Research Article

Horticultural Science and Technology. October 2020. 686-694
https://doi.org/10.7235/HORT.20200062


ABSTRACT


MAIN

  • Introduction

  • Materials and Methods

  •   Plant Materials and DNA Isolation

  •   SNP Markers and Fluidigm Genotyping

  •   Genetic Classification

  • Results

  •   SNP Marker Analysis

  •   Genetic Purity in F1 Hybrid Plants

  •   Genetic Relationship Analysis

  • Discussion

Introduction

Cucumis melo is an important vegetable crop and includes several subspecies, where introgression from unknown germplasms continues to be a critical problem in breeding programs. In recent years, melon F1 hybrids were developed and released into the seed market by various seed companies (Nguyen et al., 2019; Kishor et al., 2020). The success of any breeding program mostly depends on an adequate supply of genetically pure hybrid seeds in the seed market. Therefore, seed companies require genetic purity tests, which are verified based on the success rate of cross-pollination and the number of plants with self-pollination, assuring good quality seeds.

In the past, genetic purity testing was performed by the grow-out test (GOT), which involves characterizing representative samples of F1 hybrid seeds into true hybrid seeds or off-types based on several morphological characteristics at various stages of plant growth (Pattanaik et al., 2018). However, this method is associated with many limitations. It is time-consuming, space-demanding, and yields ambiguous classification of the genotypes. Besides that, environmental influences on morphological characteristics also make it difficult to obtain accurate morphological data. In addition to the GOT, various biochemical methods, such as isozyme analysis followed by electrophoresis, have been used for the genetic purity testing of F1 hybrids (Markova et al., 2003; Jadhav and Achar, 2016). These techniques have advantages but are associated with limited polymorphism and environmental sensitivity.

Molecular markers are considered to be an efficient tool for genetic purity analysis due to their simple, fast, and accurate applications (Cheng-Xiang et al., 2005; Luan et al., 2010; An et al., 2010; Bae et al., 2015). Simple sequence repeats (SSRs) have become the most preferred marker for genetic purity analysis in melons (Cheng-Xiang et al., 2005; Luan et al., 2010). However, regions flanking SSRs could contain insertions or deletions (INDELs) or other SSR events (Bang and Chung, 2015) and the origin of the total length variation of the SSR markers should be confirmed by sequencing before F1 genetic purity analysis. Therefore, employing single nucleotide polymorphism (SNP)-based markers for F1 genetic purity analysis can overcome the limitations of SSR markers. SNPs are the most ideal markers because of their high abundance, even distribution, and strong marker-trait associations (Hayward et al., 2012), thus capturing importance for genetic diversity studies (Heo et al., 2017; Li et al., 2019).

The present study assessed the genetic purity of hybrid seeds of various commercial melons using genome-wide SNP markers developed by our group (Kishor et al., 2020). This study aimed to validate these genome-wide SNP markers to assess the genetic purity of F1sand PT breeding lines in melons.

Materials and Methods

Plant Materials and DNA Isolation

Eight PT breeding lines, 7_PT1, 10_PT10, 35-1_PT35, 46-1_PT46, 2H104_PT104, 2H106_PT106, PT1_male, and PT1_female, and 85 F1 hybrid plants derived from the cross between PT1_male and PT1_female were used in this study. All the eight PT melon breeding lines and 85 F1 hybrid plants were developed at Changchun Jongmyo Co., Ltd., Chilkog, Republic of Korea. The young leaves of 85 F1 hybrid plants, their parents, and other PT melon breeding lines were obtained and subjected to DNA extraction. Total genomic DNA (gDNA) was isolated from leaf tissue using the SDS procedure with slight modifications (Kim et al., 1997). The quality and quantity of DNA were determined by measuring the O.D. at 260/280 nm using a DS-11 spectrophotometer (Denovix Inc., DE, USA) followed by 1.2% gel electrophoresis.

SNP Markers and Fluidigm Genotyping

A total of 96 genome-wide SNP markers were obtained from our recent study (Kishor et al., 2020) and used for the genetic purity analysis of 85 F1 hybrid melon plants and their parents (Table 1).

Table 1.

The selected 96 SNPs for genetic purity analysis

S. No SNP Id. Position Chromosome Allele Polymorphism in PT1 parents
1 M1_884520 884520 1 G/T
2 M1_975872 975872 1 A/G
3 M1_1066595 1066595 1 T/A
4 M1_1173173 1173173 1 C/T
5 M1_1296246 1296246 1 A/G
6 M1_1462130 1462130 1 T/G
7 M1_1978773 1978773 1 G/T
8 M1_2112637 2112637 1 C/T
9 M1_3013958 3013958 1 A/G Yes
10 M1_4104877 4104877 1 G/A
11 M1_4325219 4325219 1 G/A
12 M1_6614453 6614453 1 A/T Yes
13 M1_7977218 7977218 1 T/C
14 M1_8036666 8036666 1 T/A
15 M1_8793338 8793338 1 G/A
16 M1_8819586 8819586 1 C/T
17 M1_8896747 8896747 1 A/G
18 M1_9274229 9274229 1 C/T Yes
19 M1_14858123 14858123 1 G/A Yes
20 M1_14897464 14897464 1 A/G Yes
22 M1_30163614 30163614 1 T/C
23 M1_30481228 30481228 1 T/G
24 M1_30584172 30584172 1 T/C
25 M1_31094861 31094861 1 T/C
26 M1_31143403 31143403 1 G/T
27 M1_31442835 31442835 1 G/A
28 M1_31869094 31869094 1 T/G
29 M1_31872750 31872750 1 C/T
31 M2_1591748 1591748 2 G/T
32 M2_3245603 3245603 2 A/G
33 M2_15448280 15448280 2 A/T
34 M2_15448306 15448306 2 G/A
35 M2_15556647 15556647 2 A/G
36 M2_22107827 22107827 2 T/A
37 M2_25707021 25707021 2 C/T Yes
30 M2_34458684 34458684 2 G/A Yes
38 M3_86815 86815 3 T/C Yes
39 M3_2128779 2128779 3 A/T Yes
40 M3_2499868 2499868 3 G/T Yes
41 M3_2792358 2792358 3 C/T Yes
42 M3_3417026 3417026 3 C/T
43 M3_5526710 5526710 3 G/C Yes
44 M3_5568974 5568974 3 A/T Yes
45 M3_5829995 5829995 3 A/C
46 M3_6312154 6312154 3 G/A Yes
47 M3_23854097 23854097 3 G/A
48 M3_24188878 24188878 3 C/T
49 M3_28982354 28982354 3 G/A Yes
50 M3_28997081 28997081 3 C/G Yes
51 M4_1414734 1414734 4 C/T Yes
52 M4_10188263 10188263 4 G/T Yes
53 M4_15809728 15809728 4 G/A
54 M4_17312594 17312594 4 C/G
55 M4_18961613 18961613 4 C/T
56 M10_1291758 1291758 10 G/A Yes
57 M10_1537667 1537667 10 G/A
58 M10_1537793 1537793 10 G/A
59 M10_4361263 4361263 10 A/G
60 M10_4736855 4736855 10 G/A Yes
21 M10_18318803 18318803 10 A/G
61 M10_18383943 18383943 10 T/C
62 M10_18384386 18384386 10 A/T
63 M10_19109584 19109584 10 A/G
64 M10_19172671 19172671 10 T/C
65 M10_19174142 19174142 10 C/T
66 M10_19372215 19372215 10 G/A Yes
67 M10_21781267 21781267 10 T/G
68 M11_3581140 3581140 11 C/G Yes
69 M11_18095430 18095430 11 A/C Yes
70 M11_27693096 27693096 11 G/A Yes
71 M12_1557777 1557777 12 A/T Yes
72 M12_2513136 2513136 12 C/A
73 M12_6385196 6385196 12 G/A Yes
74 M12_9676532 9676532 12 G/A Yes
75 M12_9676722 9676722 12 G/A Yes
76 M12_9699659 9699659 12 G/C Yes
77 M12_9699917 9699917 12 T/C Yes
78 M12_9700013 9700013 12 A/C Yes
79 M12_11236131 11236131 12 G/C Yes
80 M12_11374398 11374398 12 T/C Yes
81 M12_11422892 11422892 12 G/C
82 M12_13049747 13049747 12 A/G Yes
83 M12_15922782 15922782 12 A/G Yes
84 M12_17012579 17012579 12 G/A
85 M12_18942955 18942955 12 A/G
86 M12_19163631 19163631 12 A/C
87 M12_20876389 20876389 12 G/A Yes
88 M12_21687813 21687813 12 A/T Yes
89 M12_22072754 22072754 12 T/C
90 M12_22234004 22234004 12 G/T
91 M12_22620200 22620200 12 C/A Yes
92 M12_22620201 22620201 12 C/A
93 M12_22647941 22647941 12 G/C Yes
94 M12_22820505 22820505 12 C/T
95 M12_22906257 22906257 12 C/T
96 M12_23157448 23157448 12 C/T

Genotyping was conducted using the Fluidigm Juno system (Fluidigm Corporation, CA, USA). The step involving pre-amplification was performed using both specific-target amplification (STA) and locus-specific primers (LSP), followed by dilution of the pre-amplified products with distilled water. PCR amplification was performed using a set of allele-specific primers (ASP). End-point reads were detected using a Biomark EP1Reader (Fluidigm Corporation, CA, USA).The SNP calling was performed according to the Fluidigm Juno protocol using the Fluidigm SNP Genotyping Analysis software.

Genetic Classification

Contamination of the 85 F1 hybrid plants with the genetic material of other varieties or species was determined using polymorphic SNP markers in the STRUCTURE 2.3.4 program (Falush et al., 2003). The burn-in period was performed using 100,000 iterations, followed by 100,000 Markov chain Monte Carlo (MCMC) iterations per run. The number of genetically distinct populations (K) was adjusted from 1 to 10, and the model was repeated three times for each K. The best K value was estimated based on the delta KK) value using STRUCTURE HARVESTER (Earl and von Holdt, 2012). Similarly, an unweighted pair group method with arithmetic average (UPGMA) tree was constructed using Cavalli-Sforza and Edwards’ (1967) genetic distance method in PowerMarker V3.25 (Liu and Muse, 2005). The UPGMA tree was constructed using the SNP assay results of 95 commercial melon cultivars (M1 to M95) from our previous study (Kishor et al., 2020), and the SNP assay results of the present study.

Results

SNP Marker Analysis

From the 96 SNP marker assay in the 85 F1 hybrid plants, their parents, and other PT melon breeding lines (Fig. 1), 89 SNP markers (92.70%) had successful DNA amplification, of which 39 SNP (43.82%) markers showed stable polymorphisms between PT1_male and PT1_female (Table 1). Therefore, these 39 polymorphic SNP markers were used to distinguish the F1 hybrid plants and their parents in the present study (Table 1). In contrast, the other 50 SNP markers could not amplify the DNA in Fluidigm genotyping but displayed no calls, monomorphism, or heterozygosity in the F1 hybrid plants and their parents.

http://static.apub.kr/journalsite/sites/kshs/2020-038-05/N0130380509/images/HST_38_05_09_F1.jpg
Fig. 1.

Image showing amplification success of SNP analysis in 85 PT1 F1 hybrid plants, their parents, and six other PT melon breeding lines. SNP calling was chatacterized as XX and YY for homozygotes, XY for heterozygotes, no calls, no template control (NTC), and invalid data.

Based on the six markers, M3_5822995, M10_4736855, M3_2738358, M3_86815, M3_2128779, and M4_17312594, F1 hybrid plant numbers PT1 F1 13, PT1 F1 15, PT1 F1 19, PT1 F1 21, PT1 F1 36, PT1 F1 39, PT1 F1 41, PT1 F1 42, PT1 F1 43, PT1 F1 60, PT1 F1 71, PT1 F1 77, and PT1 F1 83 showed amplification errors in the SNP assay. Additionally, F1 hybrid plant numbers PT1 F1 50 and PT1 F1 82 showed amplification errors in the M3_5568974 SNP marker only. These results suggest that amplification errors might be associated with outcrossing. Similarly, a genotyping error was also observed in F1 hybrid plant number PT1 F1 55 for the M1_975872 monomorphic SNP marker, which could be due to the amplification of a non-target site. In contrast, all 89 SNP markers successfully discriminated genotypes such as 7_PT1, 10_PT10, 35-1_PT35, 46-1_PT46, 2H104_PT104, and 2H106_PT106 in the SNP assay.

Genetic Purity in F1 Hybrid Plants

Contamination with other genetic material of other varieties or species was determined by using 39 polymorphic SNP markers in the model-based STRUCTURE program; this assumes many populations among 85 PT1 F1 hybrid plants. The delta K value was maximum at K=2 (Fig. S1). The individuals under the different populations with scores of more than 0.80 were classified as pure and scores of less than 0.80 as admixture plants. The results showed that F1 hybrid plant numbers PT1 F1 13, PT1 F1 15, PT1 F1 19, PT1 F1 21, PT1 F1 36, PT1 F1 39, PT1 F1 41, PT1 F1 42, PT1 F1 43, PT1 F1 60, PT1 F1 71, PT1 F1 77, and PT1 F1 83 were grouped into a separate cluster (Fig. 2), suggesting the contamination with other genetic material of other varieties due to outcrossing. Additionally, F1 hybrid plant number PT1 F1 75 showed admixed genetic material. Hence, these plants should not be considered for the selection process due to outcrossing.

http://static.apub.kr/journalsite/sites/kshs/2020-038-05/N0130380509/images/HST_38_05_09_F2.jpg
Fig. 2.

Population structure analysis of 85 PT1 F1 hybrid plants. Numbers 1 to 85 indicate the 85 PT1 F1 hybrid plants in numerical order.

Genetic Relationship Analysis

A UPGMA tree was constructed based on the SNP assay results of 96 SNP markers in 95 commercial melon cultivars (Kishor et al., 2020), and SNP assay results of the present study, which included 7_PT1, 10_PT10, 35-1_PT35, 46-1_PT46, 2H104_PT104, 2H106_PT106, PT1_males, and PT1_females, F1 hybrid plant numbers PT1 F1 01, PT1 F1 02, PT1 F1 13, PT1 F1 15, PT1 F1 19, PT1 F1 21, PT1 F1 36, PT1 F1 39, PT1 F1 41, PT1 F1 42, PT1 F1 43, PT1 F1 60, PT1 F1 71, PT1 F1 77, and PT1 F1 75, classified into eight distinct groups (Fig. 3). Most of the melon cultivars were distributed in group VIII, followed by group IV and group VII based on the 96 SNP markers. However, M37 deviated as a separate group I.

http://static.apub.kr/journalsite/sites/kshs/2020-038-05/N0130380509/images/HST_38_05_09_F3.jpg
Fig. 3.

UPGMA dendrogram based on Cavalli-Sforza and Edwards’ (1967) genetic distance using 96 SNP markers in 119 samples consisting of 95 melon cultivars (M1 to M95) from our previous study (Kishor et al., 2020), PT1 F1 hybrid plants, and PT melon breeding lines. All eight distinct groups were assigned different colors.

All the PT1 melon breeding lines and F1 hybrid plant numbers were grouped with M10 in group IV. Interestingly, all the PT1 melon breeding lines, F1 hybrid plants, and M10 were developed at the Changchun Jongmyo Co., Ltd, Company, Chilkog, Republic of Korea. Additionally, F1 hybrid plant numbers PT1 F1 13, PT1 F1 15, PT1 F1 19, PT1 F1 21, PT1 F1 36, PT1 F1 39, PT1 F1 41, PT1 F1 42, PT1 F1 43, PT1 F1 60, PT1 F1 71, and PT1 F1 77 were closely sub-grouped with the 7_PT1 melon breeding line in group IV. However, F1 hybrid plant number PT1 F1 75 deviated as a separate sub-group in group IV.

Discussion

The genetic purity of hybrids and cultivars is of great importance for the success of any breeding program. In the present study, 85 F1 hybrid plantsand eight PT melon breeding lines were evaluated using genome-wide SNP markers to assess genetic purity.

Among the various DNA-based markers, SSR markers are predominantly used for genetic purity analysis of melons (Cheng-Xiang et al., 2005; Luan et al., 2010). However, these markers are associated with INDELs or other SSR events within its flanking region (Bang and Chung, 2015). Recently, Next-generation Sequencing (NGS) technology gaining importance to generate large number of SNPs and used to develop SNP markers (Jung et al., 2020; Kishor et al., 2020). Similarly, previous studies were also reported handful of high-resolution melting (HRM)-based SNP markers for genetic purity analysis of F1 hybrids (An et al., 2010) and powdery mildew race 5-specific SNP markers in Cucumis melo (Howlader et al., 2020). Presently, however, there are very limited numbers of these SNP markers for melons. In the latest study, we reported genome-wide SNP markers via genotyping-by-sequencing (GBS) in melons (Kishor et al., 2020). This study revealed that numerous high-quality SNP markers were distributed across the 12 chromosomes in the melon genome.

Here, we used 96 high-quality SNP markers from our previous study (Kishor et al., 2020) and performed genetic purity analysis of F1 hybrids and PT breeding lines in melon via Fluidigm SNP assays. The results indicated a 92.70% success rate of DNA amplification in both the 85 F1 hybrid plantsand the eight PT melon breeding lines. Although a total of 96 SNP markers were screened, 39 SNP (43.82%) markers showed stable polymorphisms between the PT1_male and the PT1_female. Such a result was associated with highly similar genetic backgrounds between the parents, possibly due to the relatively narrow genetic base in the crop plants (Pattanaik et al., 2018).

In a breeding program, seeds can become admixed due to several reasons, such as outcrossing, pollen shedders, and physical mixtures (Pattanaik et al., 2018). Therefore, providing genetically pure hybrid seed is very important for commercially successful hybrids. A recent study reported that admixed cultivars could be classified using population structure analysis in melons (Kishor et al., 2020).

In the present study, we identified several F1 hybrid plants associated with contamination due to outcrossing based on SNP marker analysis and population structure analysis. Additionally, the SNP analysis and population structure analysis results identifying the genetic purity of F1 hybrid plants were consistent with each other. Hence, these contaminated plants are not recommended for further selection processes. Further, UPGMA analysis revealed that most of the contaminated plants were closely sub-grouped with 7_PT1, suggesting possible outcrossing with the 7_PT1 melon breeding line.

Together with DNA extraction, the Fluidigm-based SNP marker analysis presents a simple and effective approach for quality testing of melon hybrids and breeding lines. Additionally, SNP marker technology could identify genetic similarities and differences by comparing melon PT breeding lines with commercial or registered melon cultivars. Therefore, these SNP markers can help breeders protect the plant proprietary rights of new cultivars or hybrids through genetic purity testing of the melons.

Acknowledgements

This work was supported by the Korean Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Agri-Bio Industry Technology Development Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (317011-04- 3-HD020).

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