The transferability of non-wax gourd SSR marker in wax gourd. Eleven wax gourd accessions with high morphological diversity were selected to test the transferability of 1,152 SSR markers developed from other crops of the Cucurbitaceae family. 580 of them had amplification products in the tested wax gourd accessions, and each of them had clear bands in at least five of these accessions. Of them, the SSR markers developed from watermelon had the highest transferability, and 170 of 288 (59.03%) were transferrable in wax gourd, followed by 153 from melon, 142 from pumpkin and 115 from cucumber. We also checked the polymorphism of 580 SSR markers in eleven wax gourd accessions, and 42 of them were polymorphic in different accessions of wax gourd with 2, 15, 5 and 20 from cucumber, melon, watermelon and pumpkin, respectively (Table 1).
Table 1
The numbers of transferrable and polymorphic SSR markers in wax gourd.
Origins
|
Watermelon
|
Melon
|
Cucumber
|
Pumpkin
|
Total
|
SSR markers
|
288
|
288
|
288
|
288
|
1152
|
Transferable markers
|
170
|
153
|
115
|
142
|
580
|
Polymorphic markers
|
5
|
15
|
2
|
20
|
42
|
Genome wide SSR markers development in wax gourd. The analysis of transferrable markers from non-wax gourd genomes showed a low transferability of these markers, which is far from enough in the genetic study of wax gourd. Therefore, we developed the whole genome wide SSR markers from wax gourd draft genome assembly of B227. A total of 52,431 microsatellite loci were identified from wax gourd draft genome. The total sequence length of all microsatellites accounted for 0.13% of the whole genome, with an average of 55 SSR/Mb. Among different repeat types, the dinucleotides were the most common type accounting for 41.19% of the total SSR loci discovered, followed by trinucleotides (16.71%), while octonucleotides were the least frequent repeat type (3.12%) (Table 2). The SSR motif distribution with regarded to repeat numbers has also been investigated. The microsatellite frequency was decreased as the number of repeat units increased, which was more obvious for longer SSR motifs (Fig. 1). For example, the mean number of repeat motifs in heptanucleotides and octonucleotides were 3.16 and 3.14 respective, and the number of microsatellites were 6,170 and 1,637 respective (Table 2).
Table 2
Distribution of different nucleotide repeats in the wax gourd genome
Motif length
|
Number of loci identified
|
Frequency (%)
|
Mean repeat number
|
Number of loci primers designed
|
Percentage SSRs suitable for primer design (%)
|
Di -
|
21,594
|
41.19
|
11.63
|
16,339
|
75.66
|
Tri -
|
8,759
|
16.71
|
9.36
|
7,060
|
80.6
|
Tetra -
|
6,154
|
11.74
|
5.72
|
4,513
|
73.33
|
Penta -
|
6,083
|
11.6
|
4.26
|
4,809
|
79.06
|
Hexa -
|
2,034
|
3.88
|
4.32
|
1,761
|
86.58
|
Hepta -
|
6,170
|
11.77
|
3.16
|
4,868
|
78.9
|
Octo -
|
1,637
|
3.12
|
3.14
|
1,356
|
82.83
|
Tota
|
52,431
|
100
|
|
40,706
|
77.64
|
Furthermore, the repeat motifs for each type of SSRs identified in the wax gourd genome were also examined. We found that some nucleotide motifs were more prevalent than others. For example, the AT motif was dramatically overrepresented in dinucleotide motifs, and it was also the most frequent motif in the entire wax gourd genome accounting for 35.8% of the total SSR loci discovered. Similarly, the AAT, AAAT, AAAAT, AAAAAG, AAAAAAT, and AAAAAAAG were the most abundant repeats types in each class (Additional Fig. S 1). The frequency and distribution of SSR in each chromosome showed that the number of microsatellite loci was positively correlated with their chromosome size (Additional Table S 4, Fig. 2). The largest number of microsatellites were detected on chromosome 01 (5,715), followed by chromosome 08 (5,391), and the least SSR number was found on chromosome 07 (2,880). The SSR density near the centromeres is generally low, and there is also a low SSR density at the end of some chromosomes. The sequences containing microsatellite loci were screened for PCR primer design using Primer 3, and 50,298 SSR loci contained suitable flanking sites for SSR primer design. Finally, we designed 39,319 SSR primers with some SSR loci included in the same primers as compound SSRs. The exact positions of these SSRs in the wax gourd chromosomes, as well as information on repeat motifs, expected PCR product size are presented in Additional Table S 5.
The colors from blue to red indicate a gradual increase in the density of SSR markers
Genetic diversity of different phenotypes in wax gourd. We collected 8 phenotypes of 129 wax gourd accessions, and found that the range of coefficient of variation of them was from 0.11 to 0.40 (Table 3). The 129 wax gourd accessions showed relatively narrow variation in eight quantitative traits. The trait with the maximum coefficient of variation was fruit weight (0.40), followed by fruit length (0.28) and fruit diameter (0.20), while the trait with the minimum coefficient of variation was leaf length (0.11). The results showed that the coefficient of variation for fruit related traits was higher than that of all the leaf related traits, indicating that fruit related traits have a higher potential for improvement from these germplasms.
In this study, we also performed a correlation analysis for some related traits. The results showed that there was a high positive correlation between fruit related traits, and the same tendency was also observed among leaf related trait, while the correlation relationship between leaf traits and fruit traits was very weak. Among them, the positive correlation between leaf length and leaf width was the highest (0.92), followed by single fruit weight and flesh thickness (0.69) (Fig. 3).
Table 3
Analysis of variation of quantitative characters for 129 wax gourd germplasms
Trait
|
Mean
|
Max.
|
Min.
|
Range
|
S
|
CV(%)
|
Fruit length (cm)
|
41.38
|
67.00
|
2.70
|
64.30
|
11.55
|
0.28
|
Fruit diameter (cm)
|
19.97
|
36.50
|
2.27
|
34.23
|
3.90
|
0.20
|
Flesh thickness (cm)
|
4.32
|
6.50
|
2.20
|
4.30
|
0.82
|
0.19
|
Fresh weight of single fruit (kg)
|
9.37
|
20.40
|
2.59
|
17.81
|
3.79
|
0.40
|
Leaf length (cm)
|
13.81
|
17.50
|
8.34
|
9.16
|
1.47
|
0.11
|
Leaf width (cm)
|
19.32
|
26.05
|
12.50
|
13.55
|
2.51
|
0.13
|
Petiole length (cm)
|
16.61
|
25.72
|
10.14
|
15.58
|
2.85
|
0.17
|
Petiole diameter (cm)
|
7.32
|
9.85
|
3.33
|
6.51
|
0.95
|
0.13
|
FD (Fruit diameter), (Flesh thickness), FW (Fruit weight of single fruit), FL (Fruit length), LD (Leaf diameter), LL (Leaf length), PL (Petiol length), PD (Petiole diameter).
Genetic diversity analysis of wax gourd accessions. To investigate the genetic diversity of different wax gourd germplasm, 19 SSR markers developed from transcriptome of wax gourd and 48 SSR markers developed from draft genome of wax gourd in this study were used to evaluate their polymorphism in 11 tested wax gourd accessions. 21 of them were identified with good polymorphism, and they were selected together with another 11 non-wax gourd SSR markers in the genetic diversity analysis of 129 wax gourd accessions. The information of those 32 SSR markers was list in Additional Table S6. Totally, 112 alleles were detected by these 32 SSR markers (Table 4). The number of different alleles (Na) for each marker ranged from 2 to 9, with an average of 3.5. The effective number of alleles ranged from 1.142 to 6.684 with an average of 2.060 per locus. Shannon′s information index (I) for each marker ranged from 0.265 to 1.996, with an average of 0.750. Observed heterozygosity (Ho) for each marker ranged from 0.009 to 0.561, with an average of 0.230. Expected heterozygosity (He) for each marker ranged from 0.125 to 0.854, with an average of 0.427. Polymorphism Information Content (PIC) value ranged from 0.118 to 0.832, with an average of 0.370 per locus.
Table 4
Genetic diversity characteristics of 129 wax gourd accessions using 32 SSR markers
Locus
|
Na
|
Ne
|
I
|
Ho
|
He
|
PIC
|
1C096
|
2
|
1.405
|
0.463
|
0.240
|
0.289
|
0.247
|
1C189
|
7
|
4.146
|
1.577
|
0.457
|
0.762
|
0.721
|
2C053
|
5
|
1.486
|
0.602
|
0.233
|
0.328
|
0.289
|
4C020
|
6
|
4.068
|
1.482
|
0.320
|
0.757
|
0.711
|
5C190
|
3
|
2.629
|
1.022
|
0.320
|
0.622
|
0.542
|
8C070
|
3
|
2.079
|
0.776
|
0.516
|
0.521
|
0.403
|
9C051
|
2
|
1.231
|
0.335
|
0.116
|
0.188
|
0.170
|
9C071
|
5
|
1.633
|
0.802
|
0.172
|
0.389
|
0.365
|
9C255
|
2
|
1.434
|
0.480
|
0.217
|
0.304
|
0.257
|
10C006
|
3
|
1.142
|
0.267
|
0.116
|
0.125
|
0.118
|
11C204
|
2
|
1.803
|
0.637
|
0.323
|
0.447
|
0.346
|
11C239
|
5
|
2.341
|
1.126
|
0.192
|
0.575
|
0.532
|
12C147
|
7
|
4.208
|
1.582
|
0.258
|
0.765
|
0.725
|
BhSSR33868
|
2
|
1.160
|
0.265
|
0.099
|
0.138
|
0.128
|
BhSSR22321
|
6
|
1.742
|
0.857
|
0.017
|
0.428
|
0.393
|
BhSSR24640
|
3
|
1.805
|
0.674
|
0.018
|
0.448
|
0.354
|
BhSSR06554
|
2
|
1.930
|
0.675
|
0.181
|
0.484
|
0.366
|
BhSSR18918
|
2
|
1.677
|
0.593
|
0.561
|
0.405
|
0.322
|
BhSSR06950
|
9
|
6.684
|
1.996
|
0.266
|
0.854
|
0.832
|
BhSSR07727
|
4
|
1.743
|
0.766
|
0.009
|
0.428
|
0.381
|
BhSSR21714
|
3
|
1.925
|
0.718
|
0.286
|
0.483
|
0.376
|
SSR17481
|
3
|
2.057
|
0.781
|
0.419
|
0.516
|
0.404
|
RGA-UW084790
|
3
|
1.716
|
0.749
|
0.295
|
0.419
|
0.379
|
CmSSR24466
|
2
|
1.448
|
0.488
|
0.242
|
0.311
|
0.262
|
CmSSR19665
|
3
|
1.453
|
0.588
|
0.124
|
0.313
|
0.287
|
CmSSR17132
|
4
|
1.173
|
0.347
|
0.094
|
0.148
|
0.143
|
CmSSR24836
|
2
|
1.830
|
0.646
|
0.211
|
0.455
|
0.351
|
CmSSR24993
|
2
|
1.231
|
0.335
|
0.178
|
0.188
|
0.170
|
CmaSSR007847
|
2
|
1.973
|
0.686
|
0.055
|
0.495
|
0.372
|
CmaSSR005934
|
4
|
1.652
|
0.645
|
0.508
|
0.396
|
0.329
|
CmaSSR014949
|
2
|
1.319
|
0.406
|
0.063
|
0.243
|
0.212
|
WMSSR20840
|
2
|
1.797
|
0.636
|
0.244
|
0.445
|
0.345
|
Mean
|
3.5
|
2.060
|
0.750
|
0.230
|
0.427
|
0.370
|
Na number of different alleles, Ne number of effective alleles, I Shannon’s information index, Ho Observed Heterozygosity, He Expected Heterozygosity, PIC Polymorphism Information Content
Population structure and genetic diversity analysis of wax gourd germplasm. The population structure of 129 wax gourd accessions was analyzed using a model-based software STRUCTURE that employs Bayesian assignment. Evanno′s correction method[23] was applied, which showed a clear peak at K = 2 (Fig. 4A), and the 129 wax gourd accessions were divided into two main groups (Pop Ⅰ and Pop Ⅱ) based on their multi-locus genotypes (Fig. 4B). The group Pop Ⅰ contained 47 accessions, and most of them (31) were collected from the southern coastal provinces of China. The group Pop Ⅱ contained 82 accessions, and most of them (71) belonged to Yellow River Basin and Yangtze valley (Additional Table S 1).
The fingerprinting data of 129 wax gourd accessions were used to construct a dendrogram using neighbor-joining method in MEGA 6, and these accessions were classified into 2 major clusters, named cluster Ⅰ and cluster Ⅱ, respectively (Fig. 5). The cluster I could be further divided into two sub-clusters, I A and I B. The subcluster I A contained 44 accessions, including 14 accessions from Southern China and 30 from Northern China. The subcluster I B contained 19 accessions, with 4 from Northern China and 15 from Southern China. The cluster II had 24 accessions with 6 from Northern China and 18 from Southern China. The remaining materials are distributed in 8 small clusters. It is obvious that the materials from Northern China were grouped together and those from Southern China were grouped together (Fig. 5).