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Retrotranspozon temelli moleküler belirteçler kullanılarak Türk arpa (Hordeum vulgare L.) çeşitlerinin genomik karakterizasyonu

Year 2020, Volume: 33 Issue: 2, 275 - 283, 01.08.2020
https://doi.org/10.29136/mediterranean.690224

Abstract

Kökeni Doğu Akdeniz ülkelerine dayanan, hayvan yemi ve maltlık olarak tüketilen arpanın, dünyanın birçok bölgesinde tarımı ve ıslahı yapılmaktadır. Arpa çeşitlerinde genetik çeşitliliğin belirlenmesi, çeşitler arasındaki genetik ilişkilerin ortaya çıkarılması ve ıslahçı haklarının korunması için moleküler yöntemlerden faydalanılmaktadır. Bu çalışmada, arpa genomunun önemli bileşenlerinden olan BARE-1 retrotranspozonu ile ilişkili IRAP (Retrotranspozon-arası Çoğaltılmış Polimorfizm), REMAP (Retrotranspozon-Mikrosatellit Çoğaltılmış Polimorfizmi) ve iPBS (Primer Arası Bağlanma Yeri Polimorfizmi) olarak adlandırılan moleküler belirteçler kullanılarak ulusal birçok çeşidin karakterizasyonu yapılmıştır. Toplamda 211 alel olmak üzere, 3 IRAP primer çifti 49.5 REMAP primer çifti 55 ve 7 iPBS primer çifti için 107 alel tespit edilmiştir. Lokus başına ortalama 14 alel belirlenmiştir. Polimorfik Bilgi İçeriği (PIC) ortalama değeri REMAP için 0.407, IRAP için 0.454 ve IPBS için de 0.442 bulunmuştur. Genetik benzerlik değerleri 0.41 ila 0.93 arasında değişmiştir. Tüm belirteçler için ortalama fark yöntemi (UPGMA) kümeleme analizi yapılarak, çeşitler genetik benzerliklerine göre gruplandırılmışlardır. Bu çalışmada, fazla sayıda alel ve yüksek PIC değeri vermeleri, ucuz ve kolay elde edinimleri nedeniyle, transposon temelli moleküler belirteçler ile çok yakın ilişkili çeşitlerin dahi ayrımının kolay bir şekilde yapılabileceği görülmüştür. Transposon esaslı belirteçler, çeşitler arasında genetik ilişkileri belirlemenin yanı sıra genetik kaynakların korunmasında ve tohum bankalarının yönetiminde faydalı olabilecektir.

Supporting Institution

Sinop Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi

Project Number

FEF-1901-18-20

Thanks

Bu çalışma Sinop Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimince desteklenen FEF-1901-18-20 no’lu proje kapsamında gerçekleştirilmiştir.

References

  • Arystanbekkyzy M, Nadeem MA, Aktaş H, Yeken MZ, Zencirci N, Nawaz MA, Ali F, Haider MS, Tunc K, Chung G, Baloch FS (2018) Phylogenetic and taxonomic relationship of Turkish wild and cultivated emmer (Triticum turgidum ssp. dicoccoides) revealed by iPBS-retrotransposons markers. International Journal of Agriculture and Biology doi: 10.17957/IJAB/15.0876.
  • Baumel A, Ainouche M, Kalendar R, Schulman AH (2002) Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard (Poaceae). Molecular Biology Evolution 19: 1218-1227.
  • Bayat M, Amirnia R, Özkan H, Gedik A, Ates D, Tanyolac BM, Rahimi M (2018) Diversity and phylogeny of saffron (Crocus sativus L.) accessions based on IPBS markers. Genetika 50: 33-44.
  • Boyko E, Kalendar R, Korzun V (2002) A high density cytogenetic map of the Aegilops tauschii genome incorporating retrotransposons and defense related genes: insight into cereal chromosome structure and function. Plant Molecular Biology 48: 767-790.
  • Branco CJ, Vieira EA, Malone G, Kopp MM, Malone E, Bernardes A, Mistura CC, Carvalho FIF, Oliveira CA (2007) IRAP and REMAP assessments of genetic similarity in rice. Journal of Applied Genetics 48: 107-113.
  • Campbell B, LeMare S, Piperidis G, Godwin I (2011) IRAP, a retrotransposon-based marker system for the detection of somaclonal variation in barley. Molecular Breeding 27: 193-206.
  • Capy P, Gasperi G, Biemont C, Bazin C (2000) Stress and transposable elements: co-evolution or useful parasites?. Heredity 85: 101-106.
  • Chadha S, Gopalakrishna T (2005) Retrotransposon-microsatellite amplified polymorphism (REMAP) markers for genetic diversityassessment of the rice (Oryza sativa) blast pathogen (Magnaporthe grisea). Genome 48: 943-945.
  • Cömertpay G, Baloch FFS, Derya M, Andeden EE, Alsaleh A, Sürek H, Özkan H (2016) Population structure of rice varieties used in Turkish rice breeding programs determined using simple-sequence repeat and inter-primer binding site-retrotransposon data. Genetics and Molecular Research 15(1) doi: 10.4238/gmr.15017158.
  • Demirel U, Tındas İ, Yavuz C, Baloch FS, Çalıskan ME (2017) Assessing genetic diversity of potato genotypes using inter-PBS retrotransposon marker system. Plant Genetic Resources 1-9.
  • Gozukirmizi N, Yilmaz S, Marakli S, Temel A (2015) Retrotransposon-based molecular markers; Tools for variation analysis in plants Applications of Molecular Markers in Plant Genome Analysis and Breeding, Taski-Ajdukovic K. ed., Research Signpost, Kerala, 19-45.
  • Henry RJ (2013) Evolution of DNA marker technology in plants. In: Henry RJ (ed) Molecular markers in plants. Wiley-Blackwell Pub, Ames, pp. 3-20.
  • Jiang GL (2013) Moleular markers and marker-assisted breeding in plants. In: Andersen SB (ed) Agricultural and Biological Science. Plant Breeding from Laboratories to Fields doi: 10.5772/52583.
  • Kalendar R, Grob T, Regina M, Suoniemi A, Schulman AH (1999) IRAP and REMAP: Two new retrotransposon-based DNA fingerprinting techniques. Theoretical and Applied Genetics 98: 704-711.
  • Kalendar R, Schulman AH (2006) IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nature Protocols 1: 2478-2484.
  • Kalendar R, Antonius K, Smýkal P, Schulman AH (2010) iPBS: A universal method for DNA fingerprinting and retrotransposon isolation. Theoretical Applied Genetics 121: 1419-1430.
  • Mandoulakani BA, Piri Y, Darvishzadeh R, Bernoosi I, Jafari M (2011) Retroelement insertional polymorphism and genetic diversity in Medicago sativa populations revealed by IRAP and REMAP markers. Plant Molecular Biology Reporter 30: 286-296.
  • Manninen O, Kalendar R, Robinson J, Schulman AH (2000) Application of BARE-1 retrotransposon markers to the mapping of a major resistance gene for net blotch in barley. Molecular Genetics and Genomics 264: 324-334.
  • Mansour A, Jaime A, da Silva T, Edris S, Younis RAA (2010) Comparative assessment of genetic diversity in some tomato cultivars using IRAP, ISSR and RAPD molecular markers. Genes, Genomes and Genomics 4: 41-47.
  • Miller WJ, Capy P (2004) Mobile genetic elements as natural tools for genome evolution. Methods in Molecular Biology 260: 1-20.
  • Nair AS, Teo CH, Schwarzacher T, Heslop-Harrison P (2005) Genome classification of banana cultivars from South India using IRAP markers. Euphytica 144: 285-29.
  • Nemli S, Kıanoosh T, Tanyolaç MB (2015) Genetic diversity and population structure of common bean (Phaseolus vulgaris L.) accessions through retrotransposon-based interprimer binding sites (iPBSs) markers Turkish Journal of Agriculture and Forestry 39: 940-948.
  • Poczai P, Varga I, Laos M, Cseh A, Bell N, Valkonen JPT, Hyvonen J (2013) Advances in plant gene-targeted and functional markers: A review. Plant Methods 19: 6.
  • Rohlf FJ (1997) NTSYS-PC Numerical Taxonomy and Multivariate Analysis System, version 2.02i, Applied Biostatics Inc., Exeter Software, Setauket, New York.
  • Roy NS, Choi JY, Lee NSK (2015) Marker utility of transposable elements for plant genetics, breeding, and ecology: A review. Genes and Genomes 37: 141-151.
  • Sanz AM, Gonzalez SG, Syed NH, Suso MJ, Saldaña CC, Flavell AJ (2007) Genetic diversity analysis in Vicia species using retrotransposon-based SSAP markers. Molecular Genetics and Genomics 278: 433-441.
  • Singh S, Nandha S, Singh J (2017) Transposon-based genetic diversity assessment in wild and cultivated barley. The Crop Journals 4: 296-304.
  • Sipahi H, Akar T, Yildiz MA, Sayim I (2010) Determination of genetic variation and relationship in Turkish barley cultivars by hordein and RAPD markers. Turkish Journal of Field Crops 2: 108-113.
  • Sipahi H (2011) Genetic screening of Turkish barley genotypes using simple sequence repeat markers. Journal of Cell and Molecular Biology 2: 19-26.
  • Sneath PHA, Sokal RR (1973) Numerical taxonomy: The principles and practice of numerical classification. W. H. Freeman and Co., San Francisco.
  • Song W, Henry RJ (1995) Molecular analysis of the DNA polymorphism of wild barley (Hordeum spontaneum) germplasm using the polymerase chain reaction. Genetic Resources and Crop Evolution 42: 273.
  • Suoniemi A, Schmidt D, Schulman AH (1997) BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNAprocessing sites. Genetica 100: 219-230.
  • Tanhuanaa P, Kalendar R, Schulman A, Kiviharju E (2007) A major gene for grain cadmium accumulation in oat. Genome 50: 588-594.
  • Tatout C, Warwick S, Lenoir A, Deragon JM (1999) Sine insertions as clade markers for wild Crucifer species. Molecular Biology and Evolution 16: 1614-1621.
  • Teo CH, Tan SH, Ho CL, Faridah QZ, Othman YR, Heslop-Harrison JS, Kalendar R, Schulman AH (2005) Genome constitution and classification using retrotransposon-based markers in the orphan crop banana. Journal of Plant Biology 48: 96-105.
  • Vicient C, Jaaskelainen M, Kalendar R, Schulman A (2001) Active retrotransposons are a common feature of grass genome. Plant Physiology 125: 1283-1292.
  • Weber JL (1990) Informativeness of human (cC-dA)n(dG-dT)n polymorphisms. Genomics 7: 524-530.
  • Yaldız G, Çamlıca M, Nadeem MA, Nawaz MA, Baloch FS (2018) Genetic diversity assessment in Nicotiana tabacum L. with iPBS-retrotransposons. Turkish Journal of Agriculture and Forestry 42: 154-164.
  • Yıldız M, Koçak M Baloch FS (2015) Genetic bottlenecks in Turkish okra germplasm and utility of iPBS retrotransposon markers for genetic diversity assessment. Genetics and Molecular Research 14: 10588-10602.

Genomic characterization of Turkish barley (Hordeum vulgare L.) cultivars using retrotransposons-based molecular markers

Year 2020, Volume: 33 Issue: 2, 275 - 283, 01.08.2020
https://doi.org/10.29136/mediterranean.690224

Abstract

Barley, which is originated from Eastern Mediterranean countries and is consumed as animal feed and malt, is cultivated and breeding in many regions of the world. Molecular methods are benefited to determine the genetic diversity in barley cultivars, to define the genetic relationship between cultivars and to protect the breeder rights. In this study, characterization of many national cultivars was carried out using molecular markers called as IRAP (Inter retrotransposon amplified polymorphism), REMAP (Retrotransposon microsatellite polymorphism) and iPBS (inter-priming binding site) associated with BARE-1 retrotransposons that is one of the important components of barley genome. A total of 211 alleles, 49 for 3 IRAP, 55 for 5 REMAP, and 107 for 7 IPBS primer pairs were detected. An average of 14 alleles per locus was determined. Polymorphic Information Content (PIC) mean value was 0.407 for REMAP and 0.454 for IRAP and 0.442 for IPBS. Genetic similarity values ranged from 0.41 to 0.93. Cultivars were divided into groups according to their genetic similarities by performing the unweighted pair group method with arithmetic mean (UPGMA) clustering analysis for all markers. In this study, it was observed that, due to their high number of alleles and high PIC values, and their cheap and easy acquisition, it can be easily distinguished even very closely related cultivars by using transposon-based molecular markers. Transposon based markers can be useful for conservating the genetic resources and managing the seed banks beside as determining the genetic relationship between cultivars.

Project Number

FEF-1901-18-20

References

  • Arystanbekkyzy M, Nadeem MA, Aktaş H, Yeken MZ, Zencirci N, Nawaz MA, Ali F, Haider MS, Tunc K, Chung G, Baloch FS (2018) Phylogenetic and taxonomic relationship of Turkish wild and cultivated emmer (Triticum turgidum ssp. dicoccoides) revealed by iPBS-retrotransposons markers. International Journal of Agriculture and Biology doi: 10.17957/IJAB/15.0876.
  • Baumel A, Ainouche M, Kalendar R, Schulman AH (2002) Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard (Poaceae). Molecular Biology Evolution 19: 1218-1227.
  • Bayat M, Amirnia R, Özkan H, Gedik A, Ates D, Tanyolac BM, Rahimi M (2018) Diversity and phylogeny of saffron (Crocus sativus L.) accessions based on IPBS markers. Genetika 50: 33-44.
  • Boyko E, Kalendar R, Korzun V (2002) A high density cytogenetic map of the Aegilops tauschii genome incorporating retrotransposons and defense related genes: insight into cereal chromosome structure and function. Plant Molecular Biology 48: 767-790.
  • Branco CJ, Vieira EA, Malone G, Kopp MM, Malone E, Bernardes A, Mistura CC, Carvalho FIF, Oliveira CA (2007) IRAP and REMAP assessments of genetic similarity in rice. Journal of Applied Genetics 48: 107-113.
  • Campbell B, LeMare S, Piperidis G, Godwin I (2011) IRAP, a retrotransposon-based marker system for the detection of somaclonal variation in barley. Molecular Breeding 27: 193-206.
  • Capy P, Gasperi G, Biemont C, Bazin C (2000) Stress and transposable elements: co-evolution or useful parasites?. Heredity 85: 101-106.
  • Chadha S, Gopalakrishna T (2005) Retrotransposon-microsatellite amplified polymorphism (REMAP) markers for genetic diversityassessment of the rice (Oryza sativa) blast pathogen (Magnaporthe grisea). Genome 48: 943-945.
  • Cömertpay G, Baloch FFS, Derya M, Andeden EE, Alsaleh A, Sürek H, Özkan H (2016) Population structure of rice varieties used in Turkish rice breeding programs determined using simple-sequence repeat and inter-primer binding site-retrotransposon data. Genetics and Molecular Research 15(1) doi: 10.4238/gmr.15017158.
  • Demirel U, Tındas İ, Yavuz C, Baloch FS, Çalıskan ME (2017) Assessing genetic diversity of potato genotypes using inter-PBS retrotransposon marker system. Plant Genetic Resources 1-9.
  • Gozukirmizi N, Yilmaz S, Marakli S, Temel A (2015) Retrotransposon-based molecular markers; Tools for variation analysis in plants Applications of Molecular Markers in Plant Genome Analysis and Breeding, Taski-Ajdukovic K. ed., Research Signpost, Kerala, 19-45.
  • Henry RJ (2013) Evolution of DNA marker technology in plants. In: Henry RJ (ed) Molecular markers in plants. Wiley-Blackwell Pub, Ames, pp. 3-20.
  • Jiang GL (2013) Moleular markers and marker-assisted breeding in plants. In: Andersen SB (ed) Agricultural and Biological Science. Plant Breeding from Laboratories to Fields doi: 10.5772/52583.
  • Kalendar R, Grob T, Regina M, Suoniemi A, Schulman AH (1999) IRAP and REMAP: Two new retrotransposon-based DNA fingerprinting techniques. Theoretical and Applied Genetics 98: 704-711.
  • Kalendar R, Schulman AH (2006) IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nature Protocols 1: 2478-2484.
  • Kalendar R, Antonius K, Smýkal P, Schulman AH (2010) iPBS: A universal method for DNA fingerprinting and retrotransposon isolation. Theoretical Applied Genetics 121: 1419-1430.
  • Mandoulakani BA, Piri Y, Darvishzadeh R, Bernoosi I, Jafari M (2011) Retroelement insertional polymorphism and genetic diversity in Medicago sativa populations revealed by IRAP and REMAP markers. Plant Molecular Biology Reporter 30: 286-296.
  • Manninen O, Kalendar R, Robinson J, Schulman AH (2000) Application of BARE-1 retrotransposon markers to the mapping of a major resistance gene for net blotch in barley. Molecular Genetics and Genomics 264: 324-334.
  • Mansour A, Jaime A, da Silva T, Edris S, Younis RAA (2010) Comparative assessment of genetic diversity in some tomato cultivars using IRAP, ISSR and RAPD molecular markers. Genes, Genomes and Genomics 4: 41-47.
  • Miller WJ, Capy P (2004) Mobile genetic elements as natural tools for genome evolution. Methods in Molecular Biology 260: 1-20.
  • Nair AS, Teo CH, Schwarzacher T, Heslop-Harrison P (2005) Genome classification of banana cultivars from South India using IRAP markers. Euphytica 144: 285-29.
  • Nemli S, Kıanoosh T, Tanyolaç MB (2015) Genetic diversity and population structure of common bean (Phaseolus vulgaris L.) accessions through retrotransposon-based interprimer binding sites (iPBSs) markers Turkish Journal of Agriculture and Forestry 39: 940-948.
  • Poczai P, Varga I, Laos M, Cseh A, Bell N, Valkonen JPT, Hyvonen J (2013) Advances in plant gene-targeted and functional markers: A review. Plant Methods 19: 6.
  • Rohlf FJ (1997) NTSYS-PC Numerical Taxonomy and Multivariate Analysis System, version 2.02i, Applied Biostatics Inc., Exeter Software, Setauket, New York.
  • Roy NS, Choi JY, Lee NSK (2015) Marker utility of transposable elements for plant genetics, breeding, and ecology: A review. Genes and Genomes 37: 141-151.
  • Sanz AM, Gonzalez SG, Syed NH, Suso MJ, Saldaña CC, Flavell AJ (2007) Genetic diversity analysis in Vicia species using retrotransposon-based SSAP markers. Molecular Genetics and Genomics 278: 433-441.
  • Singh S, Nandha S, Singh J (2017) Transposon-based genetic diversity assessment in wild and cultivated barley. The Crop Journals 4: 296-304.
  • Sipahi H, Akar T, Yildiz MA, Sayim I (2010) Determination of genetic variation and relationship in Turkish barley cultivars by hordein and RAPD markers. Turkish Journal of Field Crops 2: 108-113.
  • Sipahi H (2011) Genetic screening of Turkish barley genotypes using simple sequence repeat markers. Journal of Cell and Molecular Biology 2: 19-26.
  • Sneath PHA, Sokal RR (1973) Numerical taxonomy: The principles and practice of numerical classification. W. H. Freeman and Co., San Francisco.
  • Song W, Henry RJ (1995) Molecular analysis of the DNA polymorphism of wild barley (Hordeum spontaneum) germplasm using the polymerase chain reaction. Genetic Resources and Crop Evolution 42: 273.
  • Suoniemi A, Schmidt D, Schulman AH (1997) BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNAprocessing sites. Genetica 100: 219-230.
  • Tanhuanaa P, Kalendar R, Schulman A, Kiviharju E (2007) A major gene for grain cadmium accumulation in oat. Genome 50: 588-594.
  • Tatout C, Warwick S, Lenoir A, Deragon JM (1999) Sine insertions as clade markers for wild Crucifer species. Molecular Biology and Evolution 16: 1614-1621.
  • Teo CH, Tan SH, Ho CL, Faridah QZ, Othman YR, Heslop-Harrison JS, Kalendar R, Schulman AH (2005) Genome constitution and classification using retrotransposon-based markers in the orphan crop banana. Journal of Plant Biology 48: 96-105.
  • Vicient C, Jaaskelainen M, Kalendar R, Schulman A (2001) Active retrotransposons are a common feature of grass genome. Plant Physiology 125: 1283-1292.
  • Weber JL (1990) Informativeness of human (cC-dA)n(dG-dT)n polymorphisms. Genomics 7: 524-530.
  • Yaldız G, Çamlıca M, Nadeem MA, Nawaz MA, Baloch FS (2018) Genetic diversity assessment in Nicotiana tabacum L. with iPBS-retrotransposons. Turkish Journal of Agriculture and Forestry 42: 154-164.
  • Yıldız M, Koçak M Baloch FS (2015) Genetic bottlenecks in Turkish okra germplasm and utility of iPBS retrotransposon markers for genetic diversity assessment. Genetics and Molecular Research 14: 10588-10602.
There are 39 citations in total.

Details

Primary Language Turkish
Subjects Agricultural Engineering
Journal Section Makaleler
Authors

Hülya Sipahi 0000-0002-7925-2766

Ayşen Yumurtacı 0000-0002-9104-7108

Project Number FEF-1901-18-20
Publication Date August 1, 2020
Submission Date February 17, 2020
Published in Issue Year 2020 Volume: 33 Issue: 2

Cite

APA Sipahi, H., & Yumurtacı, A. (2020). Retrotranspozon temelli moleküler belirteçler kullanılarak Türk arpa (Hordeum vulgare L.) çeşitlerinin genomik karakterizasyonu. Mediterranean Agricultural Sciences, 33(2), 275-283. https://doi.org/10.29136/mediterranean.690224
AMA Sipahi H, Yumurtacı A. Retrotranspozon temelli moleküler belirteçler kullanılarak Türk arpa (Hordeum vulgare L.) çeşitlerinin genomik karakterizasyonu. Mediterranean Agricultural Sciences. August 2020;33(2):275-283. doi:10.29136/mediterranean.690224
Chicago Sipahi, Hülya, and Ayşen Yumurtacı. “Retrotranspozon Temelli moleküler belirteçler kullanılarak Türk Arpa (Hordeum Vulgare L.) çeşitlerinin Genomik Karakterizasyonu”. Mediterranean Agricultural Sciences 33, no. 2 (August 2020): 275-83. https://doi.org/10.29136/mediterranean.690224.
EndNote Sipahi H, Yumurtacı A (August 1, 2020) Retrotranspozon temelli moleküler belirteçler kullanılarak Türk arpa (Hordeum vulgare L.) çeşitlerinin genomik karakterizasyonu. Mediterranean Agricultural Sciences 33 2 275–283.
IEEE H. Sipahi and A. Yumurtacı, “Retrotranspozon temelli moleküler belirteçler kullanılarak Türk arpa (Hordeum vulgare L.) çeşitlerinin genomik karakterizasyonu”, Mediterranean Agricultural Sciences, vol. 33, no. 2, pp. 275–283, 2020, doi: 10.29136/mediterranean.690224.
ISNAD Sipahi, Hülya - Yumurtacı, Ayşen. “Retrotranspozon Temelli moleküler belirteçler kullanılarak Türk Arpa (Hordeum Vulgare L.) çeşitlerinin Genomik Karakterizasyonu”. Mediterranean Agricultural Sciences 33/2 (August 2020), 275-283. https://doi.org/10.29136/mediterranean.690224.
JAMA Sipahi H, Yumurtacı A. Retrotranspozon temelli moleküler belirteçler kullanılarak Türk arpa (Hordeum vulgare L.) çeşitlerinin genomik karakterizasyonu. Mediterranean Agricultural Sciences. 2020;33:275–283.
MLA Sipahi, Hülya and Ayşen Yumurtacı. “Retrotranspozon Temelli moleküler belirteçler kullanılarak Türk Arpa (Hordeum Vulgare L.) çeşitlerinin Genomik Karakterizasyonu”. Mediterranean Agricultural Sciences, vol. 33, no. 2, 2020, pp. 275-83, doi:10.29136/mediterranean.690224.
Vancouver Sipahi H, Yumurtacı A. Retrotranspozon temelli moleküler belirteçler kullanılarak Türk arpa (Hordeum vulgare L.) çeşitlerinin genomik karakterizasyonu. Mediterranean Agricultural Sciences. 2020;33(2):275-83.

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