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Research Article

In Silico Evaluation of Restriction-Enzyme Strategies for Genotyping-By-Sequencing (GBS) in Persimmon (Diospyros Kaki Thunb.)
Sopheap Mao1
,
Hye-Gyoung Yoo1
,
Byulhana Lee1
,
Kyungho Won1*
1Pear Research Center, National Institute of Horticultural and Herbal Science, Rural Development Administration, Naju 58216, Korea
*Corresponding Author
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Abstract

Persimmon (Diospyros kaki Thunb.) is a hexaploid fruit crop for which appropriate restriction-enzyme selection is important for balancing genome complexity reduction and sequencing depth in genotyping-by-sequencing (GBS). However, no study has directly evaluated restriction-enzyme strategies through an in silico analysis for GBS library preparation and sequencing in persimmon. In this study, in silico evaluations of enzyme strategies, spefically ApeKI, PstI–MspI, ApeKI–PstI, ApeKI–EcoRI, ApeKI–MseI, ApeKI–HindIII, and ApeKI–TfiI are conducted using the reference genome of the Diospyros kaki cv. Xiaoguo-tianshi cultivar. The strategies were compared based on the predicted fragment-size distribution, number of selected fragments, genome representation, and the expected read-pair depth per selected fragment. Among the evaluated strategies, PstI–MspI generated substantially fewer retained fragments and lower genome representation but provided among the highest value for the expected sequencing depth, indicating less genome complexity and a greater concentration of sequencing reads across fewer loci. Based on these results and its previously validated application in persimmon, PstI–MspI was selected for an experimental GBS library construction using an F1 population derived from ‘Ro-19’ × ‘Taishu’. A total of 288 gDNA samples, including parental replicates, were multiplexed into 12 pooled libraries, all of which met the specified library quality criteria and were subsequently subjected to Illumina NovaSeq X 150-bp paired-end sequencing. These findings demonstrate the practical applicability of the PstI–MspI strategy for multiplexed GBS library construction and sequencing in hexaploid persimmon cultivars. However, as SNP calling, genotype accuracy, and allele-dosage estimations were not evaluated in this study and further validation is required through a downstream analysis of the generated sequencing data.

Keywords
genome representation
genotype
hexaploid
in silico analysis
sequencing depth
References
1

Akagi T, Shirasawa K, Nagasaki H, Hirakawa H, Tao R, Comai L, Henry IM (2020) The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants. PLoS Genet 16:e1008566. https://doi.org/10.1371/journal.pgen.1008566

10.1371/journal.pgen.100856632069274PMC7048303
2

Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One 6:e19379. https://doi.org/10.1371/journal.pone.0019379

10.1371/journal.pone.001937921573248PMC3087801
3

Eun MH, Han JH, Yoon JB, Lee J (2016) QTL mapping of resistance to the cucumber mosaic virus P1 strain in pepper using a genotyping-by-sequencing analysis. Hortic Environ Biotechnol 57:589-597. https://doi.org/10.1007/s13580-016-0128-3

10.1007/s13580-016-0128-3
4

Fu YB, Cheng B, Peterson GW (2014) Genetic diversity analysis of yellow mustard (Sinapis alba L.) germplasm based on genotyping by sequencing. Genet Resour Crop Evol 61:579-594. https://doi.org/10.1007/s10722-013-0058-1

10.1007/s10722-013-0058-1
5

Fu YB, Peterson GW, Dong Y (2016) Increasing genome sampling and improving SNP genotyping for genotyping-by-sequencing with new combinations of restriction enzymes. G3 (Bethesda) 6:845-856. https://doi.org/10.1534/g3.115.025775

10.1534/g3.115.02577526818077PMC4825655
6

He J, Zhao X, Laroche A, Lu ZX, Liu HK, Li Z (2014) Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding. Front Plant Sci 5:484. https://doi.org/10.3389/fpls.2014.00484

10.3389/fpls.2014.0048425324846PMC4179701
7

Heo MS, Han K, Kwon JK, Kang BC (2017) Development of SNP markers using genotyping-by-sequencing for cultivar identification in rose (Rosa hybrida). Hortic Environ Biotechnol 58:292-302. https://doi.org/10.1007/s13580-017-0268-0

10.1007/s13580-017-0268-0
8

Kagale S, Koh C, Clarke WE, Bollina V, Parkin IAP, Sharpe AG (2016) Analysis of genotyping-by-sequencing (GBS) data. Methods Mol Biol 1374:269-284. https://doi.org/10.1007/978-1-4939-3167-5_15

10.1007/978-1-4939-3167-5_15
9

Lee J, Izzah NK, Choi BS, Joh HJ, Lee SC, Perumal S, Seo J, Ahn K, Jo EJ, et al. (2016) Genotyping-by-sequencing map permits identification of clubroot resistance QTLs and revision of the reference genome assembly in cabbage (Brassica oleracea L.). DNA Res 23:29-41. https://doi.org/10.1093/dnares/dsv034

10.1093/dnares/dsv03426622061PMC4755525
10

Li H, Sun P, Wang Y, Zhang Z, Yang J, Suo Y, Han W, Diao S, Li F, et al. (2023) Allele-aware chromosome-level genome assembly of the autohexaploid Diospyros kaki Thunb. Sci Data 10:270. https://doi.org/10.1038/s41597-023-02175-2

10.1038/s41597-023-02175-237169805PMC10175270
11

Ma KB, Yang SJ, Jo YS, Kang SS, Nam M (2021) Development of kompetitive allele-specific PCR (KASP) markers for identification of persimmon (Diospyros kaki) varieties using SNPs identified by GBS. Electron J Biotechnol 49:72-81. https://doi.org/10.1016/j.ejbt.2020.11.003

10.1016/j.ejbt.2020.11.003
12

Nguyen TK, Yu J, Choi HW, In BC, Lim JH (2018) Optimization of genotyping-by-sequencing (GBS) in chrysanthemums: Selecting proper restriction enzymes for GBS library construction. Hortic Sci Technol 36:108-114. https://doi.org/10.12972/kjhst.20180012

10.12972/kjhst.20180012
13

Park S, Park YO, Park Y (2023) Population genetic analysis in persimmons (Diospyros kaki Thunb.) based on genome-wide single-nucleotide polymorphisms. Plants 12:2097. https://doi.org/10.3390/plants12112097

10.3390/plants1211209737299077PMC10255057
14

Poland JA, Brown PJ, Sorrells ME, Jannink JL (2012) Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach. PLoS One 7:e32253. https://doi.org/10.1371/journal.pone.0032253

10.1371/journal.pone.003225322389690PMC3289635
15

Poland JA, Rife TW (2012) Genotyping-by-sequencing for plant breeding and genetics. Plant Genome 5:92-102. https://doi.org/10.3835/plantgenome2012.05.0005

10.3835/plantgenome2012.05.0005
16

Rabbi I, Hamblin M, Gedil M, Kulakow P, Ferguson M, Ikpan AS, Ly D, Jannink JL (2014) Genetic mapping using genotyping-by-sequencing in the clonally propagated cassava. Crop Sci 54:1384-1396. https://doi.org/10.2135/cropsci2013.07.0482

10.2135/cropsci2013.07.0482
17

Scheben A, Batley J, Edwards D (2017) Genotyping-by-sequencing approaches to characterize crop genomes: Choosing the right tool for the right application. Plant Biotechnol J 15:149-161. https://doi.org/10.1111/pbi.12645

10.1111/pbi.1264527696619PMC5258866
18

van Orsouw NJ, Hogers RCJ, Janssen A, Yalcin F, Snoeijers S, Verstege E, Schneiders H, van der Poel H, van Oeveren J, et al. (2007) Complexity reduction of polymorphic sequences (CRoPS): A novel approach for large-scale polymorphism discovery in complex genomes. PLoS One 2:e1172. https://doi.org/10.1371/journal.pone.0001172

10.1371/journal.pone.000117218000544PMC2048665
19

Wang L, Yang J, Zhang H, Tao Q, Zhang Y, Dang Z, Zhang F, Luo Z (2022) Sequence coverage required for accurate genotyping by sequencing in polyploid species. Mol Ecol Resour 22:1417-1426. https://doi.org/10.1111/1755-0998.13558

10.1111/1755-0998.13558
20

Yonemori K, Sugiura A, Yamada M (2000) Persimmon genetics and breeding. Plant Breed Rev 19:191-225. https://doi.org/10.1002/9780470650172.ch6

10.1002/9780470650172.ch6
Information
  • Publisher :KOREAN SOCIETY FOR HORTICULTURAL SCIENCE
  • Publisher(Ko) :한국원예학회
  • Journal Title :Horticultural Science and Technology
  • Journal Title(Ko) :원예과학기술지
  • Received Date : 2026-07-15
  • Revised Date : 2026-10-06
  • Accepted Date : 2026-10-21
  • DOI :https://doi.org/10.7235/HORT.20260054
Journal Informaiton Horticultural Science and Technology Horticultural Science and Technology
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