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Research Article

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

Plant Breeding and Biotechnology 2022;10(3):163-173.
Published online: August 31, 2022

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*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

Copyright © 2022 by the Korean Society of Breeding Science

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.

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  • 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.
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.
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).
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.
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’.
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).
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.

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.

Fig. 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.
pbb-10-3-163-f1.jpg
Fig. 2
HRM curve profiles of 27 SNP primers (CMo-A01P – A27P). XX, YY and XY indicate reference SNP, alternative SNP and heterozygotes, respectively.
pbb-10-3-163-f2.jpg
Fig. 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.
pbb-10-3-163-f3.jpg
Table 1
List of F1 hybrid breeding lines and commer-cial F1 cultivars of squash used in this study.
Table 1
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
Table 2
List of SNPs used in ‘Fluidigm SNP Genotyping’ assays.
Table 2
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.

Table 3
List of primer and probe sequences used in HRM assay of SNPs.
Table 3
HRM Marker Primer (5’-3’)
Forward Reverse Probea)
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.

Table 4
Validation of ‘Fluidigm SNP Genotyping’ assay developed for the purity test.
Table 4
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
Table 5
Results of purity test in ‘Parangsae’ cultivars.
Table 5
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
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Development of SNP Markers for Identification of Squash F1 Hybrid Cultivars Using Fluidigm-Based Genotyping
Plant Breed. Biotech.. 2022;10(3):163-173.   Published online August 31, 2022
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Development of SNP Markers for Identification of Squash F1 Hybrid Cultivars Using Fluidigm-Based Genotyping
Plant Breed. Biotech.. 2022;10(3):163-173.   Published online August 31, 2022
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Development of SNP Markers for Identification of Squash F1 Hybrid Cultivars Using Fluidigm-Based Genotyping
Image Image Image
Fig. 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.
Fig. 2 HRM curve profiles of 27 SNP primers (CMo-A01P – A27P). XX, YY and XY indicate reference SNP, alternative SNP and heterozygotes, respectively.
Fig. 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.
Development of SNP Markers for Identification of Squash F1 Hybrid Cultivars Using Fluidigm-Based Genotyping

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

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)

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

HRM Marker Primer (5’-3’)
Forward Reverse Probea)
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.

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

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
Table 1 List of F1 hybrid breeding lines and commer-cial F1 cultivars of squash used in this study.
Table 2 List of SNPs used in ‘Fluidigm SNP Genotyping’ assays.

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

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

a)Bold indicates SNP.

Table 4 Validation of ‘Fluidigm SNP Genotyping’ assay developed for the purity test.
Table 5 Results of purity test in ‘Parangsae’ cultivars.