Development of SNP Markers for Identification of Squash F1 Hybrid Cultivars Using Fluidigm-Based Genotyping

Jong-Geun Park; Jeong-Eui Hong; Md Abdur Rahim; Ill-Sup Nou

doi:10.9787/PBB.2022.10.3.163

https://doi.org/10.9787/PBB.2022.10.3.163

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

Jong-Geun Park¹, Jeong-Eui Hong¹, Md Abdur Rahim², Ill-Sup Nou^1*

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, nis@sunchon.ac.kr, 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 (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract: 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

INTRODUCTION

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.

MATERIALS AND METHODS

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.

No.	Cultivars
1	SQ001
2	SQ002
3	SQ003
4	SQ004
5	SQ005
6	SQ006
7	SQ007
8	PMR Teunteunae
9	DN-A
10	DN-B
11	Nongwooae
12	Jinhanae
13	Parangsae
14	Doksuri

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:// cucurbitgenomics.org. 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 Name	Chromosome	Location (bp)	SNP [Ref/Alt]	SNP (color of dye)^a)
CMo-A01	1	3,318,442	…CAA[A/G]CCA…	A(F):G(H)
CMo-A02	1	9,837,040	…TCT[G/A]AGG…	G(F):A(H)
CMo-A03	2	8,873,265	…CTC[A/G]GCA…	A(F):G(H)
CMo-A04	2	10,378,972	…GAG[G/A]ATT…	G(F):A(H)
CMo-A05	3	5,724,100	…AGC[G/C]CCG…	G(F):C(H)
CMo-A06	3	6,119,169	…AGG[T/C]TTA…	T(F):C(H)
CMo-A07	4	8,389,880	…CTC[G/A]ATT…	G(F):A(H)
CMo-A08	4	20,447,172	…TCC[C/T]TCA…	C(F):T(H)
CMo-A09	6	3,712,655	…TTG[G/A]GTC…	G(F):A(H)
CMo-A10	6	11,115,368	…TTC[C/T]CTC…	C(F):T(H)
CMo-A11	8	2,798,704	…AGG[T/G]TAA…	T(F):G(H)
CMo-A12	8	6,252,977	…AAA[C/A]AAA…	C(F):A(H)
CMo-A13	10	6,646,259	…GCT[C/T]GAT…	C(F):T(H)
CMo-A14	11	4,451,324	…GCC[G/A]AAC…	G(F):A(H)
CMo-A15	13	8,296,803	…GAA[C/T]GCG…	C(F):T(H)
CMo-A16	15	1,475,799	…TGT[G/C]CAA…	G(F):C(H)
CMo-A17	15	9,704,446	…GGG[T/C]GTA…	T(F):C(H)
CMo-A18	16	1,782,015	…TAT[G/A]TAG…	G(F):A(H)
CMo-A19	16	3,290,808	…TTG[G/T]TGC…	G(F):T(H)
CMo-A20	16	8,018,335	…GGG[G/T]AAA…	G(F):T(H)
CMo-A21	17	2,812,397	…GAC[G/A]GTC…	G(F):A(H)
CMo-A22	17	10,502,431	…TCC[C/T]GTA…	C(F):T(H)
CMo-A23	18	1,876,608	…CAG[G/A]TCT…	G(F):A(H)
CMo-A24	18	3,666,538	…TTT[C/G]ATG…	C(F):G(H)
CMo-A25	18	10,701,307	…TCT[C/G]ACT…	C(F):G(H)
CMo-A26	20	3,466,354	…TGC[C/A]AGA…	C(F):A(H)
CMo-A27	20	4,075,437	…CTA[G/A]GCT…	G(F):A(H)

^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 Marker	Primer (5’-3’)
HRM Marker	Forward	Reverse	Probe^a)
CMo-A01P	AGTCTTCAGTGCCAACGGTGATTC	ATGGGTTTTGGAGGAGATTCTTATC	AGATTGGCAGGCAAACCATGAGCAT
CMo-A02P	ATTGCCAAAATGCCATTAAGTAAGC	GTTGTAGTTTTAGCTGCTGCTCTCA	CTGCAAATCTTACCTCTGAGGTGTTT
CMo-A03P	ATTATAGAACATGATATGCTGCCCAC	TCCCTTACGCTATCACTTGTTTGTTAA	ACATTCACAAATCTCAGCATCATCC
CMo-A04P	GTCCACCAACCCATTATGCTTTGAA	AAGAGGCAATAGTGGAAGAACTTG	AGCAAGGTGGAGAGGATTGTTTCCA
CMo-A05P	TGAGCAGCTCTTCAACCTCTTCGAA	TGCTGCAGTTTCAAATTGGGTTTCA	AGGAATGTACCAGCAGCGCCGAGA
CMo-A06P	AGCAGCAATGTTGGCAGTGGCAAA	TTCCCATGTTGACCTCAATGTCCT	AAGGCACCAATGCCTAAACCTCGCA
CMo-A07P	GATGAAGTTTCTTCAAGAACTAGTCC	GACATAGTTAATGATTTCGTCTAGCA	TCACATCAATTCGCTCGATTAAAGTT
CMo-A08P	TTGAAGTCCATGCAGCCCTTGTT	CTGTTCTTCAGCCTCAATGTGGAT	AGACCATTTCTCTCCCTCAATCACT
CMo-A09P	TGAAACTGTGTAAACTGGCTGCTCT	CATGTTGGATTTAAGAAATGGAAGAAG	TTCAAGAGGAATTGGGTCTGAAGAA
CMo-A10P	CAATTGGAGAAAGGGTTTCGCG	AGCAGCAGCGGAATGAAGTTGA	TTGAAGAACTCGATTCCCTCGAGAG
CMo-A11P	CTTATATGTGCATGGAGAAACGGC	CATGAGAGAAATGATGAAAGGACAG	TCCCTAAAACCTTAACCTCTTTCAG
CMo-A12P	GCGGTTGTTACTCATATAATGAGAAG	GAGCATCTGCAGCAAGTTCTCTC	TGTTTTCCCTTTGTTTGGGGTTGTT
CMo-A13P	GAGGTTGCACATCGGCTAGGT	CTGCAGCAAGACCTATAGGATTTGA	GAGAAGAGGCTTGATCCACAAAGTT
CMo-A14P	ATGTCAAATAAATCTGTCTCGACGC	TGGAATGGATAATCTAGAGCTACAG	TCTGGAAATGCCGAACTTTTGATACT
CMo-A15P	CGGCATTGTCGAGAGATATCGA	ATCGTGAAGAACTCCATAATGGCT	ACCGATAAGCTTCGCATTCCTGTTG
CMo-A16P	TGTACTTTCATGGAAGCTGGCGTT	TGACGGTTCGGAGGTTGGAGA	TACGGAAGATGTGCAAAGACGCCAT
CMo-A17P	GTGATATAATTCCAGTAATTTGCAGC	TTGCCCATTCTATTGCAGCTATAG	TTCATCCTACGCCCATCGGTTCAT
CMo-A18P	TCTTGATCTCTTCCATTCTGGATC	GATGAAGCAAGCGAAATTGCTACA	TCTACGTCTTATATAGATTCTGACACG
CMo-A19P	GCAGCTATCTAAGAAGGCTAAATATC	ATGATTCGAAGAAACTGCCGAATTAGT	TCCTTCTTTGGTTGGTGCAATGTGC
CMo-A20P	CCGAAACTCAACGTCAAATAATGTG	ACGGAGAGCGAAGGGCTAAATTT	TCAAGTCTTTTCCCCACCATCCAAT
CMo-A21P	TGATTGCGAAATAGTCTTTCGTTGC	TCTTGTTCAGCGTTCGAGTATCGA	GACTCGACGACGGTCACGATGA
CMo-A22P	TGCTTATCAGAGTGGCATTTATTCTG	GAGCTTAGTAAGAATGGTGATTAGAAC	AGACATACCGTCCTGTACAGTAAGA
CMo-A23P	GAAGCTTACAAACGGGTATGCAGA	ATATGTAGTGTTCTTGATAGTGTTAAT	GTGACAAAGACGCTGCCATTGTCTT
CMo-A24P	CTCACTTAAGAGGATCCAGGGTG	TGCCTTGAACACCAATGTTGCCTT	CATGACAGCATGAAAAGCATTCTCTG
CMo-A25P	TACGAGTCGCATTTCTTGACCG	ACGAAGTTAGCACAGTAATAGTCATC	TTAGGTCATTTCTGACTTTGATGCTAG
CMo-A26P	CTGTCAACTGTTTGAATACTCGGG	GTACCTAACAGTTGAAGAATCTCCA	TGGACCAGAAATGCAAGAAATGGAG
CMo-A27P	CATGTGAGCTGCTGTGGACGATT	ACAAGCGAAAGACTTCATAGCAGATAT	AGGAATAGATCTAAGCTGAATGGCT

^a)Bold indicates SNP.

RESULTS

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.

Assay	SQ001	SQ002	SQ003	SQ004	SQ005	SQ006	SQ007	PMR Teun-teunae	DN-A	DN-B	Nong-wooae	Jin-hanae	Parang-sae	Dok-suri
CMO-A01	YY	YY	XY	XY	XY	YY	YY	XY	XY	XY	XY	XY	XY	XY
CMO-A02	YY	YY	XY	XY	XY	YY	XY	XY	XY	XY	XY	XY	XY	XY
CMO-A03	YY	YY	XY	YY	YY	YY	XY	YY	XY	XY	XY	XY	XY	XY
CMO-A04	XY	YY	XY	XY	XY	YY	XX	XY	XY	XY	XY	XY	XY	XY
CMO-A05	XX	XY	XY	XY	XY	XY	XY	XY	XY	XY	XY	XY	XY	XY
CMO-A06	XX	XY	XY	XY	XX	XY	XY	XX	XX	XX	XX	XX	XX	XX
CMO-A07	XX	XX	XX	XX	XX	XX	XX	XX	XX	XX	XX	XX	YY	XY
CMO-A08	YY	YY	XY	XY	XY	YY	XY	XY	XY	XY	XY	XY	XY	XX
CMO-A09	XX	XX	XY	XY	XY	XX	XY	XX	XY	XY	XY	XY	XY	XY
CMO-A10	XY	XX	XX	XX	XX	XX	XX	XX	XY	XY	XY	XY	XY	XY
CMO-A11	YY	YY	XY	YY	YY	YY	XY	YY	YY	YY	YY	YY	YY	YY
CMO-A12	XX	XX	XY	XY	XY	XX	XY	XY	XY	XY	XY	XY	XY	XY
CMO-A13	XX	XX	XY	XX	XX	XX	XY	XX	XX	XX	XX	XX	XX	XX
CMO-A14	XX	XX	XX	XY	XX	XX	XY	XX	XY	XY	XY	XY	XY	XY
CMO-A15	XY	YY	YY	XY	YY	YY	XY	XY	XY	XY	XY	XY	YY	YY
CMO-A16	XY	XX	XX	XY	XY	XX	XY	XY	XY	XY	XY	XY	XY	XY
CMO-A17	XY	YY	XY	XY	XY	YY	XX	XY	XY	XY	XY	XY	XX	XX
CMO-A18	XY	YY	YY	XY	XY	YY	XY	XY	XY	XY	XY	XY	XX	XX
CMO-A19	YY	YY	YY	YY	YY	YY	YY	YY	XY	XY	XY	XY	YY	YY
CMO-A20	XY	YY	YY	YY	XY	YY	XY	XY	XY	XY	XY	XY	XY	XY
CMO-A21	YY	YY	XY	XY	XY	YY	XY	XY	XY	XY	XY	XY	XY	XX
CMO-A22	XY	XX	XY	XY	XY	XX	YY	XY	XY	XY	XY	XY	XY	XX
CMO-A23	XY	XY	YY	YY	YY	YY	XX	YY	XY	XY	XY	XY	XY	YY
CMO-A24	XY	XY	XX	XX	XX	XX	XY	XX	XY	XY	XY	XY	XY	XX
CMO-A25	XY	XX	XX	XY	XY	XX	XY	XY	XY	XY	XY	XY	XY	XY
CMO-A26	XX	XX	XY	XX	XX	XX	XY	XX	XX	XX	XX	XX	XX	XY
CMO-A27	YY	YY	XY	YY	YY	YY	XY	YY	XY	XY	XY	XY	XY	XY

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.

Cultivar	Lots	No. of indivi-duals	SNP marker	Heterozygote (F₁)	Purity (%)
Parangsae	LOT1	186	CMo-A03	186	100
	LOT2	189	CMo-A03	182	96.3
	LOT3	187	CMo-A03	187	100
	LOT4	189	CMo-A03	189	100

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.

DISCUSSION

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.

STATEMENT: All our experiments complied with local and national regulations.

CONFLICT OF INTEREST: The authors declare that they have no competing interests.

FUNDING: 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).

AUTHOR’S CONTRIBUTIONS: 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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