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Development of SNP Markers for Identification of Squash F1 Hybrid Cultivars Using Fluidigm-Based Genotyping
Plant Breed. Biotech. 2022;10:163-173
Published online September 1, 2022
© 2022 Korean Society of Breeding Science.

Jong-Geun Park1, Jeong-Eui Hong1, Md Abdur Rahim2, Ill-Sup Nou1*

1Department of Horticulture, Sunchon National University, Suncheon 57922, Korea
2Department of Genetics and Plant Breeding, Sher-e-Bangla Agricultural University, Dhaka 1207, Bangladesh
Corresponding author: Ill-Sup Nou,, Tel: +82-61-750-3249, Fax: +82-61-750-3208
Received July 4, 2022; Revised August 9, 2022; Accepted August 10, 2022.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Squash (Cucurbita moschata D.) is an economically important vegetable of the Cucurbitaceae family. The genetic purity of commercial hybrid seed is crucial for the success of hybrid seed production. The molecular markers like single nucleotide polymorphism (SNP) can efficiently and cost-effectively distinguish the genetic differences among F1 hybrid cultivars. Therefore, in this study, we used ‘Fluidigm SNP Genotyping’ assay using 27 SNPs to distinguish and purity analysis of registered commercial F1 hybrid cultivars and F1 breeding lines of squash. Of these, eight SNP markers, including CMo-A01, CMo-A02, CMo-A04, CMo-A05, CMo-A12, CMo-A16, CMo-A20 and CMo-A25 can successfully identified heterozygotes from the registered commercial F1 hybrid squash cultivars with 100% accuracy and partial contamination was detected for F1 hybrid squash breeding lines which resulted due to outcrossing. Moreover, the HRM analysis of a registered commercial F1 hybrid cultivar ‘Parangsae’ with CMo-A03 SNP marker showed 96.30-100% purity of the cultivar. Our results suggest that the ‘Fluidigm SNP Genotyping’ technology could be a rapid and cost-effective method for cultivar differentiation and genetic purity analysis of F1 hybrids and squash cultivars.
Keywords : Molecular marker, Genetic purity test, Squash, SNP, Fluidigm assay, High-throughput genotyping, F1 hybrid, Cultivar

Squash (Cucurbita moschata D., 2n = 2× = 40) is an important vegetable crop of the Cucurbitaceae family. As per archaeological remains, this crop was domesticated from southern Mexico and coastal Peru (Feriol et al. 2004; Jacobo-Valenzuela et al. 2011). It is an essential component in human diet in different parts of the world. In Korea, squash ranked second position in the area cultivated under Cucurbitaceous crops (Kim et al. 2016). The fruit pulp and seed are used for human consumption (Gomes et al. 2020; Mashitoa et al. 2021). The fruit pulp of squash is the richest source of carotenoids, lutein, vitamin C, dietary fiber, phenolic compounds and minerals, including K, Ca, P, Mg and Cu (Jacobo-Valenzuela et al. 2011). Besides, it has various culinary uses as a food ingredient in different preparations, including breads, pasties, soups etc. (Guiné et al. 2012). Moreover, squash has many health promoting properties, including anti-diabetic, anti-carcinogenic, and anti-microbial activities (Yadav et al. 2010; Kim et al. 2016).

The genetic purity assessment of F1 hybrid seed is essential for successful adoption of hybrid seed technology (Nandakumar et al. 2004). There are many commercial F1 hybrid squash cultivars available in the seed market of the world including Korea. Therefore, commercial seed producers need genetic purity tests to ensure quality seeds. Traditionally, grow-out-trials (GOTs) are carried out to test the seed genetic purity, which time consuming and labor expressive while molecular markers can potentially dis-criminate the genetic variation among F1 hybrid cultivars with a low cost (Yashitola et al. 2002; Ye et al. 2013; Pattanaik et al. 2018). Recently, molecular markers have already been proven as an effective approach for detection of genetic variations between F1 commercial cultivars (Jamali et al. 2019; Kim et al. 2021). For example, simple sequence repeat (SSR) markers are successfully used for differentiation of F1 cultivars in several agricultural crops, including onion (Tsukazaki et al. 2006), asparagus (Caruso et al. 2008), watermelon (Kwon et al. 2010), radish (Lee et al. 2015), pear (Pierantoni et al. 2004), melon (Kwon and Hong, 2014), pumpkin (Sim et al. 2015), and tomato (Phan et al. 2016), sunflower (Pallavi, et al. 2011). However, SSR marker has a limitation for high-throughput genotyping due to a large number of sample and markers (Guichoux et al. 2011). On the other hand, the next-generation sequencing technology allows large-scale detection of genetic variants, including single nucleotide polymorphisms (SNPs) (Serba et al. 2019; Kishor et al. 2021). Now-a-days, SNPs are considered to be the most commonly used powerful genetic markers in plant breeding for detection of genetic differences among various crop cultivars due to their co-dominant inheritance, high abundance in the genome and high- throughput genotyping (Gupta et al. 2008; Chung et al. 2014). Therefore, SNP based genotyping technology like Fluidigm SNP array has been developed for effective exploration of genetic differences in the commercial F1 hybrid cultivars, including pumpkin (Nguyen et al. 2020), melon (Kishor et al. 2020), watermelon (Park et al. 2022), chili pepper (Kim et al. 2017), tomato (Kim et al. 2020), pearl millet (Serba et al. 2019). In this study, we developed SNP type assays for a purity test of F1 cultivars/breeding line of squash for use in 27 Fluidigm SNP array which will be useful in developing a high-throughput SNP type assays for variety identification in commercial F1 hybrid squash cultivars.


Plant materials

Seven F1 hybrid breeding lines (SQ001, SQ002, SQ003, SQ004, SQ005, SQ006, and SQ007) and seven commercial F1 cultivars (PMR Teunteunae, DN-A, DN-B, Nongwooae, Jinhanae, Parangsae, and Doksuri) of squash were collec-ted from Republic of Korea were used for validation of the newly developed SNP type assays in this study (Table 1). A total of 751 individuals of the parangsae cultivars were used for F1 hybrid seed purity testing.

Table 1 . List of F1 hybrid breeding lines and commer-cial F1 cultivars of squash used in this study.

8PMR Teunteunae

Genomic DNA extraction

The genomic DNA was extracted from the young leaves of each squash commercial F1 hybrid cultivars and F1 hybrid breeding lines using the ‘DNeasy Plant Mini Kit’ (Qiagen, Wilmington, USA) according to the manufacturer’s instructions. A Nanodrop spectrophotometer was used to determine the concentration and purity of the extracted DNA samples (Thermo Scientific, Wilmington, USA) and then diluted to a concentration of 20 ng/mL with nuclease- free water.

Fluidigm SNP assay

The SNP information of was collected from ‘Cucurbit Genomics Database (CuGenDB)’ available at http:// For genotyping, seven F1 hybrid breeding lines and seven commercial F1 cultivars were used for hybrid seed purity testing (Table 1). The Fluidigm SNP assay sets used in this study were synthesized by Macrogen, Korea (Table 2). The Fluidigm assay was performed using the platform ‘Fluidigm EP1 genotyping’ (Fluidigm, USA) according to the manufacturer’s protocol. The SNPs were then called using the ‘Fludigidm EP1 Genotyping’ analysis software (version 4.8.1).

Table 2 . List of SNPs used in ‘Fluidigm SNP Genotyping’ assays.

SNP Marker NameChromosomeLocation (bp)SNP [Ref/Alt]SNP (color of dye)a)

a)F: FAM dye, H: HEX dye.

High resolution melting (HRM) analysis

SNPs were detected using high resolution melting (HRM) analysis combined with a 3’-blocked and unlabeled oligonucleotide probe (HybProbe) specific to the SNP site. The HRM primers used in this study were ordered from Macrogen (Table 3). After that, using a LightCycler 96 instrument, the gDNA was used for HRM analysis (Roche, Mannheim, Germany) according to the manufacturer’s instructions. HRM was carried out in 10 mL reaction mixtures containing 1 mL at 5ng/mL DNA, 0.1 mL of forward primer (10 pmol), 0.5 mL of reverse primer (10 pmol), 0.5 mL of probe (10 pmol), 0.3 mL of SYTO9 fluorescent dye (Invitrogen, USA), 5 mL of HS prime LP premix (GENETBIO, Korea), and 2.6 mL of DDW. HRM conditions were include three steps, first step is pre- incubation (300 seconds initial pre-incubation at 95℃), second step is 40 cycle of 3 step amplification (95℃ for 10 seconds, 64℃ to 56℃ for 15 seconds under touchdown and 72℃ for 15 seconds), and the last step is HRM reading (last step is four readings per ℃ at the final step after 60 seconds at 95℃, 60 seconds at 40℃, and 1 at 97℃). HRM data was analyzed using LightCycler 96 software (version 1.1) with a 0.2 positive/negative threshold level and 100 percent discrimination for delta Tm and curve shape.

Table 3 . List of primer and probe sequences used in HRM assay of SNPs.

HRM MarkerPrimer (5’-3’)

a)Bold indicates SNP.


We analyzed a total of 192 primer sets for the ‘Fuidigm SNP type’ assays and 29 of which had successful DNA amplification (Fig. 1, Table 2). The red, green, and blue dots represent XX (fluorescence of only the FAM dye), XY (both FAM and HEX dyes), YY (only HEX dye), respec-tively. The SNP markers and their chromosomal position of 27 successful SNPs are presented in Table 2. The primers and probe sequences of each SNP marker are present in Table 3. The marker type of each SNP type assay was divided into homozygote reference SNP (XX), homo-zygote alternative SNP (YY) and heterozygotes (XY) (Fig. 1 and Table 4). Twenty-nine SNP markers were further checked by the high resolution melting (HRM) analysis (Fig. 2) and 27 SNP markers can distinguish three different types (Table 4), including homozygote reference SNP (XX), homozygote alternative SNP (YY) and heterozygotes (XY). Eight SNP markers (CMo-A01, CMo-A02, CMo- A04, CMo-A05, CMo-A12, CMo-A16, CMo-A20 and CMo-A25) accurately distinguished hetero-zygotes (XY) from the registered commercial F1 hybrid squash cultivars while partially heterozygotes (XY) and partially homozy-gotes (XX and YY) from the analyzed squash breeding lines (Table 4). Three SNP markers (CMo-A06, CMo-A11 and CMo-A13) were distinguished all the registered com-mercial F1 hybrid squash cultivars either as homozygote reference allele (XX) or homozygote alternate allele (YY) but partially heterozygotes (XY) and partially homozygote reference allele (XX) from the squash breeding lines. However, CMo-A08 successfully identified most of the registered commercial F1 cultivars as heterozygotes (XY) except ‘Doksuri’ and CMo-A24 iden-tified the heterozygotes except ‘Doksuri’ and ‘PMR Teun-teunae’.

Table 4 . Validation of ‘Fluidigm SNP Genotyping’ assay developed for the purity test.


Figure 1. Scatter plots of 27 ‘Fluidigm SNP Genotyping’ assays. Red, blue and green dots indicated XX (fluorescence of the only FAM dye), XY (both FAM and HEX dyes) and YY (only HEX dye) types, respectively.

Figure 2. HRM curve profiles of 27 SNP primers (CMo-A01P – A27P). XX, YY and XY indicate reference SNP, alternative SNP and heterozygotes, respectively.

Further, the genetic purity of the registered commercial F1 hybrid cultivar was determined by HRM analysis using CMo-A03 SNP marker (Fig. 3). Four lots of individuals from the registered commercial F1 hybrid cultivar ‘Paransae’ were used to determine genetic purity (Table 5). The HRM analysis results revealed that LOT1 (186 heterozygotes out of 186 individuals), LOT3 (187 heterozygotes out of 187 individuals) and LOT4 (189 heterozygotes out of 189 individuals) showed 100% purity whereas LOT2 (182 heterozygotes out of 189 individuals) had 96.3% purity (Table 5).

Table 5 . Results of purity test in ‘Parangsae’ cultivars.

CultivarLotsNo. of indivi-dualsSNP markerHeterozygote (F1)Purity (%)

Figure 3. HRM curve profiles for purity test of ‘Parangsae’ F1 cultivars using CMo-A03 marker. (A) LOT1 of ‘Parangsae’, (B) LOT2, (C) LOT3 and (D) LOT4. Red and blue curves indicate genotypes of parental and F1, respectively.

The genetically pure seed crop cultivar is the key criteria for commercial cultivars including F1 hybrids. We aimed to develop SNP marker set for rapid and high-throughput genotyping system to identify and genetic purity analysis of squash cultivars based on ‘Fluidigm SNP Genotyping’ assay. The ‘Fluidigm SNP Genotyping’ assay is a cost- effective platform for distinguishing commercial crop cul-tivars (Park et al. 2022). The genetic purity of commercial F1 hybrids and cultivars is of great importance for the success of plant breeding programs (Kishor et al. 2020).

In this study, we analyzed seven registered commercial F1 hybrid cultivars and seven squash breeding lines using 27 SNP markers based on ‘Fluidigm SNP Genotyping’ assay to evaluate the genetic purity (Table 1 and 5). The results showed that all the tested SNP markers had successful DNA amplification in both the eight registered commercial F1 hybrid cultivars and the seven breeding lines of squash (Fig. 1 and Table 3). Moreover, all of those markers were analyzed using HRM analysis and all were amplified (Fig. 2). Similar results on HRM-based SNP markers for genetic purity analysis of cultivars/species were reported in melon and Capsicum species (An et al. 2010; Jeong et al. 2010). Even though total 27 SNP markers were tested, eight SNP markers (CMo-A01, CMo-A02, CMo-A04, CMo-A05, CMo-A12, CMo-A16, CMo-A20 and CMo-A25) successfully identified hetero-zygotes (XY) from the registered commercial F1 hybrid squash cultivars with 100% accuracy (Table 3). However, in case of squash breeding lines, partially heterozygotes (XY) and partially homozygotes alleles (XX and YY) were identified which suggested that those F1 hybrid squash breeding lines were contaminated due to outcrossing. Similar results were previously reported in several crop species (Pattanaik et al. 2018; Kishor et al. 2020). Then again, HRM analysis of a registered commercial F1 cultivar ‘Parangsae’ with CMo-A03 SNP marker also revealed 96.30-100% purity of the cultivar (Fig. 3 and Table 4). Our results suggested that ‘Fluidigm SNP Genotyping’ platform could be an effective approach for purity testing and identification of registered commercial squash F1 hybrids cultivars and breeding lines.


All our experiments complied with local and national regulations.


The authors declare that they have no competing interests.


This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Digital Breeding Transformation Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Project number 322071-03) and Sunchon National University Research Fund in 2020 (Grant no. 2020-0192).


I.-S.N. conceived the study. J.-G.P. and J.-E.H. con-ducted the experiments and analyzed the data. M.A.R. wrote and revised the final version of the manuscript. All authors read the final version and approved the manuscript.

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