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SNP 분자표지를 이용한 8배체 딸기( × Duch.) 유전자지도 작성

A genetic linkage map of allo-octoploid strawberry (Fragaria × ananassa Duch.) using SNP markers

Ye Rin Lee, Jundae Lee*
Korean Journal of Breeding Science 2017;49(3):119-128.
Published online: August 31, 2017

Department of Horticulture, Chonbuk National University, Jeonju 54896, Korea

*Corresponding Author : (ajfall@jbnu.ac.kr, +82-63-270-2560, +82-63-270-2581)
• Received: April 6, 2017   • Accepted: June 9, 2017

© Korean Society of Breeding Science. All rights reserved.

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • An allo-octoploid strawberry (Fragaria × ananassa Duch.) is one of the most important vegetable crops in Korea. However, there were few genomic researches of strawberry due to polyploidy and complexity of its genome. In this study, we aimed to construct a genetic linkage map of strawberry using single nucleotide polymorphism (SNP) markers that were developed through a next-generation sequencing (NGS) analysis. Two strawberry varieties, ‘Sulhyang’ and ‘Senga-sengana’, were used as a maternal and a paternal parent, respectively, and their F1 generation consisting of 94 individuals was used for construction of a genetic linkage map. A total of 19.0 Gbp (‘Sulhyang’) and 21.8 Gbp (‘Senga-sengana’) of genomic sequences were obtained through NGS analysis. Subsequently, approximately 87,000 SNPs were identified and 1,154 primer sets for high-resolution melting (HRM) analysis were designed through bioinformatic analysis. In result, a total of 224 polymorphic HRM markers were developed and 205 markers were mapped on the genetic linkage map of strawberry, which total length was 800.8 cM and the number of linkage groups were 30. This SNP-based genetic linkage map and the 224 SNP markers will be very helpful for the genomic and genetic researches of allo-octoploid strawberry.
  • Keywords: Fragaria × ananassa; HRM; genetic map; NGS; SNP
현재 국내에서 재배되고 있는 딸기(Fragaria × ananassa Duch.)는 8배체(2n=8x=56)로 F. chiloensis와 F. virginiana가 자연적으로 교배되어 만들어진 종으로 알려져 있다(Darrow 1966). 딸기의 배수체는 2배체(2n=2x=14), 4배체(2n=4x=28), 6배체(2n=6x=42) 및 8배체(2n=8x=56) 등 다양하게 분포하고 있다(Folta & Davis 2006). 그 중 2배체 종인 F. vesca에서 유전체 연구가 가장 활발하게 진행되었다(Shulaev et al. 2011, Folta 2013). 또한 딸기의 유전자지도도 F. vesca에서 simple sequence repeat (SSR) 분자표지를 이용하여 제일 먼저 작성되 었으며(Sargent et al. 2004, Monfort et al. 2006), 2011년에는 F. vesca의 유전체 전체 염기서열이 공개되기도 하였다(Shulaev et al. 2011). 결과적으로 F. vesca의 이러한 연구들은 다른 Fragaria 속에 속한 종들의 유전자지도 작성 및 유전체 분석 연구에 큰 기여를 하였으며(Rousseau-Gueutin et al. 2008, Sargent et al. 2009 & 2012, Isobe et al. 2013, Hirakawa et al. 2014), 이와 동시에 장미과(Rosaceae) 내의 비교 유전체 연구의 기초자료로 이용되기도 하였다(Vilanova et al. 2008, Jung et al. 2012).
반면에 8배체 종인 F. × ananassa의 경우, 영양번식을 하는 이형접합형 작물이기 때문에 경제적으로 중요한 작물임에도 불구하고 오랫동안 유전자지도 및 유전체 연구가 진행되지 못 하였으나, 최근에는 그 중요성이 더욱 부각되어 다양한 연구가 수행되고 있다. F. × ananassa의 최초 연관 지도는 amplified fragment length polymorphism (AFLP) 분자표지를 이용하여 작성되었고(Lerceteau-Köhler et al. 2003), 그 후에는 주로 SSR 분자표지를 이용하여 유전자지도가 만들어졌다(Rousseau-Gueutin et al. 2008, Sargent et al. 2009 & 2012, Isobe et al. 2013, Van Dijk et al. 2014). 또한 F. vesca의 유전체 정보를 이용하여 F. × ananassa의 유전체 분석 연구가 수행되기도 하였다 (Hirakawa et al. 2014). 특히 Isobe et al. (2013)은 1,856개의 SSR 분자표지를 이용하여 총 2364.1 cM의 연관거리를 가진 28개의 연관군으로 구성된 고밀도 유전자지도를 작성하였다. 그러나 SSR 분자표지를 분석하기 위해서는 폴리아크릴아마이 드 젤 전기영동을 수행해야 하기 때문에 많은 노동력과 비용이 소모되 고 또한 대량 유전자 분석 시스템(high-throughput genotyping system)으로 개발하는 것도 쉽지 않다. 따라서 SNP 분자표지 개발이 절실히 요구되고 있는 상황이다. Bassil et al. (2015)과 Sargent et al. (2016)은 8배체 딸기의 SNP 유전자지도를 보고한 바 있는데, 이는 90K Axiom® SNP array를 이용한 것이다. 대량 유전자 분석 시스템(high-throughput genotyping system) 은 대량의 분자표지를 한 번에 분석할 수 있지만 개체당 분석비용 이 비싸기 때문에 현재 상황에서 실제 육종 프로그램에 활용되기 위해서는 한 개씩 분석할 수 있는(flexible) SNP 분자표지가 필요하다.
따라서 본 연구에서는 양친의 염기서열을 분석하여 대량의 SNP를 탐색하고, 이를 이용하여 SNP 분자표지를 개발하고, 개발된 SNP 분자표지를 이용하여 8배체인 F. × ananassa의 유전자 연관 지도를 작성하고자 하였다.
식물 재료
식물 재료는 국내 품종이고 역병 이병성인 ‘설향’을 모본으로, 독일 품종이고 역병 저항성인 ‘생가생가나’를 부본으로 사용하 였고(Eikemo & Stensvand 2015), 이를 교잡한 F1 분리집단 94개체를 이용하였다.
식물 DNA 추출
교배 모본으로 사용한 양친과 94개체의 F1 식물체로부터 각각 DNA를 추출하였다. 1.5 mL microcentrifuge tube에 2-3매의 딸기 신초, 5 mm stainless steel beads 2개, DNA extraction buffer (20 mM Tris-HCl, pH 7.5; 250 mM NaCl; 25 mM EDTA; 0.5% SDS) 700 μL, 1 g PVP (polyvinylpirrolidone), 및 12.5 μL beta-mercaptoethanol을 넣은 후 Tissue Lyser II (Qiagen, Germany)를 이용하여 3분간 마쇄하였다. 그 후 70°C 의 Lab Armor beads bath (Thermo Fisher Scientific Inc., USA)에 10분간 열처리를 한 후 원심분리기(Hanil Co., Korea) 를 이용하여 4°C, 13,000 rpm에서 10분간 원심분리하였다. 상층 액 600 μL을 새로운 1.5 mL tube에 옮긴 후 동량의 chloroform: isoamyl alcohol (24:1)을 넣고 4°C, 13,000 rpm에서 10분간 원심분리하였다. 이 후 다시 상층액 500 μL을 새로운 1.5 mL tube에 옮긴 후 동량의 isopropanol을 넣고 -20°C에서 10분간 보관한 후 4°C, 13,000 rpm에서 10분간 원심분리하여 DNA pellet을 얻었다. 그 다음 70% ethanol을 700 μL 넣고 4°C, 13,000 rpm에 1분간 원심분리를 하는 방식으로 2번의 세척을 하였다. 마지막으로 0.1 μL RNase와 100 μL 멸균수를 섞은 용액을 DNA pellet이 들어있는 1.5 mL tube에 넣어 용해시킨 후 BioDrop Lite (Biochrom, England)를 이용하여 DNA 농도 를 측정하였다. 추출된 DNA는 30 ng·μL-1 농도로 맞추어 사용 하였다.
차세대 염기서열 분석 및 SNP 탐색
대량의 SNP를 탐색하기 위하여 추출된 ‘설향’과 ‘생가생가나’ 의 DNA 1 μg씩을 이용하여 차세대 염기서열 분석(next generation sequencing, NGS)을 수행하였다. 생물정보학회사인 씨더스 (Daejeon, Korea)에 의뢰하여 NGS resequencing 분석과 생물정 보분석을 수행하였다. 딸기 표준유전체 정보는 Strawberry GARDEN 홈페이지(http://sstrawberry-garden.kazusa.or.jb/)에 공개된 Fragaria × ananassa (FANhybrid_r1.2)을 이용하였다 (Hirakawa et al. 2014). 딸기 표준유전체의 총 염기서열 길이는 720 Mbp였으며, DNA 단편조각인 387,847개의 scaffold와 238,119개의 contig로 구성되어 있었다.
대량 분석된 염기서열은 전처리, 표준유전체에 정렬, SNP 탐색, non-paralogous SNP 선발, HRM 프라이머 디자인의 과정 을 거쳐 분석되었다. 전처리 과정은 solexaQA (v.1.13) package 의 DynamicTrim과 LengthSort 프로그램을 사용하여 분석하였 고(Cox et al. 2010), 전처리 과정을 통과한 cleaned reads를 BWA (0.6.1-r104) 프로그램을 사용하여 표준유전체에 mapping하였다(Li & Durbin 2009). 그 결과 생성된 BAM format의 파일을 SAMtools (0.1.16) 프로그램을 사용하여 raw SNP를 탐색하였고 동시에 consensus sequence를 추출하였다 (Li et al. 2009). 그 다음 ‘설향’과 ‘생가생가나’의 SNP를 비교 분석하기 위해 SEEDERS in-house script를 이용하여 샘플간 통합 SNP matrix를 작성하였다(Kim et al. 2014). 마지막으로 다형성 SNP의 flanking sequence (400 bp)를 추출하고, 딸기 표준유전체에 BLASTN (2.2.26+)을 수행하여 서열 비교를 통 해 딸기 표준유전체에 한 번만 나타나는 SNP를 선발하였고, target SNP를 증폭할 수 있는 high-resolution melting (HRM) 분석용 프라이머를 디자인하였다(Table 1).
Table 1
List of HRM primer sets used in this study.
Table 1
Marker name LG Position (cM) Forward primer Reverse primer Fragaria vesca genome
Chromosome Position (bp)
FAN-SCF420 1 0.0 GGAATACCGAGACTCGTCACT ACGACAATCCTATTCAATCCACTG 6 30689884 - 30690283
FAN-SCF813 1 9.0 TGAATAAAGGGTTTTCAGTTTTGA TCAGACATCTCTCAAGGTCGT 6 30647220 - 30647488
FAN-SCF384 1 10.2 ACCAACAACTCAAACTTCAAAAGT GGCATTTTGTTTTATGACGAGACT 6 31592825 - 31593037
FAN-SCF132 1 11.4 AGTGGAGGACATATTCAAGTGTCT GTTGCGGTGCTTAAGAGTGC 6 31401845 - 31402250
FAN-SCF418 1 12.8 TCGAAATTCACACGAAGAAGA TTGGACTATACTCTGCAATACTG 6 31347505 - 31347862
FAN-SCF892 1 15.4 TGGGCCACTCTGTCTATGTT TCGGCTTGTCCATCCTTTCC 6 31458344 - 31458618
FAN-CON1139 1 21.4 TGCAGGGGACTCTGAGATCA TGGAAGTCCAGGAGTACTTGG 6 28464183 - 28464582
FAN-SCF833 1 38.6 TGAAGTGCTGCAATCCTGGT AACGGTGGGCTAGATTTGGG 6 25589098 - 25589497
FAN-SCF174 1 66.7 TGTTGTAGTGAGAGCCGTCG ACCAAAGGGCGATGATCCTC 6 15978363 - 15978658
FAN-SCF715 1 71.4 TGAAGGAAGAGGAGGAAGTGT TGGTTTAGGGTGTTTTAGGGGT 6 14511815 - 14512028
FAN-CON1046 1 78.9 CTAGCCTGGTCAACCGACTC TCCCATTAGTGTGGTTTAGCGA 6 10237152 - 10237439
FAN-SCF338 1 102.0 TGCATACATTTTCTGTGCGA CCAGGTAACGATAAGGGCCC 6 7114971 - 7115361
FAN-SCF735 1 103.8 TGCGACAGGATAATCATGACGT ACCCCTACTCCATCATCCCC 6 7158469 - 7158868
FAN-SCF858 1 113.4 AGAATCTAGACTGATGGATCGT ACCGACGTAATAACAACCACCA 6 4581492 - 4581890
FAN-SCF33 2 0.0 CTACTGCCCACAACTCGGTG CCAATGGCTACAATCTGCGC 7 16926268 - 16926609
FAN-SCF607 2 68.5 CTCGTGAAATTAGCATCCGTCA TCTCTATGCACACTGACAACCA 4 24082358 - 24082757
FAN-SCF377 2 70.8 GCTGATGAAACATAGTAATTCCGA TGCGGACAAAATGGATATTTTCT 4 26910570 - 26910801
FAN-SCF468 2 72.0 ACCAAAATCATAAGCAAAACCAGA TCAGAAGGAAGCTTTCTAGAGCA 4 26624055 - 26624454
FAN-SCF399 2 72.0 ACCTCTCAAATGTGGTTCTCA CCCTCTAGGACCTTCTGATGC 4 26707960 - 26708303
FAN-SCF405 2 72.0 TCCACAAGAACCTAAAAGCAT GGTGAAGGTAAATGAAAAATCTGT 4 26145596 - 26145936
FAN-SCF719 2 72.0 AGCCTCCCAGCCTAACAGTA CATGATTCAATGACGCGGCC 4 26613015 - 26613412
FAN-SCF39 2 73.1 CATGGCAGGCTAGCTTCGAT TGGACCGACTTCCTCAACAG 4 26359016 - 26359415
FAN-SCF52 2 74.7 TGCTCTTTCGAAATGCCAGTG TCACACACTGCTTAGCAACTGA 4 26652718 - 26653117
FAN-SCF956 2 77.0 ACTCTTAGCTAAATGGTTCTTGCT ACCCTATATTGCAAGAACACCT 4 24693600 - 24693962
FAN-SCF824 2 79.2 TTCCCTATATATGTTTCCCATGCA ATGCGCACCACCGAGCTTAG 4 24568435 - 24568834
FAN-SCF309 2 80.8 CCAATAAAACTTGCGGCGGT TGGTGGGTGCAATTTGATTG 4 24851133 - 24851532
FAN-SCF637 2 81.9 ACTAGGAGGAAGGGGCCTTT ACACCAAGATCAGCATCATCA 4 26476579 - 26476858
FAN-SCF578 2 86.0 GCTATTCAGCGGGGTAAGCA GCTTAGTGTCAAAAATGCAGCC 4 25972133 - 25972532
FAN-CON1146 2 88.7 AGTCTCCAAAGCCTGAAACCA CCAGGTATTCGGTCAGTTGCT 4 25186628 - 25187027
FAN-SCF6 2 89.2 TCGCAAATGGCTTTTTAGTTGC AGACTGTATTTGTCTGAGACGA 4 25176196 - 25176558
FAN-SCF131 2 89.8 ACAATCAAACGTGAGGGGCT CCAACGATGAATGTGAAGAACAGA 4 25687752 - 25688153
FAN-SCF909 2 94.5 ACCCCTAACTAAGTCATCACCG CCTTGTTGGGTCTGGGCTAA 6 21985116 - 21985169
FAN-SCF945 2 96.7 TGCTGCACATTGTTCAGTCT TGTTTATTCAGTGTTCATCAGATG 4 23040342 - 23040504
FAN-SCF595 2 98.5 TGGTGCAGGATGAGAATGAGG CACCTGGTGACGGTCATTCA 4 22850917 - 22851309
FAN-SCF345 2 102.6 AAGGGGACGGTACAGCTACT GGTGCCATCCATTGAGGCTA 4 22292764 - 22293102
FAN-SCF163 2 103.9 GGTTCGCTATGGCTAGGTCC AGCTCAAGTTGTGGGTTCCA 4 22363717 - 22364056
FAN-SCF231 2 104.5 ACTGCTTTCCTCTGTGGTTT AGAGGCATACAACACTGAGTCT 4 22008076 - 22008475
FAN-SCF294 2 106.8 GTGCCTTCAAGGAGCTGGAT CCCTGCCATAAATTGTTAAATGCA 4 22202764 - 22203163
FAN-SCF402 2 109.1 GCACGCCTCCTCAGTTACAT CGAGCTGACGTTTAAGTCCCA 4 22640567 - 22640947
FAN-SCF108 3 0.0 AGGGTGTGGAATATCATCACTCA ATGGGCCTTTGTACCGGTTT 6 21857853 - 21858252
FAN-SCF389 3 22.4 TCTAGCGCAATCTGCACCAA TCAATGAAACGTACGGCGGT 6 21995951 - 21996362
FAN-SCF166 3 26.1 TACACCGACAGGGAGGTTGA TTGATCCGAGCGAGCTTACC 6 22152785 - 22153184
FAN-SCF59 3 26.1 GGCTCTGACTTCGGCTTCTT TCTGCTGGCATTTTGTGTGC 6 22245209 - 22245570
FAN-SCF51 3 26.1 CAGGAGGGACTGGAAAGACA TGTGTTGTCATCCCACTAAGCA 6 22330843 - 22331242
FAN-SCF135 3 26.7 TCGTTGGAACCTCAGGTTGG AGGGATATTGATGATATTGTTGCA 6 21563283 - 21563682
FAN-SCF185 3 27.2 GTGCTACCGCTGAGGACAAT CTAACATCCTCCTGGGCGTG 6 21786636 - 21787035
FAN-SCF105 3 27.2 TTTCGCTTCCAAGTCACCAAC AGTTTGTCAAAATCTCACCTCC 6 21410739 - 21411138
FAN-SCF215 3 27.8 CGTCGTAGTGGATGATCCTT TGACAAATGACAACCCATGTT 6 22851692 - 22852092
FAN-SCF58 3 28.3 TGGGGTAAGTGCATTTGGTCA CCAAAGCCACTCCTCTTCGT 6 23743188 - 23743587
FAN-SCF83 3 28.3 ATGGGCTCCAGCTCTAGTCA AGAGAGAGTTCAAAAGAGGGGT 6 23220621 - 23221030
FAN-SCF115 3 28.3 GGTGGACATGAATATGAGAAAGCA GGAAAGACCCCTACAAAAACCC 6 23209827 - 23210225
FAN-SCF173 3 28.9 ACCAGACTACCACTGTGCCA AAGCAGGGTCGACAACTACC 6 23136810 - 23137047
FAN-SCF388 3 30.7 ACCCTGCCTCCTGTTCAAAC CACGGCTATACCCAAAGAGCT 6 24286158 - 24286551
FAN-SCF437 3 33.2 TTCTTGCAGGCAGACCAGTC GGTGCACTGAGGTGGTTTCT 6 22909018 - 22909416
FAN-SCF238 3 33.8 AGCTGTCTAAAAAGCTCTTCGGA TCGCAACAGAAGTGACTCCC 6 21833742 - 21834148
FAN-SCF450 3 33.8 CCGAAACATGGTCGATCCGT CCCAACCTTGCTGCTATTGC 6 22164873 - 22165272
FAN-CON1153 3 34.4 CCCATACGCGTGGCTTCAAT ACTTATCCTCTTCTTTGATGTGGG 6 21433559 - 21433813
FAN-SCF438 3 43.0 TTCCCAGATCACTCTGCTGA GGGTTGTTAAGGTATAGTACTTGG 6 13401292 - 13401547
FAN-SCF436 3 47.2 ACCAATTTATCGAAACTTCACCGA TCGATCAAAGACTTGCTATCGA 6 14629000 - 14629399
FAN-SCF246 3 48.3 TGAGGAGTAGGAGCTGTCCA TTAGCTAAGCAGGGTTGGCG 6 10601014 - 10601413
FAN-SCF165 3 50.1 GCACAAAATATTGCTGATTAGGCA TGCAGTTTCTTATATGTTGTACGG 6 14765076 - 14765398
FAN-SCF79 3 59.0 TGTTGTTTCTTCCTCCTTGGT AGTTGGTAAAAGTCTAGGTATGGA 6 11024227 - 11024401
FAN-SCF233 3 60.1 CCAACTCAAATGTGAAGTTGTGGA TCCGCCCAGACTCAAAACTT 6 9124886 - 9125097
FAN-CON1070 3 65.5 GCAAGCTTTGGCACTCATCG TCCCTCAGGAGATGGTTGGT 6 9045076 - 9045396
FAN-SCF443 4 0.0 TCACAACGCTTGTCAAAGGC ACCAGAAAGAGTATCCTCGTCT - -
FAN-SCF172 4 3.5 TTGAGGCTCATTGACAACGT TGAGGGAGAGAGATAGAGGTGC 6 3493659 - 3493907
FAN-SCF316 4 6.5 ACAGCCTCAATCAGCTGCTT TAAGTTTGGGCGATGATCCT 6 2504998 - 2505390
FAN-SCF769 4 30.7 CCGAAGAGATGAGCAACGGT GGGACTTGATCATTTGCCGG 6 9394367 - 9394762
FAN-SCF656 4 32.5 ACATATCGCTGCTGGACCAC AGCTAGCAACTTCCGACCAA 6 10822978 - 10823285
FAN-SCF119 4 34.6 CCATCATTTTACAAACGCTACACA ACGTGATCTTGCAACGGGAA 6 11132414 - 11132696
FAN-SCF569 4 35.3 TGACTTTTCGTCCATGTTGTCA GACCGAAACGTATCCATAAATAAA 6 10946444 - 10946849
FAN-SCF570 4 37.7 ACGAAAGTGGAGGTGCTCTG TGAGCGAGATGAAAACGTCG 3 5315532 - 5315808
FAN-SCF273 4 43.8 TCTCAAACGTCTTGACGCCT TGAGTGCTATTGTGGCGACT 6 18744174 - 18744235
FAN-CON1062 4 43.8 ACACCACGATATGCAACCACT ATTTCAAATGGGCCGGGTCA 6 14238062 - 14238401
FAN-SCF183 4 44.3 ACCATCGACGATGAGGAAGC CGGGGATGCTGGTCATACTT 3 31695069 - 31695130
FAN-SCF69 4 48.8 AGCGGTCCATTGACTCAACA TGTTGTAAAGCTTGTCGGGA 6 14700987 - 14701320
FAN-CON1071 5 0.0 TTGAGGTTTAAGCCGAGATGACT GTACTAACAGAGGCGGCGTT 6 27458588 - 27458625
FAN-CON1087 5 6.2 TGCAGATGTTGACTCTGCGT TTTTTACGTCCGCCGACTGA 4 32415681 - 32415732
FAN-SCF290 5 7.3 TTCACTCAAAAATTCGTGTGTT ACTTCTTACACTGACAAAACTCGA 4 32269269 - 32269656
FAN-SCF168 5 35.6 TCAACAGAGAAGATGCTTGGT ACACTCCCTTTTCCTAGCACA 6 9735998 - 9736029
FAN-CON1123 5 37.6 TCGTCACATCTACAATTGGCTCT GCGATGGTTCTAAGGTGTTCG 4 23313914 - 23314024
FAN-SCF965 5 39.3 TGTGGAGAAGTTCTAAGAATCCCG CGATCAAAGATGAAACCTAATTCC 6 1903611 - 1903648
FAN-CON1056 5 42.8 AGTGATTGGGCTGCATCAAA GCTAGGTACAGTTGCCCTGG 4 21935175 - 21935548
FAN-CON1143 5 47.0 TGGTAGCCTTCCCAATGCAG ATAAGCCAACCCCCGAGTTG 5 11569758 - 11570157
FAN-CON1128 6 0.0 TCGACATAGTGAACGGTGGC CAAATGGATTTGCGGCACCA 7 423374 - 423768
FAN-CON1053 6 1.1 GCTGCAACAAAATTGGCTTCC AGGAGACTCTAAGGTATTGATCGA 7 344631 - 344903
FAN-CON1059 6 2.3 CCTAGCTCGGCAATCAGAGG TGCAGTCAGGAGCAGTTGAG 6 30104705 - 30104918
FAN-CON1097 6 15.3 ACGTTGACGCGTAATTTGGA TGCAGTTGGTGAATGTTTGA 7 6499804 - 6500187
FAN-SCF474 6 19.3 TCGTGGGCTTAAGTTCTCAAGA CTCGCCAAGAGTATTCTCCA - -
FAN-CON1122 6 21.0 AGGACTTCTTTGATAGTACCGACA TCTTCCCAACCCACAAGCAA 7 7598690 - 7599115
FAN-SCF145 6 23.4 GCAATTTCTAGAGAGGCAAGCA TGCTTTTAAAGACAGAGAAGACA 7 5750781 - 5750840
FAN-CON1073 6 33.3 ACCCGTGAAACAATAGGGAAC AAAGCGATATACATAACATGCACC 7 11248187 - 11248367
FAN-SCF961 6 34.6 TGTGCGAATTTAATTTCCACACA GAGGAAGCATGAGCATCGGA 1 2921841 - 2921960
FAN-SCF139 6 41.1 TGCAACTCAACCAAGCCAGA AGTTCACACTTTGTAGCCACTGA 7 12594627 - 12594928
FAN-SCF730 7 0.0 ACCGGGTAACTTTGTTGGCA ACTCACCAATATCCGATGTGGT 2 10795356 - 10795437
FAN-CON1134 7 1.7 ATCTGAGGTTTTAATTCTCATGC TGGTATTGTACAAGGAAAGAGAGG 7 2111414 - 2111539
FAN-CON1145 7 2.8 TGGAAGCACTAGCCTTTGTCA GCAAAAGGAAAGAAATAACGTGGG 5 8070408 - 8070518
FAN-CON1115 7 23.5 TCATTTCCTGGCAGCTACTGA TCAGAAACCAAACTAGCCAAT 5 11182526 - 11182760
FAN-SCF756 7 32.7 GCTACTGCATTTTAGCACTTTGC TTGATAAACCAGTCTTATAACGCG 5 24691171 - 24691218
FAN-CON1066 8 0.0 CCTTTTGGGCCCATGACTTG GGTGTGATTAATCGGCCCGA 5 8945836 - 8946223
FAN-SCF378 8 0.0 CAAGAGGCAGCTTCTCTGGT GACTCCACAAAGCCTGGTCA 5 9274286 - 9274540
FAN-CON1069 8 9.7 TTGTCGTCTTAATCAAATGATTGT ACAGCTATATCCTCCACATGT 5 5735911 - 5736310
FAN-SCF698 8 12.0 GCCGAAAACCGTGGTTTGAG ACCGTGCTTGTTGTACTCGT 6 4563039 - 4563335
FAN-SCF27 8 12.5 CTGTGACTCTGTGAGGGCAG TGCAGACATGAAATGCACCA 5 5272835 - 5273234
FAN-CON1117 8 15.4 GTTTCACCATCCAAGCTGCG TCAACCCACAACCACCAACT 5 4918889 - 4919285
FAN-SCF227 8 16.0 TCACCGAGAGTAGCCTAGGA AGATGCCAGGTTACTAGTTACT 5 4858458 - 4858855
FAN-SCF63 8 25.0 GTTCTGATTGCTAGCATCTGCA TCCTGATGCGCTACTCTTGT 4 32169627 - 32169969
FAN-SCF480 8 27.3 AGGATAACAATCTGTAGTGGGGA TCACCTTAGGCCTCAGCAAA 5 2964486 - 2964724
FAN-CON1015 8 30.2 AGATTGTTGGACGCCTCTAGTC TCCTCATCAGATAAGGTGTGCA 5 1708322 - 1708548
FAN-SCF260 8 30.8 TCTCTTGAACACATTGAACCACA TCTATAATCGGGTTCTTGGGT 5 1188138 - 1188537
FAN-SCF434 9 0.0 ACGTTGGTTTCTAAGGGTGT CGATCACAAGGTCCTGAGAGA 6 35969226 - 35969306
FAN-SCF279 9 2.2 CCACCTGGGGGTTGAAAGTA CCGCTTTGCATAACTTTCGGT 2 15447700 - 15447757
FAN-SCF326 9 2.9 TGGTTTTTCGCATTTGTTGGT GGGGACGCTTGAAGAACACA 6 2118937 - 2119014
FAN-SCF846 9 7.7 TGTCAGACACAAGTTAGCTTTTGA AATCACGGTCCAACACTCGT 6 35292089 - 35292279
FAN-SCF847 9 18.4 GGAGCTTCTTTATTTTTACTCGCA TGCAAAATCCGGACAGCAAC 6 32014064 - 32014193
FAN-SCF88 9 26.8 TCATACTTGTTGCTGTAGTCACA GGGTTCTGTTTGTGGTTGGG 6 17704328 - 17704727
FAN-CON1099 9 30.6 AGGAGAAGGAATTGGATCACA AGGTTCTTTCTTGGACTGGCA 6 28740651 - 28740822
FAN-SCF369 10 0.0 TCAAGTTCACGTTTTACAGTGTCA TGCATTCTCACCTTCTCAACT 2 14150750 - 14151150
FAN-CON1089 10 15.0 GGCACAACCTGTTTGAGTTT AGTCCATTCTGCTCCAGTCT 2 3613082 - 3613418
FAN-CON1105 10 19.8 GCGATGACCCAATTTTCGGG GCCACATCTGTCACATATGCC 2 10339151 - 10339546
FAN-SCF154 10 20.4 TGCCAGCGAATGAGTAGCAG ACTGCTTATTTGCTTTGGCCA 2 10167626 - 10168044
FAN-SCF559 10 21.0 GCAAATTGAGTACAGTGACCGA CCTTGGGTTTGCCTGTAGAA 2 10088172 - 10088571
FAN-SCF1 10 29.6 GATACAGACAGGTTTGTGCGC TATGGTCTCGCAGAAGCCAC 2 10333513 - 10333912
FAN-SCF605 11 0.0 TGCCCCTCCTCTCGTTTTTC TGACTTCGAACTTGACCTCCA 6 18216050 - 18216445
FAN-SCF807 11 18.9 TGCAATAAAGCAAAGTTCAAAAAT AGTTCTTTTGCTTCGTACACT 6 28062711 - 28062898
FAN-CON1016 11 24.9 AGGGAATGGAGGTATAAGAGTT CCTTTCTGGTAGCAGCCTTACT 6 26052420 - 26052541
FAN-SCF67 12 0.0 AAGGGTGGTTTTAGGGCTGG CGGGCTTTCACCGATCTCTA 4 7444429 - 7444493
FAN-SCF203 12 1.6 ATTTGCGAACCTGTGACCAC AGAAAATGATGGCGCTTCAACA 3 8554786 - 8554880
FAN-SCF235 12 6.6 ACCCACTATGGAGTTAGCTCT TCCAACAAACACCTAGGGCA 6 3571899 - 3572282
FAN-SCF129 12 11.4 TGGCGATACAAGTTCGTAAACA AGTGAGGCTGATTTAGAGTTTCCA 5 14764214 - 14764422
FAN-SCF2 12 23.9 TCGAGAGGTGTATAAAATGCTCT TGTTTGTATGCATAACAAGCTGA 4 5849067 - 5849236
FAN-SCF548 13 0.0 CCTGGGGTGTTCAACTAGGC ATCTGCCGTGTGATGCAAGA 5 10208591 - 10208636
FAN-SCF112 13 5.1 TGTAGGAGACAAAGCGTAGTGA TGAGTAGCAGCAGACTGTAACA 6 11944125 - 11944534
FAN-SCF34 13 5.7 ATTTGAAACGCGTGGATCGAG GTCTCCAGCACCACCAGGAA 2 1358158 - 1358239
FAN-SCF84 13 7.3 CTTGAGGCCGACGATCTGTT AGATTCTGCCATCCCGCATC 3 1653607 - 1653678
FAN-SCF483 13 12.8 GGGCATTGGGATCTCAGCTT CGTCGCCTAGTGACAAGGAA 6 9565771 - 9566099
FAN-SCF760 13 15.1 CAATGACCCTCACTCACCCC ACCCTACACCATGATCCTCCA 6 9911049 - 9911186
FAN-SCF263 13 22.8 TGTTTAACGGATCATAAATGCAT GTCATTCGCGACTCGGTACA 6 12241307 - 12241731
FAN-CON1039 14 0.0 GAGATGAGGATGCAGGCCAA TGCTCACTCTATCGATGGGA 6 20401011 - 20401336
FAN-CON1142 14 6.8 AGTTCATCCGAAGCTGTGCA ACTGGATATACCGCGCATGT 6 12579743 - 12579956
FAN-CON1137 14 10.9 TCACCTGGAAATTCAGAGGA TGCCAAAGGTCGAAAATGCG 6 13012521 - 13012836
FAN-SCF289 14 22.2 ACCCTGAAATGGTTAACGTGGT GCCATATGCCTTACTCATATCT 6 20589193 - 20589212
FAN-SCF905 15 0.0 GGAGGGCTGTTCTTCAGTGG TCTTACGCAGTGCAGTCCTG 3 22549713 - 22549953
FAN-CON1076 15 1.2 GGTGCAATCCAATGAGCAGG ACTCAGGCTCGGTAGACCAT 3 22549227 - 22549626
FAN-SCF362 15 6.1 CTGGATCGCTACCTGCACTC TTAGGAGGGGTCTTCCGTGA 2 6880598 - 6880965
FAN-SCF66 15 17.8 TCCCAGCGCTTCAATCCTTT CGTTTTGGATACGTCTTGGGG 3 29379153 - 29379406
FAN-SCF367 15 20.2 CAGGAGGAAGCTCATGTGGG TGTGCATGGAACCTAAAGCTCT 3 29119775 - 29119894
FAN-SCF423 16 0.0 ATGGGTGCTGCGTGCTAGCA CGTTCTCCACGGATCCTAATCC 7 257891 - 257922
FAN-SCF197 16 9.4 CATCAAAAGAGGGGTCCGCT ACAGTAACCCATGCGATACAGT 1 21455604 - 21455985
FAN-SCF516 16 11.3 TAATCAGACCGCTCCCTCCT TATGGAGCCCATAGCCAGGT 4 28252096 - 28252225
FAN-SCF286 16 16.9 TGGACGTATGCTGGAGCCTA GGAGTACTCAGTGCAGCCTG 4 24247781 - 24248036
FAN-SCF553 16 19.9 TGGTCATTTCACTTGTGAATTGT ACCTTCAAAACAGAAGTTGACA 4 23988922 - 23989203
FAN-SCF98 17 0.0 CAGCAGGAGGTTACTCTGGC AGCAAGTTAGTAACGCACCT 1 18718021 - 18718404
FAN-SCF917 17 8.9 ACTTGGACCTACCCCTACCC GACAGGTTAGTGGAATGGCCA 1 17493100 - 17493499
FAN-CON1054 17 13.7 GGTCATCGCTCACCACTTCA CGCCATAAAATAGTAAGCGGGG 1 15672766 - 15673145
FAN-SCF382 17 18.6 TGCACATAATTGTTGTTTGGCA GGGTGTGTGTGGTACCATGT 1 12566836 - 12567235
FAN-SCF229 18 0.0 GCACAGGTTTTGGTGCATGT ACACGACACGACAACACGTA 2 27529617 - 27529951
FAN-SCF194 18 10.6 CTATGGCTGTCTCCGGCAAT TATGGCGCTTTCGAGACCAA 2 26588193 - 26588592
FAN-SCF354 18 17.0 TCCGTGCGCACATCTATATGA TAGATGGATTGGGCGCCATG 2 25907743 - 25908140
FAN-SCF872 19 0.0 TCCTGGGATTGTTAGAGTCTGT CAAAATGCACGTCAGATTATATGA 2 22312544 - 22312930
FAN-SCF89 19 3.9 TGCCCATACTAAAAGTGATCATGG TGTTCCTGGAGCTTTCATCCA 2 21320143 - 21320464
FAN-SCF5 19 7.1 ACCCAATTCAAGGACCTGCA TCACCATTACGCATTTACACGA 2 21507575 - 21507885
FAN-SCF178 19 8.8 ACTCGACAATCTGTGGTGGT TCATGCCCTTAATCGCCCTT 2 21361719 - 21362108
FAN-SCF216 19 9.9 AGCTTCTGAGTACGAAATGCA ACAACGGATCTTAAAAGAAGGCA 2 21093928 - 21094332
FAN-SCF804 19 10.5 AGCAACCCGAGAAACCTTGT TGGAGAAGCCTTTTTAATCAAGTG 2 21257000 - 21257326
FAN-SCF359 19 11.0 GTACAGGGGTAAACGCGCTA CTGGGAGACTGGGATGTTTGA 2 20767369 - 20767760
FAN-SCF385 19 12.8 ACTGTCTTCGTTTCACAACACA ATGACCTTTCGCTTGCAAGG 2 21702155 - 21702552
FAN-SCF782 19 15.1 AAGAAAAGACCCACCCCACA ATGGAATCAAAACACTGTGTTCGT 2 21709041 - 21709329
FAN-CON1144 19 16.9 AGCCATGAGTTCTCAATCACTGT CACGTAAAACCCCGAAACCC 2 21747548 - 21747947
FAN-CON984 20 0.0 AGGAATCATACATTAGTTTCTCCT TGCTTAGGGAACTGCAAATCCT 3 5608121 - 5608363
FAN-SCF55 20 0.6 AGGGTTGGCCTTGAAATCGA GACCAGTGCGAACCTCTTGA 3 5867946 - 5868345
FAN-SCF11 20 16.1 GCTTCCAGAACCCTGAAGCT AGTCAGGCTGTTGGGTAAGG 7 14002188 - 14002310
FAN-SCF625 21 0.0 ACGAAATACTGGCTCTGCTAGG ACCCCAACAATTCCAAGCCA 7 8259563 - 8259843
FAN-CON1032 21 4.2 TCGTTAAGTGTATGATTCTCTCCA GTGTGTTAAATTTATGTCGTACCA 7 6515448 - 6515725
FAN-SCF14 21 8.4 CCTCCCTAGCCTCCATCTCA CTCGATCATCACCTTGGTCCA 2 11289091 - 11289202
FAN-SCF871 22 0.0 TGTGTGACTTTCTGTATGTTCA CCAGGAACTTGTTGAGGAAAGC 3 9173551 - 9173871
FAN-SCF767 22 3.0 ACATGTTTCTGTGTCAGTTGGT CCCTTCCGCTGATCTCTGTC 3 7351198 - 7351590
FAN-SCF97 22 6.5 TCCTTTGACATGATTGCCACG AGTCAATGTCAACCTCCACTGG 3 6958967 - 6959366
FAN-CON1118 22 8.3 GCATTTGCTTTCAAGAGCGC GCGTAAGTTTGTCCTGCAGG 3 7052910 - 7053309
FAN-CON1141 23 0.0 ACGATTCGACGCCTTTCGAT ATTGGAGGAGGAGCATGCTT 3 15482868 - 15483267
FAN-CON1063 23 2.2 TCAGAAGAAATATAGCGTGGGT AAGAATCCCACTCCAACAGCT 3 22688348 - 22688574
FAN-SCF587 23 3.5 GTCTGCAAAAGCAGGCAAGT AGTCGTTGGATTTTTCTTTTAGGA 3 31471188 - 31471587
FAN-SCF915 23 3.5 CGCGCCAATTGATGAAGGAG TCGAGCCTGTCATGGTCATG 3 31438822 - 31439221
FAN-SCF793 23 5.7 CCCACCCGAGCCCTTAAAAT CGTCTGTGCCTATGTCGAGT 3 23487528 - 23487821
FAN-CON1085 23 6.8 GGACCTTGCCTTCCATGGAA GGTTCTGAAACAAGATCCCCG 3 15926347 - 15926746
FAN-SCF287 23 7.4 TGCATGTGCCGTTTTCACAG ACCAATGAAACCACGAACCT 3 16140230 - 16140639
FAN-SCF924 23 8.0 TCTGTCGGACAGTCTAAATGG TGAACCCCTTCTACTGCAAAACT 3 16134694 - 16135091
FAN-SCF78 24 0.0 GACCATTCGTCGCGGATAGT ACAGCACGTTCTGAGTTGAT 7 23293212 - 23293479
FAN-CON1025 24 6.8 TGAACCGGAAACTCTTACGT TGGACCAAATCAAAACACACA 7 21504945 - 21505040
FAN-CON1154 25 0.0 GCTGAGTCCCTCGCATGTTA ACATCACTTCAGCTGCACCA 5 16151073 - 16151358
FAN-CON1130 25 1.2 GCAGGTGGATGTCCGATTCA GATTTGACGATCGGCCAAGC 5 16155482 - 16155766
FAN-SCF593 25 2.4 CCACTGCTGGAAGCCGAAAC AGGATGGAATTTATCGCCCGA 4 7038979 - 7039083
FAN-SCF252 25 6.1 ACATGTCAAACGGTGAAAGCA TATGCGAGCGAGCTAGATGC 3 24661969 - 24662295
FAN-SCF10 26 0.0 GGGCATTGTGGGTGCAAATA CGGTGATCAAATTTGGGCCA 2 10258308 - 10258711
FAN-SCF458 26 4.2 ACCTAGTTTGAGCTATCTGGCA CGTCCCACAAGTAGCTAGCT 2 3725132 - 3725214
FAN-SCF126 27 0.0 TCAACAGTTCAACACACTGA TGTAAGCACTAGAACACTGGA 4 1265833 - 1266095
FAN-SCF164 27 0.0 TGCAGGCATTGGATCGTCAT TCATTTGCAATGAAGCATCGCT 4 1524501 - 1524913
FAN-SCF390 27 2.4 TCTAGGGTTTGAGGACGCAA GCATACAATTGGCACGTCCG 4 1770856 - 1771285
FAN-SCF383 27 3.1 CCGTCTCCCATCACTCCTCT GGCGACTCAAGAGTTACCGA 3 11683723 - 11683764
FAN-SCF520 28 0.0 TGAGGATGTGCAACGTCTCA TCGGGTTTCCAGATGTCGAC 2 19491481 - 19491897
FAN-SCF896 28 2.5 ATCTGAAGAGGCAAGCAGGG CCAACTGATCTGCTCCTCCG 2 21512966 - 21513328
FAN-SCF76 29 0.0 GGATGAGAGGAACCGAGGGA TTGAGATCTCGCACCGTTCC 7 7221386 - 7221749
FAN-SCF743 29 1.2 GCTCCAGCTTAGTCCATGCA GCCTGAAGAAACACACACCG 4 15153915 - 15154307
FAN-CON1082 30 0.0 TCCGTCCATTGTGAAGAGGC AGATCAACTTCAGAATCTGAGAA 3 196716 - 196972
FAN-SCF259 30 1.1 TGGGGTTGCACTTGAACAGT GCGTTTCTTGCTTCAACGGT 3 117480 - 117879
HRM 분석
HRM 반응액은 genomic DNA 10 ng, 10x PCR buffer 2.0 μL, 2.5 mM dNTP mixture 1.0 μL, 0.1 units Taq DNA polymerase (Transgen Biotech, China), SYTOⓇ 9 green fluorescent nucleic acid stain (Life TechnologiesTM, USA) 1.0 μL, 각각의 프라이머(Table 1) 10 pmole·μL-1, 그리고 멸균 된 3차 증류수로 총 20.0 μL로 맞추었다. PCR 반응은 Biometra Tadvanced (Biometra, Germany)를 이용하였으며, 95°C에서 5분간 초기 denaturation을 수행한 뒤, 95°C에서 10초간 denaturation, 60°C에서 20초간 annealing 및 extension 과정을 40회 반복하였다. 마지막으로 72°C에서 15초 동안 full extension 반응을 시켰다. PCR 산물은 LightCyclerⓇ Real-Time PCR (Roche, Switzerland)를 이용하여 65°C에서 97°C까지 0.03%씩 온도를 올리면서 각 온도에서 SYTO 9의 형광을 측정하여 melting curve를 그렸고, LightCyclerⓇ v.1.1.0.1320 프로그램(Roche, Switzerland)을 사용하여 유전 자형을 분석하였다(Wittwer et al. 2003).
유전자지도 작성
유전자지도 작성은 JoinMapⓇ 4.1 프로그램을 이용하여 수행 하였다(Van Ooijen 2006). 연관 그룹은 LOD≥3.0 조건에서 최대 거리 30 cM을 기준으로 하였고, 지도 거리는 Kosambi (1944) mapping function을 이용하여 계산하였으며, 집단 옵션 은 CP[outbreeder full-sib family, 연관상(linkage phases)을 전 혀 모르고 이형접합(heterozygous)과 동형접합(homozygous)이 불균일하게(heterogeneously) 분포되어 있는 양친을 교배한 집 단]를 사용하였다(Van Ooijen 2011). 최종 유전자 연관 지도는 MAPCHART v.2.1 프로그램을 사용하여 그렸다(Voorrips 2002).
양친의 대량 염기서열 분석
유전자지도 작성용 교배 모본으로 사용된 ‘설향’과 ‘생가생가 나’를 NGS resequencing한 결과, ‘생가생가나’는 총 30.7 Gbp 의 염기서열을, ‘설향’은 총 25.3 Gbp의 염기서열을 해독하였다 (Table 2). 이 염기서열에서 필요없는 부분을 잘라낸(trimming) 결과, ‘생가생가나’는 총 21.8 Gbp, ‘설향’은 총 19.0 Gbp의 염기서열이 남았다(Table 2). 이는 딸기 표준유전체의 길이인 720 Mbp의 약 30배(‘생가생가나’) 및 약 26.5배(‘설향’)에 해당 하는 것이었다(Table 2). 가공된 trimmed reads를 이용하여 딸기 표준유전체인 F. × ananassa (FANhybrid_r1.2) 염기서열에 mapping한 결과, ‘생가생가나’는 총 표준유전체의 약 76%인 530 Mbp를, ‘설향’은 약 76.5%인 534 Mbp를 커버하였다 (Table 2).
Table 2
Statistics of raw reads, trimmed reads, and mapped reads of next generation resequencing data.
Table 2
Plant material Use Raw reads Trimmed reads Mapped reads
Total length (bp) Total length (bp) Trimmed/ Raw Genome coverage The number of mapped reads (%) Mapped region of reference genome (bp) Reference genome coverage
‘Senga-Sengana’ Male 30,682,186,222 21,846,702,806 71.2% ≒ 193,915,797 530,090,126 76.0%
parent 30.34X (75.9%)
‘Sulhyang’ Female 25,256,529,246 19,080,792,008 75.5% ≒ 169,416,290 533,936,123 76.5%
parent 26.50X (77.0%)
대량 SNP 탐색 및 HRM용 프라이머 디자인
분석된 ‘생가생가나’와 ‘설향’의 염기서열을 비교하여 총 421,480개의 SNP을 탐색하였으며 그 중 양친 모두 동형접합형 인(homozygous) SNP는 206,019개로 가장 많았다(Table 3). 그러나 본 연구에서는 F1 분리집단을 유전자지도 작성용 집단으 로 사용하였기 때문에 사용할 수 없는 SNP였으며 이 SNP를 사용하기 위해서는 F2 분리집단을 이용하여야 한다. 그 이유는 F1 집단 모든 개체에서 이형접합형인(heterozygous) 유전자형만 나타나기 때문이다. 나머지 세 가지 경우(동형 대 이형, 이형 대 동형 및 이형 대 이형)의 SNP는 F1 분리집단에서 사용할 수 있다(Table 3). 동형 대 이형 및 이형 대 동형의 SNP는 각각 53,933개 및 74,367개가 탐색되었고, 이 SNP는 F1 분리집단에 서 1:1로 분리되기 때문에 pseudo-testcross mapping에 사용될 수 있는 것이다(Grattapaglia & Sederoff 1994). 이형 대 이형의 SNP는 87,161개가 탐색되었고, 이 SNP는 F1 분리집단에서 1:2:1로 분리되기 때문에 순계를 이용한 F2 분리집단과 같은 분리를 보인다. 따라서 본 연구에서는 이 네 가지 경우의 SNP 중에서 이형 대 이형 SNP를 이용하여 유전자지도를 작성하였다 (Fig. 1).
Table 3
Summary of single nucleotide polymorphism (SNP) detection and high resolution melting (HRM) primer design between strawberry cultivars ‘Sulhyang’ and ‘Senga-sengana.’
Table 3
SNP type The number of polymorphic SNP The number of non-paralogous SNP The number of HRM candidates The number of HRM primer sets designed
‘Senga-sengana’ ‘Sulhyang’ Total Intergenic region Genic region
CDSz Intron Exon
Homozygous Homozygous 206,019 8,128 6,750 2,324 1,212 504 416 192
Homozygous Heterozygous 53,933 2,896 2,347 652 341 134 112 65
Heterozygous Homozygous 74,367 4,220 3,460 1,338 676 298 247 117
Heterozygous Heterozygous 87,161 5,604 4,460 1,154 443 458 181 72
Total 421,480 20,848 17,017 5,468 2,672 1,394 956 446

zCDS indicates a coding sequence region except intron and exon regions.

Fig. 1
A genetic linkage map consisting of 205 single nucleotide polymorphism (SNP) markers using an F1 sgregating population of strawberry cultivars 'suhyang' x ;Senga-sengana'.
KJBS-49-119_F1.gif
탐색된 총 87,161개의 이형 대 이형 SNP 중에서 non-paralogous SNP의 수는 5,604개였다(Table 3). 본 연구에서 사용한 딸기는 8배체이기 때문에 paralog들이 다수 존재해서 분자표지 개발하 는데 원하지 않는 paralog 부분이 PCR될 수 있기 때문에 이러한 경우를 없애기 위해 non-paralogous SNP만을 선발하였다. 그 중 HRM용 프라이머를 디자인할 수 있는 SNP는 총 4,460개였 고, 실제로 총 1,154개의 HRM 분석용 프라이머를 디자인하여 실험에 사용하였다(Table 3).
HRM 분석용 프라이머를 디자인한 1,154개의 SNP를 유전체 의 위치에 따라 분류해 보면, 유전자 사이(intergenic)에 존재하 는 것이 443개였고, 유전자 내부(genic)에 존재하는 것이 711개 였다(Table 3). 유전자 내부에서도 인트론(intron) 상에 존재하 는 것이 181개, 엑손(exon) 상에 존재하는 것이 72였고, 단백질 로 번역되지 않는 지역(untranslated region, UTR)에 존재하는 것이 458개였다(Table 3).
SNP 분자표지 개발
총 1,154개의 HRM 분석용 프라이머를 디자인하여 SNP 분자 표지 개발에 사용하였다(Table 1, Wittwer et al. 2003). 양친으 로 사용된 ‘설향’과 ‘생가생가나’ 그리고 94개의 F1 개체를 이용 하여 실제로 다형성이 존재하는지 확인하기 위하여 HRM 분석 을 수행하였다. 그 결과 1,154개의 프라이머 조합 중 다형성 (polymorphic)이 나타난 것은 224개였으며, 나머지 930개는 다형성이 없었거나(monomorphic), 두 개 이상의 단편이 증폭되 어 melting curve가 복잡해 유전자형을 구분할 수 없었다. 고추 (Capsicum spp.)의 경우 HRM 분자표지 개발 성공 확률이 80% 인 것에 비하여 훨씬 낮은 성공률(19.4%)을 보였는데(Lee et al. 2013), 단형성(monomorphic)으로 나타난 것은 생물정보 분석에서 거짓양성(false positive)이 선발되기 때문이고, 복잡한 melting curve가 나타난 것은 딸기 표준유전체의 정보가 아직 불완전해 paralog를 완벽하게 제거하지 못하였기 때문인 것으로 생각된다(Hirakawa et al. 2014).
Ge et al. (2013)은 96개의 EST 염기서열로부터 총 116개의 SNP 분자표지를 개발하여 16개의 중국 딸기 재배 품종을 구분하 는데 이용하였는데, 본 연구에서는 NGS resequencing을 이용하 여 이보다 많은 SNP 분자표지를 개발하였다(Table 1). 최근에 Bassil et al. (2015)과 Sargent et al. (2016)은 90 K Axiom® SNP array를 이용하여 각각 6,594개 및 8,407개의 SNP 분자표 지를 개발하였다. 하지만 이는 high-throughput whole genome genotyping 분석 방법으로 한 번에 9만개의 SNP를 동시에 분석 하는 것이라 본 연구에서 개발한 한 개씩 따로 분석할 수 있는 즉 형질과 연관되어 있다면 육종에 바로 사용할 수 있는 SNP 분자표지와는 다른 것이라 하겠다.
8배체 딸기 유전자지도 작성
개발된 224개의 HRM 분자표지 중 205개는 딸기 유전자 연관지도 상에 위치하였다(Fig. 1, Table 1). 총 연관거리는 800.8 cM이었으며, 8배체 딸기의 경우 28개의 연관군이 작성되 어야 하지만 본 실험에는 30개의 연관군으로 나타났다(Fig. 1). 이는 유전자지도 작성 시 사용된 분자표지의 수가 적었기 때문인 것으로 판단된다. Bassil et al. (2015)과 Sargent et al. (2016)은 총 연관거리가 각각 2,050 cM 및 1,820 cM인 28개의 연관군으 로 구성된 8배체 딸기 유전자지도를 작성한 바 있다. 따라서 본 연구에서 만들어진 유전자지도는 아직 딸기 유전체 전체를 커버하지 못하고 있기 때문에 새로운 분자표지를 유전자지도에 추가해야 할 필요성이 있다. 딸기 유전체 상의 위치를 알고 있는 기 개발된 SSR 또는 SNP 분자표지를 추가함으로써 유전자지도 연관군에 염색체 번호를 부여할 필요도 있다. 하지만 본 연구에서 개발된 SNP 분자표지는 HRM 분석 방법으로 쉽게 분석할 수 있고, non-paralogous SNP 만을 선발하여 개발한 것으로 단인자 멘델 유전에 따라 단순하게 분리하기 때문에 8배체 딸기 유전체 분석 및 다양한 유전 연구에 있어서 큰 도움이 될 수 있을 것이라 생각된다. 또한 본 연구에서 작성한 딸기 유전자지도는 F1 식물체 의 역병 저항성 분석을 통해 QTL 분석에 이용될 수 있을 것이다.
딸기(Fragaria × ananassa Duch.)는 국내에서 중요한 과채류 작물 중 하나이다. 그러나 현재 재배되고 있는 딸기는 다배체이며 유전체가 복잡하기 때문에 유전체 및 유전학적 연구는 아직 미미한 실정이다. 본 실험에서는 차세대 염기서열 분석(next generation sequencing, NGS) 방법을 이용하여 대량의 SNP 분자표지를 개발하였으며, 이를 이용하여 8배체 딸기 유전자지 도를 작성하였다. 식물재료로는 ‘설향’(모친)과 ‘생가생가나’(부 친)를 양친으로 사용하였고, 이들의 F1 분리집단 94개체를 유전 자지도 작성에 사용하였다. NGS 분석 결과, ‘설향’과 ‘생가생가 나’에서 각각 19.0 Gbp와 21.8 Gbp의 염기서열을 얻었다. 양친 간의 분석된 총 SNP수는 약 87,000개였으며, 그 중 프라이머를 디자인한 SNP의 수는 1,154개였다. High resolution melting (HRM) 분석을 통하여 다형성을 확인한 결과, 1,154개의 프라이 머 중 224개에서만 다형성을 보였으며, 이 중 205개의 HRM 분자표지는 딸기 유전자지도 상에 위치시켰다. 총 연관거리는 800.8 cM이였으며, 30개의 연관군으로 이루어졌다. 본 연구에 서 개발된 SNP 분자표지 및 이를 기반으로 하여 작성된 유전자지 도는 8배체 딸기의 유전체 및 다양한 유전 연구에 큰 도움을 줄 수 있을 것으로 생각된다.
본 논문은 농림수산식품기술기획평가원 수출전략기술개발사 업(과제번호: 315047-3)의 지원에 의해 이루어졌습니다.
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A genetic linkage map of allo-octoploid strawberry (Fragaria × ananassa Duch.) using SNP markers
Korean. J. Breed. Sci.. 2017;49(3):119-128.   Published online September 1, 2017
DOI: https://doi.org/10.9787/KJBS.2017.49.3.119
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A genetic linkage map of allo-octoploid strawberry (Fragaria × ananassa Duch.) using SNP markers
Korean. J. Breed. Sci.. 2017;49(3):119-128.   Published online September 1, 2017
DOI: https://doi.org/10.9787/KJBS.2017.49.3.119
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A genetic linkage map of allo-octoploid strawberry (Fragaria × ananassa Duch.) using SNP markers
Image
Fig. 1 A genetic linkage map consisting of 205 single nucleotide polymorphism (SNP) markers using an F1 sgregating population of strawberry cultivars 'suhyang' x ;Senga-sengana'.

Fig. 1

A genetic linkage map of allo-octoploid strawberry (Fragaria × ananassa Duch.) using SNP markers

List of HRM primer sets used in this study.

Marker name LG Position (cM) Forward primer Reverse primer Fragaria vesca genome
Chromosome Position (bp)
FAN-SCF420 1 0.0 GGAATACCGAGACTCGTCACT ACGACAATCCTATTCAATCCACTG 6 30689884 - 30690283
FAN-SCF813 1 9.0 TGAATAAAGGGTTTTCAGTTTTGA TCAGACATCTCTCAAGGTCGT 6 30647220 - 30647488
FAN-SCF384 1 10.2 ACCAACAACTCAAACTTCAAAAGT GGCATTTTGTTTTATGACGAGACT 6 31592825 - 31593037
FAN-SCF132 1 11.4 AGTGGAGGACATATTCAAGTGTCT GTTGCGGTGCTTAAGAGTGC 6 31401845 - 31402250
FAN-SCF418 1 12.8 TCGAAATTCACACGAAGAAGA TTGGACTATACTCTGCAATACTG 6 31347505 - 31347862
FAN-SCF892 1 15.4 TGGGCCACTCTGTCTATGTT TCGGCTTGTCCATCCTTTCC 6 31458344 - 31458618
FAN-CON1139 1 21.4 TGCAGGGGACTCTGAGATCA TGGAAGTCCAGGAGTACTTGG 6 28464183 - 28464582
FAN-SCF833 1 38.6 TGAAGTGCTGCAATCCTGGT AACGGTGGGCTAGATTTGGG 6 25589098 - 25589497
FAN-SCF174 1 66.7 TGTTGTAGTGAGAGCCGTCG ACCAAAGGGCGATGATCCTC 6 15978363 - 15978658
FAN-SCF715 1 71.4 TGAAGGAAGAGGAGGAAGTGT TGGTTTAGGGTGTTTTAGGGGT 6 14511815 - 14512028
FAN-CON1046 1 78.9 CTAGCCTGGTCAACCGACTC TCCCATTAGTGTGGTTTAGCGA 6 10237152 - 10237439
FAN-SCF338 1 102.0 TGCATACATTTTCTGTGCGA CCAGGTAACGATAAGGGCCC 6 7114971 - 7115361
FAN-SCF735 1 103.8 TGCGACAGGATAATCATGACGT ACCCCTACTCCATCATCCCC 6 7158469 - 7158868
FAN-SCF858 1 113.4 AGAATCTAGACTGATGGATCGT ACCGACGTAATAACAACCACCA 6 4581492 - 4581890
FAN-SCF33 2 0.0 CTACTGCCCACAACTCGGTG CCAATGGCTACAATCTGCGC 7 16926268 - 16926609
FAN-SCF607 2 68.5 CTCGTGAAATTAGCATCCGTCA TCTCTATGCACACTGACAACCA 4 24082358 - 24082757
FAN-SCF377 2 70.8 GCTGATGAAACATAGTAATTCCGA TGCGGACAAAATGGATATTTTCT 4 26910570 - 26910801
FAN-SCF468 2 72.0 ACCAAAATCATAAGCAAAACCAGA TCAGAAGGAAGCTTTCTAGAGCA 4 26624055 - 26624454
FAN-SCF399 2 72.0 ACCTCTCAAATGTGGTTCTCA CCCTCTAGGACCTTCTGATGC 4 26707960 - 26708303
FAN-SCF405 2 72.0 TCCACAAGAACCTAAAAGCAT GGTGAAGGTAAATGAAAAATCTGT 4 26145596 - 26145936
FAN-SCF719 2 72.0 AGCCTCCCAGCCTAACAGTA CATGATTCAATGACGCGGCC 4 26613015 - 26613412
FAN-SCF39 2 73.1 CATGGCAGGCTAGCTTCGAT TGGACCGACTTCCTCAACAG 4 26359016 - 26359415
FAN-SCF52 2 74.7 TGCTCTTTCGAAATGCCAGTG TCACACACTGCTTAGCAACTGA 4 26652718 - 26653117
FAN-SCF956 2 77.0 ACTCTTAGCTAAATGGTTCTTGCT ACCCTATATTGCAAGAACACCT 4 24693600 - 24693962
FAN-SCF824 2 79.2 TTCCCTATATATGTTTCCCATGCA ATGCGCACCACCGAGCTTAG 4 24568435 - 24568834
FAN-SCF309 2 80.8 CCAATAAAACTTGCGGCGGT TGGTGGGTGCAATTTGATTG 4 24851133 - 24851532
FAN-SCF637 2 81.9 ACTAGGAGGAAGGGGCCTTT ACACCAAGATCAGCATCATCA 4 26476579 - 26476858
FAN-SCF578 2 86.0 GCTATTCAGCGGGGTAAGCA GCTTAGTGTCAAAAATGCAGCC 4 25972133 - 25972532
FAN-CON1146 2 88.7 AGTCTCCAAAGCCTGAAACCA CCAGGTATTCGGTCAGTTGCT 4 25186628 - 25187027
FAN-SCF6 2 89.2 TCGCAAATGGCTTTTTAGTTGC AGACTGTATTTGTCTGAGACGA 4 25176196 - 25176558
FAN-SCF131 2 89.8 ACAATCAAACGTGAGGGGCT CCAACGATGAATGTGAAGAACAGA 4 25687752 - 25688153
FAN-SCF909 2 94.5 ACCCCTAACTAAGTCATCACCG CCTTGTTGGGTCTGGGCTAA 6 21985116 - 21985169
FAN-SCF945 2 96.7 TGCTGCACATTGTTCAGTCT TGTTTATTCAGTGTTCATCAGATG 4 23040342 - 23040504
FAN-SCF595 2 98.5 TGGTGCAGGATGAGAATGAGG CACCTGGTGACGGTCATTCA 4 22850917 - 22851309
FAN-SCF345 2 102.6 AAGGGGACGGTACAGCTACT GGTGCCATCCATTGAGGCTA 4 22292764 - 22293102
FAN-SCF163 2 103.9 GGTTCGCTATGGCTAGGTCC AGCTCAAGTTGTGGGTTCCA 4 22363717 - 22364056
FAN-SCF231 2 104.5 ACTGCTTTCCTCTGTGGTTT AGAGGCATACAACACTGAGTCT 4 22008076 - 22008475
FAN-SCF294 2 106.8 GTGCCTTCAAGGAGCTGGAT CCCTGCCATAAATTGTTAAATGCA 4 22202764 - 22203163
FAN-SCF402 2 109.1 GCACGCCTCCTCAGTTACAT CGAGCTGACGTTTAAGTCCCA 4 22640567 - 22640947
FAN-SCF108 3 0.0 AGGGTGTGGAATATCATCACTCA ATGGGCCTTTGTACCGGTTT 6 21857853 - 21858252
FAN-SCF389 3 22.4 TCTAGCGCAATCTGCACCAA TCAATGAAACGTACGGCGGT 6 21995951 - 21996362
FAN-SCF166 3 26.1 TACACCGACAGGGAGGTTGA TTGATCCGAGCGAGCTTACC 6 22152785 - 22153184
FAN-SCF59 3 26.1 GGCTCTGACTTCGGCTTCTT TCTGCTGGCATTTTGTGTGC 6 22245209 - 22245570
FAN-SCF51 3 26.1 CAGGAGGGACTGGAAAGACA TGTGTTGTCATCCCACTAAGCA 6 22330843 - 22331242
FAN-SCF135 3 26.7 TCGTTGGAACCTCAGGTTGG AGGGATATTGATGATATTGTTGCA 6 21563283 - 21563682
FAN-SCF185 3 27.2 GTGCTACCGCTGAGGACAAT CTAACATCCTCCTGGGCGTG 6 21786636 - 21787035
FAN-SCF105 3 27.2 TTTCGCTTCCAAGTCACCAAC AGTTTGTCAAAATCTCACCTCC 6 21410739 - 21411138
FAN-SCF215 3 27.8 CGTCGTAGTGGATGATCCTT TGACAAATGACAACCCATGTT 6 22851692 - 22852092
FAN-SCF58 3 28.3 TGGGGTAAGTGCATTTGGTCA CCAAAGCCACTCCTCTTCGT 6 23743188 - 23743587
FAN-SCF83 3 28.3 ATGGGCTCCAGCTCTAGTCA AGAGAGAGTTCAAAAGAGGGGT 6 23220621 - 23221030
FAN-SCF115 3 28.3 GGTGGACATGAATATGAGAAAGCA GGAAAGACCCCTACAAAAACCC 6 23209827 - 23210225
FAN-SCF173 3 28.9 ACCAGACTACCACTGTGCCA AAGCAGGGTCGACAACTACC 6 23136810 - 23137047
FAN-SCF388 3 30.7 ACCCTGCCTCCTGTTCAAAC CACGGCTATACCCAAAGAGCT 6 24286158 - 24286551
FAN-SCF437 3 33.2 TTCTTGCAGGCAGACCAGTC GGTGCACTGAGGTGGTTTCT 6 22909018 - 22909416
FAN-SCF238 3 33.8 AGCTGTCTAAAAAGCTCTTCGGA TCGCAACAGAAGTGACTCCC 6 21833742 - 21834148
FAN-SCF450 3 33.8 CCGAAACATGGTCGATCCGT CCCAACCTTGCTGCTATTGC 6 22164873 - 22165272
FAN-CON1153 3 34.4 CCCATACGCGTGGCTTCAAT ACTTATCCTCTTCTTTGATGTGGG 6 21433559 - 21433813
FAN-SCF438 3 43.0 TTCCCAGATCACTCTGCTGA GGGTTGTTAAGGTATAGTACTTGG 6 13401292 - 13401547
FAN-SCF436 3 47.2 ACCAATTTATCGAAACTTCACCGA TCGATCAAAGACTTGCTATCGA 6 14629000 - 14629399
FAN-SCF246 3 48.3 TGAGGAGTAGGAGCTGTCCA TTAGCTAAGCAGGGTTGGCG 6 10601014 - 10601413
FAN-SCF165 3 50.1 GCACAAAATATTGCTGATTAGGCA TGCAGTTTCTTATATGTTGTACGG 6 14765076 - 14765398
FAN-SCF79 3 59.0 TGTTGTTTCTTCCTCCTTGGT AGTTGGTAAAAGTCTAGGTATGGA 6 11024227 - 11024401
FAN-SCF233 3 60.1 CCAACTCAAATGTGAAGTTGTGGA TCCGCCCAGACTCAAAACTT 6 9124886 - 9125097
FAN-CON1070 3 65.5 GCAAGCTTTGGCACTCATCG TCCCTCAGGAGATGGTTGGT 6 9045076 - 9045396
FAN-SCF443 4 0.0 TCACAACGCTTGTCAAAGGC ACCAGAAAGAGTATCCTCGTCT - -
FAN-SCF172 4 3.5 TTGAGGCTCATTGACAACGT TGAGGGAGAGAGATAGAGGTGC 6 3493659 - 3493907
FAN-SCF316 4 6.5 ACAGCCTCAATCAGCTGCTT TAAGTTTGGGCGATGATCCT 6 2504998 - 2505390
FAN-SCF769 4 30.7 CCGAAGAGATGAGCAACGGT GGGACTTGATCATTTGCCGG 6 9394367 - 9394762
FAN-SCF656 4 32.5 ACATATCGCTGCTGGACCAC AGCTAGCAACTTCCGACCAA 6 10822978 - 10823285
FAN-SCF119 4 34.6 CCATCATTTTACAAACGCTACACA ACGTGATCTTGCAACGGGAA 6 11132414 - 11132696
FAN-SCF569 4 35.3 TGACTTTTCGTCCATGTTGTCA GACCGAAACGTATCCATAAATAAA 6 10946444 - 10946849
FAN-SCF570 4 37.7 ACGAAAGTGGAGGTGCTCTG TGAGCGAGATGAAAACGTCG 3 5315532 - 5315808
FAN-SCF273 4 43.8 TCTCAAACGTCTTGACGCCT TGAGTGCTATTGTGGCGACT 6 18744174 - 18744235
FAN-CON1062 4 43.8 ACACCACGATATGCAACCACT ATTTCAAATGGGCCGGGTCA 6 14238062 - 14238401
FAN-SCF183 4 44.3 ACCATCGACGATGAGGAAGC CGGGGATGCTGGTCATACTT 3 31695069 - 31695130
FAN-SCF69 4 48.8 AGCGGTCCATTGACTCAACA TGTTGTAAAGCTTGTCGGGA 6 14700987 - 14701320
FAN-CON1071 5 0.0 TTGAGGTTTAAGCCGAGATGACT GTACTAACAGAGGCGGCGTT 6 27458588 - 27458625
FAN-CON1087 5 6.2 TGCAGATGTTGACTCTGCGT TTTTTACGTCCGCCGACTGA 4 32415681 - 32415732
FAN-SCF290 5 7.3 TTCACTCAAAAATTCGTGTGTT ACTTCTTACACTGACAAAACTCGA 4 32269269 - 32269656
FAN-SCF168 5 35.6 TCAACAGAGAAGATGCTTGGT ACACTCCCTTTTCCTAGCACA 6 9735998 - 9736029
FAN-CON1123 5 37.6 TCGTCACATCTACAATTGGCTCT GCGATGGTTCTAAGGTGTTCG 4 23313914 - 23314024
FAN-SCF965 5 39.3 TGTGGAGAAGTTCTAAGAATCCCG CGATCAAAGATGAAACCTAATTCC 6 1903611 - 1903648
FAN-CON1056 5 42.8 AGTGATTGGGCTGCATCAAA GCTAGGTACAGTTGCCCTGG 4 21935175 - 21935548
FAN-CON1143 5 47.0 TGGTAGCCTTCCCAATGCAG ATAAGCCAACCCCCGAGTTG 5 11569758 - 11570157
FAN-CON1128 6 0.0 TCGACATAGTGAACGGTGGC CAAATGGATTTGCGGCACCA 7 423374 - 423768
FAN-CON1053 6 1.1 GCTGCAACAAAATTGGCTTCC AGGAGACTCTAAGGTATTGATCGA 7 344631 - 344903
FAN-CON1059 6 2.3 CCTAGCTCGGCAATCAGAGG TGCAGTCAGGAGCAGTTGAG 6 30104705 - 30104918
FAN-CON1097 6 15.3 ACGTTGACGCGTAATTTGGA TGCAGTTGGTGAATGTTTGA 7 6499804 - 6500187
FAN-SCF474 6 19.3 TCGTGGGCTTAAGTTCTCAAGA CTCGCCAAGAGTATTCTCCA - -
FAN-CON1122 6 21.0 AGGACTTCTTTGATAGTACCGACA TCTTCCCAACCCACAAGCAA 7 7598690 - 7599115
FAN-SCF145 6 23.4 GCAATTTCTAGAGAGGCAAGCA TGCTTTTAAAGACAGAGAAGACA 7 5750781 - 5750840
FAN-CON1073 6 33.3 ACCCGTGAAACAATAGGGAAC AAAGCGATATACATAACATGCACC 7 11248187 - 11248367
FAN-SCF961 6 34.6 TGTGCGAATTTAATTTCCACACA GAGGAAGCATGAGCATCGGA 1 2921841 - 2921960
FAN-SCF139 6 41.1 TGCAACTCAACCAAGCCAGA AGTTCACACTTTGTAGCCACTGA 7 12594627 - 12594928
FAN-SCF730 7 0.0 ACCGGGTAACTTTGTTGGCA ACTCACCAATATCCGATGTGGT 2 10795356 - 10795437
FAN-CON1134 7 1.7 ATCTGAGGTTTTAATTCTCATGC TGGTATTGTACAAGGAAAGAGAGG 7 2111414 - 2111539
FAN-CON1145 7 2.8 TGGAAGCACTAGCCTTTGTCA GCAAAAGGAAAGAAATAACGTGGG 5 8070408 - 8070518
FAN-CON1115 7 23.5 TCATTTCCTGGCAGCTACTGA TCAGAAACCAAACTAGCCAAT 5 11182526 - 11182760
FAN-SCF756 7 32.7 GCTACTGCATTTTAGCACTTTGC TTGATAAACCAGTCTTATAACGCG 5 24691171 - 24691218
FAN-CON1066 8 0.0 CCTTTTGGGCCCATGACTTG GGTGTGATTAATCGGCCCGA 5 8945836 - 8946223
FAN-SCF378 8 0.0 CAAGAGGCAGCTTCTCTGGT GACTCCACAAAGCCTGGTCA 5 9274286 - 9274540
FAN-CON1069 8 9.7 TTGTCGTCTTAATCAAATGATTGT ACAGCTATATCCTCCACATGT 5 5735911 - 5736310
FAN-SCF698 8 12.0 GCCGAAAACCGTGGTTTGAG ACCGTGCTTGTTGTACTCGT 6 4563039 - 4563335
FAN-SCF27 8 12.5 CTGTGACTCTGTGAGGGCAG TGCAGACATGAAATGCACCA 5 5272835 - 5273234
FAN-CON1117 8 15.4 GTTTCACCATCCAAGCTGCG TCAACCCACAACCACCAACT 5 4918889 - 4919285
FAN-SCF227 8 16.0 TCACCGAGAGTAGCCTAGGA AGATGCCAGGTTACTAGTTACT 5 4858458 - 4858855
FAN-SCF63 8 25.0 GTTCTGATTGCTAGCATCTGCA TCCTGATGCGCTACTCTTGT 4 32169627 - 32169969
FAN-SCF480 8 27.3 AGGATAACAATCTGTAGTGGGGA TCACCTTAGGCCTCAGCAAA 5 2964486 - 2964724
FAN-CON1015 8 30.2 AGATTGTTGGACGCCTCTAGTC TCCTCATCAGATAAGGTGTGCA 5 1708322 - 1708548
FAN-SCF260 8 30.8 TCTCTTGAACACATTGAACCACA TCTATAATCGGGTTCTTGGGT 5 1188138 - 1188537
FAN-SCF434 9 0.0 ACGTTGGTTTCTAAGGGTGT CGATCACAAGGTCCTGAGAGA 6 35969226 - 35969306
FAN-SCF279 9 2.2 CCACCTGGGGGTTGAAAGTA CCGCTTTGCATAACTTTCGGT 2 15447700 - 15447757
FAN-SCF326 9 2.9 TGGTTTTTCGCATTTGTTGGT GGGGACGCTTGAAGAACACA 6 2118937 - 2119014
FAN-SCF846 9 7.7 TGTCAGACACAAGTTAGCTTTTGA AATCACGGTCCAACACTCGT 6 35292089 - 35292279
FAN-SCF847 9 18.4 GGAGCTTCTTTATTTTTACTCGCA TGCAAAATCCGGACAGCAAC 6 32014064 - 32014193
FAN-SCF88 9 26.8 TCATACTTGTTGCTGTAGTCACA GGGTTCTGTTTGTGGTTGGG 6 17704328 - 17704727
FAN-CON1099 9 30.6 AGGAGAAGGAATTGGATCACA AGGTTCTTTCTTGGACTGGCA 6 28740651 - 28740822
FAN-SCF369 10 0.0 TCAAGTTCACGTTTTACAGTGTCA TGCATTCTCACCTTCTCAACT 2 14150750 - 14151150
FAN-CON1089 10 15.0 GGCACAACCTGTTTGAGTTT AGTCCATTCTGCTCCAGTCT 2 3613082 - 3613418
FAN-CON1105 10 19.8 GCGATGACCCAATTTTCGGG GCCACATCTGTCACATATGCC 2 10339151 - 10339546
FAN-SCF154 10 20.4 TGCCAGCGAATGAGTAGCAG ACTGCTTATTTGCTTTGGCCA 2 10167626 - 10168044
FAN-SCF559 10 21.0 GCAAATTGAGTACAGTGACCGA CCTTGGGTTTGCCTGTAGAA 2 10088172 - 10088571
FAN-SCF1 10 29.6 GATACAGACAGGTTTGTGCGC TATGGTCTCGCAGAAGCCAC 2 10333513 - 10333912
FAN-SCF605 11 0.0 TGCCCCTCCTCTCGTTTTTC TGACTTCGAACTTGACCTCCA 6 18216050 - 18216445
FAN-SCF807 11 18.9 TGCAATAAAGCAAAGTTCAAAAAT AGTTCTTTTGCTTCGTACACT 6 28062711 - 28062898
FAN-CON1016 11 24.9 AGGGAATGGAGGTATAAGAGTT CCTTTCTGGTAGCAGCCTTACT 6 26052420 - 26052541
FAN-SCF67 12 0.0 AAGGGTGGTTTTAGGGCTGG CGGGCTTTCACCGATCTCTA 4 7444429 - 7444493
FAN-SCF203 12 1.6 ATTTGCGAACCTGTGACCAC AGAAAATGATGGCGCTTCAACA 3 8554786 - 8554880
FAN-SCF235 12 6.6 ACCCACTATGGAGTTAGCTCT TCCAACAAACACCTAGGGCA 6 3571899 - 3572282
FAN-SCF129 12 11.4 TGGCGATACAAGTTCGTAAACA AGTGAGGCTGATTTAGAGTTTCCA 5 14764214 - 14764422
FAN-SCF2 12 23.9 TCGAGAGGTGTATAAAATGCTCT TGTTTGTATGCATAACAAGCTGA 4 5849067 - 5849236
FAN-SCF548 13 0.0 CCTGGGGTGTTCAACTAGGC ATCTGCCGTGTGATGCAAGA 5 10208591 - 10208636
FAN-SCF112 13 5.1 TGTAGGAGACAAAGCGTAGTGA TGAGTAGCAGCAGACTGTAACA 6 11944125 - 11944534
FAN-SCF34 13 5.7 ATTTGAAACGCGTGGATCGAG GTCTCCAGCACCACCAGGAA 2 1358158 - 1358239
FAN-SCF84 13 7.3 CTTGAGGCCGACGATCTGTT AGATTCTGCCATCCCGCATC 3 1653607 - 1653678
FAN-SCF483 13 12.8 GGGCATTGGGATCTCAGCTT CGTCGCCTAGTGACAAGGAA 6 9565771 - 9566099
FAN-SCF760 13 15.1 CAATGACCCTCACTCACCCC ACCCTACACCATGATCCTCCA 6 9911049 - 9911186
FAN-SCF263 13 22.8 TGTTTAACGGATCATAAATGCAT GTCATTCGCGACTCGGTACA 6 12241307 - 12241731
FAN-CON1039 14 0.0 GAGATGAGGATGCAGGCCAA TGCTCACTCTATCGATGGGA 6 20401011 - 20401336
FAN-CON1142 14 6.8 AGTTCATCCGAAGCTGTGCA ACTGGATATACCGCGCATGT 6 12579743 - 12579956
FAN-CON1137 14 10.9 TCACCTGGAAATTCAGAGGA TGCCAAAGGTCGAAAATGCG 6 13012521 - 13012836
FAN-SCF289 14 22.2 ACCCTGAAATGGTTAACGTGGT GCCATATGCCTTACTCATATCT 6 20589193 - 20589212
FAN-SCF905 15 0.0 GGAGGGCTGTTCTTCAGTGG TCTTACGCAGTGCAGTCCTG 3 22549713 - 22549953
FAN-CON1076 15 1.2 GGTGCAATCCAATGAGCAGG ACTCAGGCTCGGTAGACCAT 3 22549227 - 22549626
FAN-SCF362 15 6.1 CTGGATCGCTACCTGCACTC TTAGGAGGGGTCTTCCGTGA 2 6880598 - 6880965
FAN-SCF66 15 17.8 TCCCAGCGCTTCAATCCTTT CGTTTTGGATACGTCTTGGGG 3 29379153 - 29379406
FAN-SCF367 15 20.2 CAGGAGGAAGCTCATGTGGG TGTGCATGGAACCTAAAGCTCT 3 29119775 - 29119894
FAN-SCF423 16 0.0 ATGGGTGCTGCGTGCTAGCA CGTTCTCCACGGATCCTAATCC 7 257891 - 257922
FAN-SCF197 16 9.4 CATCAAAAGAGGGGTCCGCT ACAGTAACCCATGCGATACAGT 1 21455604 - 21455985
FAN-SCF516 16 11.3 TAATCAGACCGCTCCCTCCT TATGGAGCCCATAGCCAGGT 4 28252096 - 28252225
FAN-SCF286 16 16.9 TGGACGTATGCTGGAGCCTA GGAGTACTCAGTGCAGCCTG 4 24247781 - 24248036
FAN-SCF553 16 19.9 TGGTCATTTCACTTGTGAATTGT ACCTTCAAAACAGAAGTTGACA 4 23988922 - 23989203
FAN-SCF98 17 0.0 CAGCAGGAGGTTACTCTGGC AGCAAGTTAGTAACGCACCT 1 18718021 - 18718404
FAN-SCF917 17 8.9 ACTTGGACCTACCCCTACCC GACAGGTTAGTGGAATGGCCA 1 17493100 - 17493499
FAN-CON1054 17 13.7 GGTCATCGCTCACCACTTCA CGCCATAAAATAGTAAGCGGGG 1 15672766 - 15673145
FAN-SCF382 17 18.6 TGCACATAATTGTTGTTTGGCA GGGTGTGTGTGGTACCATGT 1 12566836 - 12567235
FAN-SCF229 18 0.0 GCACAGGTTTTGGTGCATGT ACACGACACGACAACACGTA 2 27529617 - 27529951
FAN-SCF194 18 10.6 CTATGGCTGTCTCCGGCAAT TATGGCGCTTTCGAGACCAA 2 26588193 - 26588592
FAN-SCF354 18 17.0 TCCGTGCGCACATCTATATGA TAGATGGATTGGGCGCCATG 2 25907743 - 25908140
FAN-SCF872 19 0.0 TCCTGGGATTGTTAGAGTCTGT CAAAATGCACGTCAGATTATATGA 2 22312544 - 22312930
FAN-SCF89 19 3.9 TGCCCATACTAAAAGTGATCATGG TGTTCCTGGAGCTTTCATCCA 2 21320143 - 21320464
FAN-SCF5 19 7.1 ACCCAATTCAAGGACCTGCA TCACCATTACGCATTTACACGA 2 21507575 - 21507885
FAN-SCF178 19 8.8 ACTCGACAATCTGTGGTGGT TCATGCCCTTAATCGCCCTT 2 21361719 - 21362108
FAN-SCF216 19 9.9 AGCTTCTGAGTACGAAATGCA ACAACGGATCTTAAAAGAAGGCA 2 21093928 - 21094332
FAN-SCF804 19 10.5 AGCAACCCGAGAAACCTTGT TGGAGAAGCCTTTTTAATCAAGTG 2 21257000 - 21257326
FAN-SCF359 19 11.0 GTACAGGGGTAAACGCGCTA CTGGGAGACTGGGATGTTTGA 2 20767369 - 20767760
FAN-SCF385 19 12.8 ACTGTCTTCGTTTCACAACACA ATGACCTTTCGCTTGCAAGG 2 21702155 - 21702552
FAN-SCF782 19 15.1 AAGAAAAGACCCACCCCACA ATGGAATCAAAACACTGTGTTCGT 2 21709041 - 21709329
FAN-CON1144 19 16.9 AGCCATGAGTTCTCAATCACTGT CACGTAAAACCCCGAAACCC 2 21747548 - 21747947
FAN-CON984 20 0.0 AGGAATCATACATTAGTTTCTCCT TGCTTAGGGAACTGCAAATCCT 3 5608121 - 5608363
FAN-SCF55 20 0.6 AGGGTTGGCCTTGAAATCGA GACCAGTGCGAACCTCTTGA 3 5867946 - 5868345
FAN-SCF11 20 16.1 GCTTCCAGAACCCTGAAGCT AGTCAGGCTGTTGGGTAAGG 7 14002188 - 14002310
FAN-SCF625 21 0.0 ACGAAATACTGGCTCTGCTAGG ACCCCAACAATTCCAAGCCA 7 8259563 - 8259843
FAN-CON1032 21 4.2 TCGTTAAGTGTATGATTCTCTCCA GTGTGTTAAATTTATGTCGTACCA 7 6515448 - 6515725
FAN-SCF14 21 8.4 CCTCCCTAGCCTCCATCTCA CTCGATCATCACCTTGGTCCA 2 11289091 - 11289202
FAN-SCF871 22 0.0 TGTGTGACTTTCTGTATGTTCA CCAGGAACTTGTTGAGGAAAGC 3 9173551 - 9173871
FAN-SCF767 22 3.0 ACATGTTTCTGTGTCAGTTGGT CCCTTCCGCTGATCTCTGTC 3 7351198 - 7351590
FAN-SCF97 22 6.5 TCCTTTGACATGATTGCCACG AGTCAATGTCAACCTCCACTGG 3 6958967 - 6959366
FAN-CON1118 22 8.3 GCATTTGCTTTCAAGAGCGC GCGTAAGTTTGTCCTGCAGG 3 7052910 - 7053309
FAN-CON1141 23 0.0 ACGATTCGACGCCTTTCGAT ATTGGAGGAGGAGCATGCTT 3 15482868 - 15483267
FAN-CON1063 23 2.2 TCAGAAGAAATATAGCGTGGGT AAGAATCCCACTCCAACAGCT 3 22688348 - 22688574
FAN-SCF587 23 3.5 GTCTGCAAAAGCAGGCAAGT AGTCGTTGGATTTTTCTTTTAGGA 3 31471188 - 31471587
FAN-SCF915 23 3.5 CGCGCCAATTGATGAAGGAG TCGAGCCTGTCATGGTCATG 3 31438822 - 31439221
FAN-SCF793 23 5.7 CCCACCCGAGCCCTTAAAAT CGTCTGTGCCTATGTCGAGT 3 23487528 - 23487821
FAN-CON1085 23 6.8 GGACCTTGCCTTCCATGGAA GGTTCTGAAACAAGATCCCCG 3 15926347 - 15926746
FAN-SCF287 23 7.4 TGCATGTGCCGTTTTCACAG ACCAATGAAACCACGAACCT 3 16140230 - 16140639
FAN-SCF924 23 8.0 TCTGTCGGACAGTCTAAATGG TGAACCCCTTCTACTGCAAAACT 3 16134694 - 16135091
FAN-SCF78 24 0.0 GACCATTCGTCGCGGATAGT ACAGCACGTTCTGAGTTGAT 7 23293212 - 23293479
FAN-CON1025 24 6.8 TGAACCGGAAACTCTTACGT TGGACCAAATCAAAACACACA 7 21504945 - 21505040
FAN-CON1154 25 0.0 GCTGAGTCCCTCGCATGTTA ACATCACTTCAGCTGCACCA 5 16151073 - 16151358
FAN-CON1130 25 1.2 GCAGGTGGATGTCCGATTCA GATTTGACGATCGGCCAAGC 5 16155482 - 16155766
FAN-SCF593 25 2.4 CCACTGCTGGAAGCCGAAAC AGGATGGAATTTATCGCCCGA 4 7038979 - 7039083
FAN-SCF252 25 6.1 ACATGTCAAACGGTGAAAGCA TATGCGAGCGAGCTAGATGC 3 24661969 - 24662295
FAN-SCF10 26 0.0 GGGCATTGTGGGTGCAAATA CGGTGATCAAATTTGGGCCA 2 10258308 - 10258711
FAN-SCF458 26 4.2 ACCTAGTTTGAGCTATCTGGCA CGTCCCACAAGTAGCTAGCT 2 3725132 - 3725214
FAN-SCF126 27 0.0 TCAACAGTTCAACACACTGA TGTAAGCACTAGAACACTGGA 4 1265833 - 1266095
FAN-SCF164 27 0.0 TGCAGGCATTGGATCGTCAT TCATTTGCAATGAAGCATCGCT 4 1524501 - 1524913
FAN-SCF390 27 2.4 TCTAGGGTTTGAGGACGCAA GCATACAATTGGCACGTCCG 4 1770856 - 1771285
FAN-SCF383 27 3.1 CCGTCTCCCATCACTCCTCT GGCGACTCAAGAGTTACCGA 3 11683723 - 11683764
FAN-SCF520 28 0.0 TGAGGATGTGCAACGTCTCA TCGGGTTTCCAGATGTCGAC 2 19491481 - 19491897
FAN-SCF896 28 2.5 ATCTGAAGAGGCAAGCAGGG CCAACTGATCTGCTCCTCCG 2 21512966 - 21513328
FAN-SCF76 29 0.0 GGATGAGAGGAACCGAGGGA TTGAGATCTCGCACCGTTCC 7 7221386 - 7221749
FAN-SCF743 29 1.2 GCTCCAGCTTAGTCCATGCA GCCTGAAGAAACACACACCG 4 15153915 - 15154307
FAN-CON1082 30 0.0 TCCGTCCATTGTGAAGAGGC AGATCAACTTCAGAATCTGAGAA 3 196716 - 196972
FAN-SCF259 30 1.1 TGGGGTTGCACTTGAACAGT GCGTTTCTTGCTTCAACGGT 3 117480 - 117879

Statistics of raw reads, trimmed reads, and mapped reads of next generation resequencing data.

Plant material Use Raw reads Trimmed reads Mapped reads
Total length (bp) Total length (bp) Trimmed/ Raw Genome coverage The number of mapped reads (%) Mapped region of reference genome (bp) Reference genome coverage
‘Senga-Sengana’ Male 30,682,186,222 21,846,702,806 71.2% ≒ 193,915,797 530,090,126 76.0%
parent 30.34X (75.9%)
‘Sulhyang’ Female 25,256,529,246 19,080,792,008 75.5% ≒ 169,416,290 533,936,123 76.5%
parent 26.50X (77.0%)

Summary of single nucleotide polymorphism (SNP) detection and high resolution melting (HRM) primer design between strawberry cultivars ‘Sulhyang’ and ‘Senga-sengana.’

SNP type The number of polymorphic SNP The number of non-paralogous SNP The number of HRM candidates The number of HRM primer sets designed
‘Senga-sengana’ ‘Sulhyang’ Total Intergenic region Genic region
CDSz Intron Exon
Homozygous Homozygous 206,019 8,128 6,750 2,324 1,212 504 416 192
Homozygous Heterozygous 53,933 2,896 2,347 652 341 134 112 65
Heterozygous Homozygous 74,367 4,220 3,460 1,338 676 298 247 117
Heterozygous Heterozygous 87,161 5,604 4,460 1,154 443 458 181 72
Total 421,480 20,848 17,017 5,468 2,672 1,394 956 446

zCDS indicates a coding sequence region except intron and exon regions.

Table 1 List of HRM primer sets used in this study.
Table 2 Statistics of raw reads, trimmed reads, and mapped reads of next generation resequencing data.
Table 3 Summary of single nucleotide polymorphism (SNP) detection and high resolution melting (HRM) primer design between strawberry cultivars ‘Sulhyang’ and ‘Senga-sengana.’

CDS indicates a coding sequence region except intron and exon regions.

Table 1

Table 2

Table 3

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