm)
(cm)
(cm)
(cm)
(cm)
(cm)
(cm)
Cus 125/71
CS
330
2.9
21.5
14.9
12.7
16.9
10.5
7.5
11.9
Long Green
CS
210
6.1
17.3
16.9
12.5
17.1
6.5
4.9
17.3
JL-3 Dhillon
CS
500
1.6
36.5
15.5
14.8
16.1
8.5
8.7
10.4
Nanhai 2 Aouri
CS
230
3.6
16.9
15.3
13.9
15.9
6.5
9.3
9.7
Nepal Local
CS
590
2.9
22.5
20.4
15.5
16.1
9.5
8.2
9.9
Shantung Thorny
CS
360
1.3
35.9
14.8
15.5
18.3
9.5
12.1
10.3
AAUC-2
CS
790
3.3
24.3
18.7
15.7
20.1
8.5
14.5
15.3
Perundurai
CS
400
2.5
23.9
18.3
12.7
17.5
12.5
8.5
12.5
IC-4079
CS
390
2.5
26.9
21.1
14.3
16.5
5.5
9.9
9.9
M
422.22
2.96
25.07
17.32
14.17
17.16
8.61
9.28
11.91
SD
182.12
1.38
7.06
2.41
1.29
1.34
2.21
2.73
2.71
CV %
43%
47%
28%
14%
9%
8%
26%
29%
23%
N-78
CM
1390
2.9
32.9
21.3
14.9
13.3
10.5
9.3
12.7
Mysore
CM
290
3.3
21.3
24.2
8.2
12.7
5.5
5.6
7.5
CM-1
CM
820
7.1
33.7
39.3
14.3
16.3
8.5
12.2
8.6
Olakkur
CM
640
3.9
22.7
26.3
13.5
13.3
5.5
8.5
12.3
Mayilam
CM
770
5.6
30.3
27.9
13.7
15.5
6.5
7.7
11.9
Muskmelon Orange
CM
520
3.1
14
35.3
11.9
14.5
9.5
9.1
13.6
Pudukkottai
CM
795
5.3
46.3
18.9
12.1
14.4
6.5
7.9
11.7
Theni
CM
850
2.6
18.9
39.3
14.9
17.7
8.5
6.5
14.3
Andaman
CM
1350
3.2
40.7
29.9
14.6
17.9
12.5
10.6
14.7
M
825
4.11
28.97
29.15
13.12
15.06
8.16
8.6
825
SD
355.82
1.53
10.61
7.46
2.15
1.91
2.39
2.01
355.82
CV %
43%
37%
37%
26%
16%
13%
29%
23%
43%
(Note) M: Mean, SD: Standard Deviation, CV: Coefficient of Variance, CS:C. sativus, CM: C. melo.Table 5
Characteristics of the 18 accessions on 23 qualitative traits.
Accession
Species
PL
SS
SC
SPD
SPT
LS
LM
LC
Cus 125/71
CS
Indeterminate
Angular
Dark green
Medium
Hard
Entire
SPINY
Dark green
Long Green
CS
Indeterminate
Angular
Light green
Medium
Hard
Entire
SPINY
Green
JL-3 Dhillon
CS
Indeterminate
Angular
Dark green
Dense
Intermediate
Entire
SPINY
Dark green
Nanhai 2 Aouri
CS
Indeterminate
Angular
Dark green
Medium
Hard
Entire
SPINY
Dark green
Nepal Local
CS
Indeterminate
Angular
Dark green
Dense
Intermediate
Cordate
OTHERS
Dark green
Shantung Thorny
CS
Indeterminate
Angular
Dark green
Medium
Hard
Entire
SPINY
Dark green
AAUC-2
CS
Indeterminate
Angular
Green
Sparse
Intermediate
Entire
SPINY
Dark green
Perundurai
CS
Indeterminate
Angular
Dark green
Dense
Intermediate
Entire
SPINY
Green
IC-4079
CS
Indeterminate
Angular
Dark green
Dense
Intermediate
Entire
SPINY
Dark green
N-78
CM
Indeterminate
Angular
Green
Medium
Hard
Entire
SPINY
Light green
Mysore
CM
Indeterminate
Angular
Green
Medium
Hard
Triangular
SPINY
Light green
CM-1
CM
Indeterminate
Angular
Dark green
Medium
Hard
Entire
SPINY
Green
Olakkur
CM
Indeterminate
Angular
Green
Medium
Hard
Reniform
UNDULATE
Light green
Mayilam
CM
Indeterminate
Angular
Green
Medium
Hard
Pentalobate
LOBATE
Light green
Muskmelon Orange
CM
Indeterminate
Angular
Green
Medium
Hard
Pentalobate
LOBATE
Light green
Pudukkottai
CM
Indeterminate
Angular
Dark green
Medium
Hard
Reniform
ENTIRE
Light green
Theni
CM
Indeterminate
Angular
Green
Medium
Hard
Reniform
ENTIRE
Light green
Andaman
CM
Indeterminate
Angular
Green
Medium
Intermediate
Cordate
SPINY
Light green
Accession
Species
LG
LPD
LPT
FC
FS
SFS
BEFS
Cus 125/71
CS
Intermediate
Dense
Soft
Yellow
Elongated
Rounded
Pointed
Long Green
CS
Intermediate
Sparse
Soft
Yellow
Elongated
Rounded
Rounded
JL-3 Dhillon
CS
Intermediate
Dense
Soft
Yellow
Elongated
Pointed
Pointed
Nanhai 2 Aouri
CS
Glossy
Dense
Soft
Yellow
Elongated
Rounded
Rounded
Nepal Local
CS
Glossy
Medium
Hard
Yellow
Elongated
Rounded
Rounded
Shantung Thorny
CS
Glossy
Dense
Soft
Yellow
Elongated
Pointed
Pointed
AAUC-2
CS
Glossy
Dense
Soft
Yellow
Others
Flattened
Flattened
Perundurai
CS
Dull
Dense
Soft
Yellow
Elongated
Flattened
Rounded
IC-4079
CS
Intermediate
Dense
Soft
Yellow
Elongated
Rounded
Pointed
N-78
CM
Intermediate
Medium
Hard
Yellow
Elongated
Pointed
Rounded
Mysore
CM
Intermediate
Dense
Soft
Yellow
Elliptical
Pointed
Rounded
CM-1
CM
Intermediate
Dense
Soft
Yellow
Pyriform
Rounded
Rounded
Olakkur
CM
Intermediate
Medium
Intermediate
Yellow
Others
Flattened
Flattened
Mayilam
CM
Intermediate
Dense
Soft
Yellow
Pyriform
Pointed
Depressed
Muskmelon Orange
CM
Dull
Dense
Soft
Yellow
Globular
Flattened
Flattened
Pudukkottai
CM
Intermediate
Dense
Soft
Yellow
Elongated
Rounded
Rounded
Theni
CM
Intermediate
Sparse
Intermediate
Yellow
Globular
Flattened
Depressed
Andaman
CM
Intermediate
Medium
Hard
Yellow
Elongated
Pointed
Rounded
Accession
Species
FSL
FST
FSH
PFSC
SFSC
SSCP
FMC
SC
Cus 125/71
CS
Glossy
Spiny
Hard
Dark green
Pale green
Cream
Spotted
White
Long Green
CS
Intermediate
Rough
Hard
Brown
White
Green
Striped
Green
JL-3 Dhillon
CS
Intermediate
Spiny
Hard
Pale green
Yellow
Cream
Striped
White
Nanhai 2 Aouri
CS
Matt
Spiny
Hard
Pale green
Yellow
Cream
Striped
White
Nepal Local
CS
Glossy
Spiny
Intermediate
Brown
Absent
Absent
Striped
White
Shantung Thorny
CS
Intermediate
Spiny
Hard
Yellow
Absent
Absent
Spotted
White
AAUC-2
CS
Matt
Spiny
Hard
Brown
Orange
Cream
Netted
White
Perundurai
CS
Intermediate
Spiny
Hard
Pale green
Green
White
Netted
Cream
IC-4079
CS
Matt
Rough
Hard
Brown
Yellow
Others
Spotted
White
N-78
CM
Glossy
Smooth
Intermediate
Green
Yellow
Cream
Spotted
White
Mysore
CM
Matt
Smooth
Intermediate
Yellow
Absent
Absent
Spotted
White
CM-1
CM
Glossy
Smooth
Hard
Dark green
Pale green
Cream
Spotted
White
Olakkur
CM
Intermediate
Smooth
Soft
Pale green
Yellow
Cream
Spotted
Cream
Mayilam
CM
Intermediate
Smooth
Hard
Yellow
Dark green
White
Netted
Cream
Muskmelon Orange
CM
Glossy
Others
Hard
Yellow
Dark green
Cream
Short streaked
White
Pudukkottai
CM
Glossy
Smooth
Hard
Yellow
Pale green
Orange
Netted
White
Theni
CM
Intermediate
Smooth
Soft
Yellow
Dark green
Cream
Short streaked
White
Andaman
CM
Intermediate
Smooth
Soft
Yellow
Pale green
Orange
Spotted
White
Note. CS: C. sativus, CM: C. melo.Figure 1
Photographs of fruits ofCucumis sativus accessions used in the present study. Scale: 5 cm.Figure 2
Photographs of fruits ofCucumis melo accessions used in the present study. Scale: 5 cm.The other eight quantitative traits did not show significant difference as fruit weight. We divided these traits into two groups, namely, fruit-related traits and stem, and leaf-related traits. The first group included fruit length, fruit girth, and peduncle length. According to the results of fruit girth and length, it was shown that the fruit size ofC. melo was bigger than C. sativus except one cucumber accession “Shantung Thorny,” which was bigger than C. melo “Mysore”. “CM-1” and “Andaman” were the biggest ones in melon accessions. The second group were the traits including leaf width, leaf length, number of primary branches, intermodal length, and leaf petiole length, which showed smaller variance than fruit-related characters.By Principal Component Analysis (PCA) and k-means cluster analysis, eight quantitative values were covered by three principle components (PC1, PC2, and PC3). Contribution of variables in each principle components was calculated (Table6). Fruit length and fruit girth dominated in PC1 and PC2, respectively. Five characters, number of primary branches, internodal length, leaf petiole length, leaf length, and leaf width, had the similar contribution to PC3. Based on the result of principle component analysis, eighteen accessions were classified into three groups (Figure 3). Group 1 contained nine accessions (seven C. sativus accessions: AAUC-2, Perundurai, Nepal Local, IC-4079, Cus125/71, Long Green, and Nanhai2 Aouri and two C. meloaccessions: Olakkur and Mysore) and Group2 contained six accessions (twoC. sativus accessions: Shantung Thorny and  JL-3 Dhillon and four C. melo accessions: Andaman, N-78, Pudukkottai, and Mayilam). Group 3 contained three C. melo accessions (CM-1, Theni, and Muskmelon Orange). The relative dispersion among accessions within Group2 was greater than the other two. Group 3 contained the least number of accessions, that is, C. melo cultivars only. Group 1 was mainly constituted of C. sativusaccessions, while Group 2 and Group 3 contained most of C. melo accessions. The dispersion among C. melo accessions was greater than that among C. sativusaccessions.Table 6
Eight quantitative characters contribution to three principle components.
Quantitative character
PC1
PC2
PC3
No. of primary branches
0.041
0.027
0.457
Internodal length
0.086
0.046
0.422
Leaf petiole length
−0.047
−0.001
0.452
Leaf length
0.053
0.016
0.385
Leaf width
0.017
−0.029
0.487
Peduncle length
−0.008
0.068
−0.135
Fruit length
0.935
0.329
−0.065
Fruit girth
−0.332
0.939
0.007(a) Distribution of eighteen accessions on the first and second principle components; (b) distribution of eighteen accessions on the first and third principle components.
(a)
(b)Characteristics on twenty-three qualitative traits of eighteen accessions are shown in Table5. Traits observed were varied from stem characters, leaf characters, fruit characters and seed characters. Same growth patterns were shown in all the plants observed and all of them were indeterminate. For stem characters, all of the accessions had angular stems with different degrees of green color. C. sativus “Long Green” was the only one with light green stem, which was lighter than the others. Besides, C. sativus “AAUC-2” displayed sparse stem pubescence and C. sativus “JL-3 Dhillon”, C. sativus “Nepal Local”, “Perundurai,” and “IC-4079” showed dense stem pubescence. Other accessions were between them. For leaf characters, five types of leaf shape were recorded, namely, entire, cordate, triangular, reniform, and pentalobate. Among them, the entire shape was common, including ten accessions showing entire leaves with spiny margin. All accessions had green leaves with different density, and C. sativus “Perundurai” was the only one without leaf glossiness. Pubescence was also observed on leaves as on stems in all accessions, and the leaf pubescence was softer than those on stem. The pubescence trait is closely related to plant defense system. It is under investigation for map-based cloning by SSR markers. The biggest diversity appeared on the fruit-related characteristics, as shown with the results of quantitative characteristics. Except C. sativus “AAUC-2”, the fruit shapes of all the C. sativus accessions were elongated. For C. melo accessions, fruit shapes of C. melo “Mayilam” and “CM-1” were pyriform. C. melo “Mysore” was only elliptical and C. melo “Muskmelon Orange” and “Theni” were globular. Fruit characters were further recorded on fruit skin luster, texture, and hardness, which are related to the appearance and taste. C. sativus “Nanhai 2 Aouri”, “AAUC-2”, “IC-4079”, and C. melo “Mysore” did not have fruit skin luster. Fruit skin textures of all the C. melo accessions were smooth and those of C. sativus accessions were rough or spiny. Three C. melo accessions, “Olakkur”, “Theni”, and “Andaman,” had soft fruit skins. C. sativus “Nepal local”, C. melo “N-78”, and “Mysore” had harder fruit skin than C. melo “Olakkur”, “Theni”, and “Andaman”, but softer than the others. Besides, all of accessions had yellow flowers and white or cream seeds, expect that the seeds of “Long Green” were green.


### 3.2. Phylogenetic Analysis

The lengths ofmatK and rbcL genes ranged from 750 to 1067 bp, sequences were submitted to DNA Database of Japan (DDBJ). The aligned data matrix combined with matK and rbcL gene was 1,728 bp long. Based on the aligned matrix, phylogenetic trees were made by Neighbor-joining and maximum likelihood method (Figures 4 and 5).Figure 4
Neighbor-joining tree for 18 accessions in this study based on combined sequence ofrbcL and matK genes. The tree is rooted on Cucumis hirsutus. Bootstrap values based on 1000 replicates above branches.Figure 5
Maximum likelihood tree for 18 accessions in this study based on combined sequence ofrbcL and matK genes. The tree is rooted on Cucumis hirsutus. Bootstrap values based on 1000 replicates above branches.On the topology ofC. sativus, “JL-3 Dhillon” showed divergence from other accessions. Fruit morphologies of “JL-3 Dhillon”, the longest fruit length and yellow fruit skin, differed from other C. sativus (Tables 4 and 5). In a previous phylogenetic study of Cucumis, some special morphological traits were circumscribed within some certain clades [5]. Species of the Asian/Australian clade had more of less ellipsoid fruits that stay green or turn yellow-orange at maturity. C. ritchiei/C. maderaspatanusclade was characterized by smooth and round fruits that turn red at maturity [5]. Also here in the present study, this correlation between morphological traits and clade divergence at molecular level was shown in the case of “JL-3 Dhillon”.CGN23965 “I.C 4079” has the scientific nameC. sativusgroupGherkin, while CGN24494 “Nepal local” has the scientific name C. sativusvar.sikkimensis(TANU and CGN). In the phylogenetic tree, “I.C 4079” and “Nepal local” did not show big divergence with other accessions, both of them were nested in the same clade. Two Chinese C. sativus accessions “Cus 125/71” and “Nanhai 2 Aouri” were nested in the same clade as well as the accessions from different areas, India and Nepal. One of reasons for narrow molecular divergence could be due to the accessions derived from narrow areas of India and Himalaya.According to the previous works and present study, comparing withC. sativus, genetic diversity inC. meloseems to be much bigger. African melon group has 30 species that is divided into six subgroups [2], and ancient melon varieties were distributed throughout South Asia, which could be the first genetic diversity center [7]. C. meloaccessions studied in the present study were mainly introduced from three institutions, namely, India Institute of Vegetable Research (IIVR), Tamil Nadu Agricultural University (TNAU), and National Bureau of Plant Genetic Resources (NBPGR), USA. They distributed all around India, but in the phylogenetic tree, the accessions from different places nested in the same clade with each other. A big divergent node was found with C. melo “CM1”, branching with the other accessions. Special morphological traits were also found in accession “CM1”, such as the longest intermodal length and unique fruit color (Figure 2). “Muskmelon Orange” branch was close to “CM1”, and they were grouped into Cluster 2 in the cluster analysis (Figure 3).The fruit shapes ofC. melo “Muskmelon Orange” and “Theni” were globular, being distinguished from the otherC. melo accessions. This discrepancy was not supported by the phylogenetic results. Besides, C. melo “Mayilam” branch was the biggest divergent node. It shared the same group with “N-78” in cluster analysis in spite of that they were far from each other in phylogenetic trees. So in C. melo, correlations between morphological traits and phylogenetic tree were not as supportive as those in Cucumis sativus. It could be concluded that C. melo holds bigger diversity in morphology than C. sativus, which has caused more complex factors for analyzing phylogenetic relationships.


## 3.1. Quantitative and Qualitative Characterization

Fruit appearances of eighteen accessions are shown in Figures1 and 2, and their morphological performances are shown in Tables 4 and 5. For the nine quantitative characters, there was a large divergence among the values of fruit weight.   C. melo “N78” and C. melo “Andaman” had the biggest fruit weights, 1,390 g and 1,350 g, respectively. These two values exceeded all the other accessions ranging from 210 g of C. sativus “Long Green” to 850 gC. melo “Theni.” C. melo cultivars had bigger fruit weights than those of C. sativus cultivars. C. meloaccessions were almost above 500 g and the mean was 825 g. On the other hand, C. sativusaccessions were 420 g on average and 400 g less than C. melo. The exceptions were C. melo “Mysore” and C. sativus “AAUC-2”, being the lightest C. melo and the heaviest C. sativus, with the weights of 290 g and 790 g, respectively (Table 4). López-Sesé et al. reported that the mean fruit weight for a collection of 125 Spanish accessions was 1.5 kg, while the mean fruit weight of Villaconejos landraces also from Spain was 2.08 kg [17]. It was also reported that the variability in fruit weight among Spanish varieties was 0.8 to 4.95 kg [18]. The range of fruit weight in the present study was 0.29 to 1.39 kg, not as heavy as the Spanish group. The difference might be due to the different collection region, as well as the different breeding process in the long history of cultivation between the Mediterranean region and south Asian region.Table 4
The values on 9 quantitative characters of 18 accessions.
Accession
Species
FW
PL
FL
FG
LL
LW
NPB
IL
LPL
(g)
(cm)
(cm)
(cm)
(cm)
(cm)
(cm)
(cm)
Cus 125/71
CS
330
2.9
21.5
14.9
12.7
16.9
10.5
7.5
11.9
Long Green
CS
210
6.1
17.3
16.9
12.5
17.1
6.5
4.9
17.3
JL-3 Dhillon
CS
500
1.6
36.5
15.5
14.8
16.1
8.5
8.7
10.4
Nanhai 2 Aouri
CS
230
3.6
16.9
15.3
13.9
15.9
6.5
9.3
9.7
Nepal Local
CS
590
2.9
22.5
20.4
15.5
16.1
9.5
8.2
9.9
Shantung Thorny
CS
360
1.3
35.9
14.8
15.5
18.3
9.5
12.1
10.3
AAUC-2
CS
790
3.3
24.3
18.7
15.7
20.1
8.5
14.5
15.3
Perundurai
CS
400
2.5
23.9
18.3
12.7
17.5
12.5
8.5
12.5
IC-4079
CS
390
2.5
26.9
21.1
14.3
16.5
5.5
9.9
9.9
M
422.22
2.96
25.07
17.32
14.17
17.16
8.61
9.28
11.91
SD
182.12
1.38
7.06
2.41
1.29
1.34
2.21
2.73
2.71
CV %
43%
47%
28%
14%
9%
8%
26%
29%
23%
N-78
CM
1390
2.9
32.9
21.3
14.9
13.3
10.5
9.3
12.7
Mysore
CM
290
3.3
21.3
24.2
8.2
12.7
5.5
5.6
7.5
CM-1
CM
820
7.1
33.7
39.3
14.3
16.3
8.5
12.2
8.6
Olakkur
CM
640
3.9
22.7
26.3
13.5
13.3
5.5
8.5
12.3
Mayilam
CM
770
5.6
30.3
27.9
13.7
15.5
6.5
7.7
11.9
Muskmelon Orange
CM
520
3.1
14
35.3
11.9
14.5
9.5
9.1
13.6
Pudukkottai
CM
795
5.3
46.3
18.9
12.1
14.4
6.5
7.9
11.7
Theni
CM
850
2.6
18.9
39.3
14.9
17.7
8.5
6.5
14.3
Andaman
CM
1350
3.2
40.7
29.9
14.6
17.9
12.5
10.6
14.7
M
825
4.11
28.97
29.15
13.12
15.06
8.16
8.6
825
SD
355.82
1.53
10.61
7.46
2.15
1.91
2.39
2.01
355.82
CV %
43%
37%
37%
26%
16%
13%
29%
23%
43%
(Note) M: Mean, SD: Standard Deviation, CV: Coefficient of Variance, CS:C. sativus, CM: C. melo.Table 5
Characteristics of the 18 accessions on 23 qualitative traits.
Accession
Species
PL
SS
SC
SPD
SPT
LS
LM
LC
Cus 125/71
CS
Indeterminate
Angular
Dark green
Medium
Hard
Entire
SPINY
Dark green
Long Green
CS
Indeterminate
Angular
Light green
Medium
Hard
Entire
SPINY
Green
JL-3 Dhillon
CS
Indeterminate
Angular
Dark green
Dense
Intermediate
Entire
SPINY
Dark green
Nanhai 2 Aouri
CS
Indeterminate
Angular
Dark green
Medium
Hard
Entire
SPINY
Dark green
Nepal Local
CS
Indeterminate
Angular
Dark green
Dense
Intermediate
Cordate
OTHERS
Dark green
Shantung Thorny
CS
Indeterminate
Angular
Dark green
Medium
Hard
Entire
SPINY
Dark green
AAUC-2
CS
Indeterminate
Angular
Green
Sparse
Intermediate
Entire
SPINY
Dark green
Perundurai
CS
Indeterminate
Angular
Dark green
Dense
Intermediate
Entire
SPINY
Green
IC-4079
CS
Indeterminate
Angular
Dark green
Dense
Intermediate
Entire
SPINY
Dark green
N-78
CM
Indeterminate
Angular
Green
Medium
Hard
Entire
SPINY
Light green
Mysore
CM
Indeterminate
Angular
Green
Medium
Hard
Triangular
SPINY
Light green
CM-1
CM
Indeterminate
Angular
Dark green
Medium
Hard
Entire
SPINY
Green
Olakkur
CM
Indeterminate
Angular
Green
Medium
Hard
Reniform
UNDULATE
Light green
Mayilam
CM
Indeterminate
Angular
Green
Medium
Hard
Pentalobate
LOBATE
Light green
Muskmelon Orange
CM
Indeterminate
Angular
Green
Medium
Hard
Pentalobate
LOBATE
Light green
Pudukkottai
CM
Indeterminate
Angular
Dark green
Medium
Hard
Reniform
ENTIRE
Light green
Theni
CM
Indeterminate
Angular
Green
Medium
Hard
Reniform
ENTIRE
Light green
Andaman
CM
Indeterminate
Angular
Green
Medium
Intermediate
Cordate
SPINY
Light green
Accession
Species
LG
LPD
LPT
FC
FS
SFS
BEFS
Cus 125/71
CS
Intermediate
Dense
Soft
Yellow
Elongated
Rounded
Pointed
Long Green
CS
Intermediate
Sparse
Soft
Yellow
Elongated
Rounded
Rounded
JL-3 Dhillon
CS
Intermediate
Dense
Soft
Yellow
Elongated
Pointed
Pointed
Nanhai 2 Aouri
CS
Glossy
Dense
Soft
Yellow
Elongated
Rounded
Rounded
Nepal Local
CS
Glossy
Medium
Hard
Yellow
Elongated
Rounded
Rounded
Shantung Thorny
CS
Glossy
Dense
Soft
Yellow
Elongated
Pointed
Pointed
AAUC-2
CS
Glossy
Dense
Soft
Yellow
Others
Flattened
Flattened
Perundurai
CS
Dull
Dense
Soft
Yellow
Elongated
Flattened
Rounded
IC-4079
CS
Intermediate
Dense
Soft
Yellow
Elongated
Rounded
Pointed
N-78
CM
Intermediate
Medium
Hard
Yellow
Elongated
Pointed
Rounded
Mysore
CM
Intermediate
Dense
Soft
Yellow
Elliptical
Pointed
Rounded
CM-1
CM
Intermediate
Dense
Soft
Yellow
Pyriform
Rounded
Rounded
Olakkur
CM
Intermediate
Medium
Intermediate
Yellow
Others
Flattened
Flattened
Mayilam
CM
Intermediate
Dense
Soft
Yellow
Pyriform
Pointed
Depressed
Muskmelon Orange
CM
Dull
Dense
Soft
Yellow
Globular
Flattened
Flattened
Pudukkottai
CM
Intermediate
Dense
Soft
Yellow
Elongated
Rounded
Rounded
Theni
CM
Intermediate
Sparse
Intermediate
Yellow
Globular
Flattened
Depressed
Andaman
CM
Intermediate
Medium
Hard
Yellow
Elongated
Pointed
Rounded
Accession
Species
FSL
FST
FSH
PFSC
SFSC
SSCP
FMC
SC
Cus 125/71
CS
Glossy
Spiny
Hard
Dark green
Pale green
Cream
Spotted
White
Long Green
CS
Intermediate
Rough
Hard
Brown
White
Green
Striped
Green
JL-3 Dhillon
CS
Intermediate
Spiny
Hard
Pale green
Yellow
Cream
Striped
White
Nanhai 2 Aouri
CS
Matt
Spiny
Hard
Pale green
Yellow
Cream
Striped
White
Nepal Local
CS
Glossy
Spiny
Intermediate
Brown
Absent
Absent
Striped
White
Shantung Thorny
CS
Intermediate
Spiny
Hard
Yellow
Absent
Absent
Spotted
White
AAUC-2
CS
Matt
Spiny
Hard
Brown
Orange
Cream
Netted
White
Perundurai
CS
Intermediate
Spiny
Hard
Pale green
Green
White
Netted
Cream
IC-4079
CS
Matt
Rough
Hard
Brown
Yellow
Others
Spotted
White
N-78
CM
Glossy
Smooth
Intermediate
Green
Yellow
Cream
Spotted
White
Mysore
CM
Matt
Smooth
Intermediate
Yellow
Absent
Absent
Spotted
White
CM-1
CM
Glossy
Smooth
Hard
Dark green
Pale green
Cream
Spotted
White
Olakkur
CM
Intermediate
Smooth
Soft
Pale green
Yellow
Cream
Spotted
Cream
Mayilam
CM
Intermediate
Smooth
Hard
Yellow
Dark green
White
Netted
Cream
Muskmelon Orange
CM
Glossy
Others
Hard
Yellow
Dark green
Cream
Short streaked
White
Pudukkottai
CM
Glossy
Smooth
Hard
Yellow
Pale green
Orange
Netted
White
Theni
CM
Intermediate
Smooth
Soft
Yellow
Dark green
Cream
Short streaked
White
Andaman
CM
Intermediate
Smooth
Soft
Yellow
Pale green
Orange
Spotted
White
Note. CS: C. sativus, CM: C. melo.Figure 1
Photographs of fruits ofCucumis sativus accessions used in the present study. Scale: 5 cm.Figure 2
Photographs of fruits ofCucumis melo accessions used in the present study. Scale: 5 cm.The other eight quantitative traits did not show significant difference as fruit weight. We divided these traits into two groups, namely, fruit-related traits and stem, and leaf-related traits. The first group included fruit length, fruit girth, and peduncle length. According to the results of fruit girth and length, it was shown that the fruit size ofC. melo was bigger than C. sativus except one cucumber accession “Shantung Thorny,” which was bigger than C. melo “Mysore”. “CM-1” and “Andaman” were the biggest ones in melon accessions. The second group were the traits including leaf width, leaf length, number of primary branches, intermodal length, and leaf petiole length, which showed smaller variance than fruit-related characters.By Principal Component Analysis (PCA) and k-means cluster analysis, eight quantitative values were covered by three principle components (PC1, PC2, and PC3). Contribution of variables in each principle components was calculated (Table6). Fruit length and fruit girth dominated in PC1 and PC2, respectively. Five characters, number of primary branches, internodal length, leaf petiole length, leaf length, and leaf width, had the similar contribution to PC3. Based on the result of principle component analysis, eighteen accessions were classified into three groups (Figure 3). Group 1 contained nine accessions (seven C. sativus accessions: AAUC-2, Perundurai, Nepal Local, IC-4079, Cus125/71, Long Green, and Nanhai2 Aouri and two C. meloaccessions: Olakkur and Mysore) and Group2 contained six accessions (twoC. sativus accessions: Shantung Thorny and  JL-3 Dhillon and four C. melo accessions: Andaman, N-78, Pudukkottai, and Mayilam). Group 3 contained three C. melo accessions (CM-1, Theni, and Muskmelon Orange). The relative dispersion among accessions within Group2 was greater than the other two. Group 3 contained the least number of accessions, that is, C. melo cultivars only. Group 1 was mainly constituted of C. sativusaccessions, while Group 2 and Group 3 contained most of C. melo accessions. The dispersion among C. melo accessions was greater than that among C. sativusaccessions.Table 6
Eight quantitative characters contribution to three principle components.
Quantitative character
PC1
PC2
PC3
No. of primary branches
0.041
0.027
0.457
Internodal length
0.086
0.046
0.422
Leaf petiole length
−0.047
−0.001
0.452
Leaf length
0.053
0.016
0.385
Leaf width
0.017
−0.029
0.487
Peduncle length
−0.008
0.068
−0.135
Fruit length
0.935
0.329
−0.065
Fruit girth
−0.332
0.939
0.007(a) Distribution of eighteen accessions on the first and second principle components; (b) distribution of eighteen accessions on the first and third principle components.
(a)
(b)Characteristics on twenty-three qualitative traits of eighteen accessions are shown in Table5. Traits observed were varied from stem characters, leaf characters, fruit characters and seed characters. Same growth patterns were shown in all the plants observed and all of them were indeterminate. For stem characters, all of the accessions had angular stems with different degrees of green color. C. sativus “Long Green” was the only one with light green stem, which was lighter than the others. Besides, C. sativus “AAUC-2” displayed sparse stem pubescence and C. sativus “JL-3 Dhillon”, C. sativus “Nepal Local”, “Perundurai,” and “IC-4079” showed dense stem pubescence. Other accessions were between them. For leaf characters, five types of leaf shape were recorded, namely, entire, cordate, triangular, reniform, and pentalobate. Among them, the entire shape was common, including ten accessions showing entire leaves with spiny margin. All accessions had green leaves with different density, and C. sativus “Perundurai” was the only one without leaf glossiness. Pubescence was also observed on leaves as on stems in all accessions, and the leaf pubescence was softer than those on stem. The pubescence trait is closely related to plant defense system. It is under investigation for map-based cloning by SSR markers. The biggest diversity appeared on the fruit-related characteristics, as shown with the results of quantitative characteristics. Except C. sativus “AAUC-2”, the fruit shapes of all the C. sativus accessions were elongated. For C. melo accessions, fruit shapes of C. melo “Mayilam” and “CM-1” were pyriform. C. melo “Mysore” was only elliptical and C. melo “Muskmelon Orange” and “Theni” were globular. Fruit characters were further recorded on fruit skin luster, texture, and hardness, which are related to the appearance and taste. C. sativus “Nanhai 2 Aouri”, “AAUC-2”, “IC-4079”, and C. melo “Mysore” did not have fruit skin luster. Fruit skin textures of all the C. melo accessions were smooth and those of C. sativus accessions were rough or spiny. Three C. melo accessions, “Olakkur”, “Theni”, and “Andaman,” had soft fruit skins. C. sativus “Nepal local”, C. melo “N-78”, and “Mysore” had harder fruit skin than C. melo “Olakkur”, “Theni”, and “Andaman”, but softer than the others. Besides, all of accessions had yellow flowers and white or cream seeds, expect that the seeds of “Long Green” were green.


## 3.2. Phylogenetic Analysis

The lengths ofmatK and rbcL genes ranged from 750 to 1067 bp, sequences were submitted to DNA Database of Japan (DDBJ). The aligned data matrix combined with matK and rbcL gene was 1,728 bp long. Based on the aligned matrix, phylogenetic trees were made by Neighbor-joining and maximum likelihood method (Figures 4 and 5).Figure 4
Neighbor-joining tree for 18 accessions in this study based on combined sequence ofrbcL and matK genes. The tree is rooted on Cucumis hirsutus. Bootstrap values based on 1000 replicates above branches.Figure 5
Maximum likelihood tree for 18 accessions in this study based on combined sequence ofrbcL and matK genes. The tree is rooted on Cucumis hirsutus. Bootstrap values based on 1000 replicates above branches.On the topology ofC. sativus, “JL-3 Dhillon” showed divergence from other accessions. Fruit morphologies of “JL-3 Dhillon”, the longest fruit length and yellow fruit skin, differed from other C. sativus (Tables 4 and 5). In a previous phylogenetic study of Cucumis, some special morphological traits were circumscribed within some certain clades [5]. Species of the Asian/Australian clade had more of less ellipsoid fruits that stay green or turn yellow-orange at maturity. C. ritchiei/C. maderaspatanusclade was characterized by smooth and round fruits that turn red at maturity [5]. Also here in the present study, this correlation between morphological traits and clade divergence at molecular level was shown in the case of “JL-3 Dhillon”.CGN23965 “I.C 4079” has the scientific nameC. sativusgroupGherkin, while CGN24494 “Nepal local” has the scientific name C. sativusvar.sikkimensis(TANU and CGN). In the phylogenetic tree, “I.C 4079” and “Nepal local” did not show big divergence with other accessions, both of them were nested in the same clade. Two Chinese C. sativus accessions “Cus 125/71” and “Nanhai 2 Aouri” were nested in the same clade as well as the accessions from different areas, India and Nepal. One of reasons for narrow molecular divergence could be due to the accessions derived from narrow areas of India and Himalaya.According to the previous works and present study, comparing withC. sativus, genetic diversity inC. meloseems to be much bigger. African melon group has 30 species that is divided into six subgroups [2], and ancient melon varieties were distributed throughout South Asia, which could be the first genetic diversity center [7]. C. meloaccessions studied in the present study were mainly introduced from three institutions, namely, India Institute of Vegetable Research (IIVR), Tamil Nadu Agricultural University (TNAU), and National Bureau of Plant Genetic Resources (NBPGR), USA. They distributed all around India, but in the phylogenetic tree, the accessions from different places nested in the same clade with each other. A big divergent node was found with C. melo “CM1”, branching with the other accessions. Special morphological traits were also found in accession “CM1”, such as the longest intermodal length and unique fruit color (Figure 2). “Muskmelon Orange” branch was close to “CM1”, and they were grouped into Cluster 2 in the cluster analysis (Figure 3).The fruit shapes ofC. melo “Muskmelon Orange” and “Theni” were globular, being distinguished from the otherC. melo accessions. This discrepancy was not supported by the phylogenetic results. Besides, C. melo “Mayilam” branch was the biggest divergent node. It shared the same group with “N-78” in cluster analysis in spite of that they were far from each other in phylogenetic trees. So in C. melo, correlations between morphological traits and phylogenetic tree were not as supportive as those in Cucumis sativus. It could be concluded that C. melo holds bigger diversity in morphology than C. sativus, which has caused more complex factors for analyzing phylogenetic relationships.


## 4. Conclusion

EighteenCucumis accessions derived from south Asia, which represent the most diverse genetic resources, were evaluated morphologically and at the molecular level. It was shown that, although large morphological diversity was found among C. melo and C. sativus accessions, genetic diversity based on the molecular analysis showed larger diversity in C. melo than in C. sativus. Thus, it would be necessary to investigate phylogenetic relationships at the molecular level among Cucumis species using accessions collected more widely in Asia, Africa, and Australia continents. Also, the accessions studied here are new germplasms and could be alternative candidates for breeding programs in this area.



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*Source: 134134-2012-10-14.xml*