izvestiiatimacadИзвестия Тимирязевской сельскохозяйственной академииIZVESTIYA OF TIMIRYAZEV AGRICULTURAL ACADEMY0021-342XФГБОУ ВО РГАУ-МСХА имени К.А. Тимирязева10.26897/0021-342X-2022-6-79-93izvestiiatimacad-349Research ArticleГЕНЕТИКА, БИОТЕХНОЛОГИЯ, СЕЛЕКЦИЯ И СЕМЕНОВОДСТВОGENETICS, BIOTECHNOLOGY, BREEDING AND SEED PRODUCTIONКонкурентная аллель-специфичная ПЦР (KASP): особенности, интерпретация результатовCompetitive allele specifi c PCR (KASP): features, the interpretation of the resultsНикитинаЕ. А.NikitinaE. A.

Никитина Екатерина Александровна, младший научный сотрудник

127550, г. Москва, ул. Тимирязевская, 42 

Ekaterina A. Nikitina, Junior Research Associate

42 Timiryazevskaya Str., Moscow, 127434

shhket@gmail.comАрхиповА. В.ArkhipovA. A.

Архипов Андрей Владимирович, младший научный сотрудник

 127550, г. Москва, ул. Тимирязевская, 42

Andrey V. Arkhipov, Junior Research Associate

42 Timiryazevskaya Str., Moscow, 127434

batler51@yandex.ruМиньковаЯ. В.Min’kovaYa. V.

Минькова Яна Вадимовна, младший научный сотрудник

127550, г. Москва, ул. Тимирязевская, 42

Yana V. Min’kova, Junior Research Associate

42 Timiryazevskaya Str., Moscow, 127434

flleurs@mail.ruЯновскийА. С.YanovskiyA. S.

Яновский Алексей Сергеевич, канд. с.-х. наук, в.н.с., Отдел селекции и семеноводства пшеницы и тритикале

350012,  г. Краснодар, Центральная усадьба КНИИСХ

Aleksey S. Yanovskiy, PhD (Ag), Senior Research Associate

Krasnodar, Tsentral’naya usad’ba KNIISKh,  350012

yanovskij81@list.ruКоробковаВ. А.KorobkovaV. A.

Коробкова Варвара Александровна, младший научный сотрудник

 127550,  г. Москва, ул. Тимирязевская, 42

Varvara A. Korobkova, Junior Research Associate

42 Timiryazevskaya Str., Moscow, 127434

bowlingistka@gmail.comСамаринаМ. А.SamarinaM. A.

Самарина Мария Алексеевна, младший научный сотрудник

 127550,  г. Москва, ул. Тимирязевская, 42 

Mariya A. Samarina, Junior Research Associate

42 Timiryazevskaya Str., Moscow, 127434

Samarina.homa@yandex.ruЧерноокА. Г.ChernookA. G.

Черноок Анастасия Геннадьевна, младший научный сотрудник

 127550,  г. Москва, ул. Тимирязевская, 42

Anastasiya G. Chernook, Junior Research Associate

42 Timiryazevskaya Str., Moscow, 127434

irbis-sibri@yandex.ruКрупинП. Ю.KrupinP. Yu.

Крупин Павел Юрьевич, канд. биол. наук, в.н.с.

127550, г. Москва, ул. Тимирязевская, 42

Pavel Yu. Krupin, PhD (Bio), Senior Research Associate

42 Timiryazevskaya Str., Moscow, 127434

pavelkroupin1985@gmail.comКарловГ. И.KarlovG. I.

Карлов Геннадий Ильич, д-р биол. наук, академик РАН, директор

127550, г. Москва, ул. Тимирязевская, 42

Gennadiy I. Karlov, DSc (Bio), RAS Academician (Full Member), Director

42 Timiryazevskaya Str., Moscow, 127434

karlov@iab.ac.ruДивашукМ. Г.DivashukM. G.

Дивашук Михаил Георгиевич, канд. биол. наук, в.н.с. 

127550, г. Москва, ул. Тимирязевская, 42; e-mail: 

Mikhail G. Divashuk, PhD (Bio), Senior Research Associate

42 Timiryazevskaya Str., Moscow, 127434

divashuk@gmail.comФГБНУ «Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии»РоссияAll-Russian Research Institute of Agricultural BiotechnologyRussian FederationФГБНУ «Национальный центр зерна им. П.П. Лукьяненко»РоссияNational Center of Grain Named after P.P. LukyanenkoRussian FederationФГБНУ «Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии»; Российский государственный аграрный университет – МСХА имени К.А. ТимирязеваРоссияAll-Russian Research Institute of Agricultural BiotechnologyRussian Federation202231032023167993Copyright © Никитина Е.А., Архипов А.В., Минькова Я.В., Яновский А.С., Коробкова В.А., Самарина М.А., Черноок А.Г., Крупин П.Ю., Карлов Г.И., Дивашук М.Г., 20232023Никитина Е.А., Архипов А.В., Минькова Я.В., Яновский А.С., Коробкова В.А., Самарина М.А., Черноок А.Г., Крупин П.Ю., Карлов Г.И., Дивашук М.Г.Nikitina E.A., Arkhipov A.A., Min’kova Y.V., Yanovskiy A.S., Korobkova V.A., Samarina M.A., Chernook A.G., Krupin P.Y., Karlov G.I., Divashuk M.G.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://izvestiia.timacad.ru/jour/article/view/349

Использование конкурентной аллель-специфичной ПЦР становится популярным методом для массового генотипирования сельскохозяйственных культур. Разработка маркеров на SNP в полиплоидных организмах требует дополнительной проверки и тестирования при разработке праймеров для дальнейшего использования. Предлагается алгоритм проверки и интерпретации работоспособности KASP-маркеров на пшенице с помощью существующих геномных сборок мягкой пшеницы. С помощью описанного алгоритма было проанализировано 6 KASP-маркеров на гены VIVIPAROUS-1, MOTHER OF FT AND TFL1, TGW6-A1, PSY-A1, Dreb-B1 и локус устойчивости к листовой ржавчине Lr14a. Наиболее перспективными KASP-маркерами по результатам биоинформатического анализа и ПЦР оказались маркеры на гены VIVIPAROUS-1, MOTHER OF FT AND TFL1.

The use of competitive allele-specific PCR is becoming a popular method for mass crop genotyping. The development of markers for SNPs in polyploid organisms requires additional validation and testing when developing primers for further use. In this work, we propose an algorithm for testing and interpreting the performance of KASP markers on wheat using existing common wheat genomic assemblies. Using the described algorithm, six KASP-markers on VIVIPAROUS-1, MOTHER OF FT AND TFL1, TGW6-A1, PSY-A1, Dreb-B1 and leaf rust resistance locus Lr14a were analyzed. The most promising KASP markers by bioinformatic analysis and PCR were the markers for VIVIPAROUS-1, MOTHER OF FT AND TFL1 genes.

KASPтвердая пшеницамягкая пшеницавыравнивание последовательностейконкурентная аллель-специфичная ПЦРмолекулярный маркерKASPdurum wheatcommon wheatsequence alignmentcompetitive allelespecific PCRmolecular markerReferencesAyalew H., Tsang P.W., Chu C., Wang J., Liu S., Chen C., Ma X.F. Comparison of TaqMan, KASP and rhAmp SNP genotyping platforms in hexaploid wheat // PLOS ONE. – 2019. – Vol. 14. – № 5. – P. e0217222.Ayalew H., Tsang P.W., Chu C., Wang J., Liu S., Chen C., Ma X.F. Comparison of TaqMan, KASP and rhAmp SNP genotyping platforms in hexaploid wheat. PLOS ONE. 2019; 14 (5): e0217222.Brinton J., Ramirez-Gonzalez R.H., Simmonds J., Wingen L., Orford S., Griffiths S. & 10 Wheat Genome Project, Haberer G., Spannagl M., Walkowiak S., Pozniak C. & Uauy C. A haplotype-led approach to increase the precision of wheat breeding // Communications Biology. – 2020. – Vol. 3. – № 1. – P. 712.Brinton J., Ramirez-Gonzalez R.H., Simmonds J., Wingen L., Orford S., S. Griffiths, 10 Wheat Genome Project, Haberer G., Spannagl M., Walkowiak S., Pozniak C., Uauy C. A haplotype-led approach to increase the precision of wheat breeding. Communications Biology. 2020; 3 (1): 712.Darrier B., Colas I., Rimbert H., Choulet F., Bazile J., Sortais A., Jenczewski E., Sourdille P. Location and Identification on Chromosome 3B of Bread Wheat of Genes Affecting Chiasma Number // Plants. – 2022. – Vol. 11. – № 17. – P. 2281.Darrier B., Colas I., Rimbert H., Choulet F., Bazile J., Sortais A., Jenczewski E., Sourdille P. Location and Identification on Chromosome 3B of Bread Wheat of Genes Affecting Chiasma Number. Plants. 2022; 11 (17): 2281.Duan X., Yu H., Ma J., Sun W., Zhao Y., Yang R., Ning T., Li Q., Q. Liu, Guo T., Yan M., Tian J., Chen J. A major and stable QTL controlling wheat thousand grain weight: identification, characterization, and CAPS marker development // Molecular Breeding. – 2020. – Vol. 40. – № 7. – P. 68.Duan X., Yu H., W. Sun Ma J., Zhao Y., Yang R., Ning T., Li Q., Liu Q., Guo T., Yan M., Tian J., Chen J. A major and stable QTL controlling wheat thousand grain weight: identification, characterization, and CAPS marker development. Molecular Breeding. 2020; 40 (7): 68.Food and Agroculture Organization of the United Nations Selected indicators in Russion Federation // Сайт FAOSTAT. – 2020. – URL: https://www.fao.org/faostat/en/#country/185.Food and Agriculture Organization of the United Nations. Selected indicators in Russion Federation, 2020. [Electronic source]. URL: https://www.fao.org/faostat/en/#country/185Hanig M., Gao F., Liu J., Wen W., Zhang Y., Rasheed A., Xia X., He Z. & Cao S. TaTGW6-A1, an ortholog of rice TGW6, is associated with grain weight and yield in bread wheat // Molecular Breeding. – 2016. – Vol. 36. – № 1. – P. 1.Hanig M., Gao F., Liu J., Wen W., Zhang Y., A Rasheed., Xia X., He Z., Cao S. TaTGW6-A1, an ortholog of rice TGW6, is associated with grain weight and yield in bread wheat. Molecular Breeding. 2016; 36 (1): 1.He X.Y., He Z.H., Zhang L.P, Sun D.J., Morris C.F., Fuerst E.P. & Xia X.C. Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat // Theoretical and Applied Genetics. – 2007. – Vol. 115. – № 1. – Pр. 47–58.He X.Y., He Z.H., Zhang L.P., Sun D.J., Morris C.F., Fuerst E.P., Xia X.C. Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat. Theoretical and Applied Genetics. 2007; 115 (1): 47–58.He X.Y., Zhang Y.L., He Z.H., Wu Y.P., Xiao Y.G., Ma C.X. & Xia X.C. Characterization of phytoene synthase 1 gene (Psy1) located on common wheat chromosome 7A and development of a functional marker // Theoretical and Applied Genetics. – 2008. – Vol. 116. – № 2. – Pр. 213–221.He. X.Y., Zhang Y.L., He Z.H., Wu Y.P., Xiao Y.G., Ma C.X., Xia X.C. Characterization of phytoene synthase 1 gene (Psy1) located on common wheat chromosome 7A and development of a functional marker. Theoretical and Applied Genetics. 2008; 116 (2): 213–221.Kaur B., Mavi G.S., Gill M.S., Saini D.K. Utilization of KASP technology for wheat improvement // Cereal Research Communications. – 2020. – Vol. 48. – № 4. – Pр. 409–421.Kaur B., Mavi G.S., Gill M.S., Saini D.K. Utilization of KASP technology for wheat improvement. Cereal Research Communications. 2020; 48 (4): 409–421.Kumar S., Kumar М., Mir R.R., Kumar R. & Kumar S. Advances in Molecular Markers and Their Use in Genetic Improvement of Wheat // Physiological, Molecular, and Genetic Perspectives of Wheat Improvement. – Cham: Springer International Publishing, 2021. – Pр. 139–174.Kumar S., Kumar M., Mir R.R., Kumar R. Advances in Molecular Markers and Their Use in Genetic Improvement of Wheat. Physiological, Molecular, and Genetic Perspectives of Wheat Improvement. Cham: Springer International Publishing, 2021: 139–174.Lei L., Zhu X., Wang S., Zhu M., Carver B.F., Yan L. TaMFT-A1 Is Associated with Seed Germination Sensitive to Temperature in Winter Wheat // PLoS ONE. – 2013. – Vol. 8. – № 9. – P. e73330.Lei L., Zhu X., Wang S., Zhu M., Carver B.F., Yan L. TaMFT-A1 Is Associated with Seed Germination Sensitive to Temperature in Winter Wheat. PLoS ONE. 2013; 8 (9): e73330.Poland J.A., Brown P.J., Sorrells M.E., Jannink J. – L. Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach // PLoS ONE. – 2012. – Vol. 7. – № 2. – P. e32253.Poland J.A., Brown P.J., Sorrells M.E., Jannink J. – L. Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach. PLoS ONE. 2012; 7 (2): e32253Ramirez-Gonzalez R.H., Uauy C., Caccamo M. PolyMarker: A fast polyploid primer design pipeline: Fig. 1 // Bioinformatics. – 2015. – Vol. 31. – № 12. – Pр. 2038–2039.Ramirez-Gonzalez R.H., Uauy C., Caccamo M. PolyMarker: A fast polyploid primer design pipeline: Fig. 1. Bioinformatics. 2015; 31 (12): 2038–2039.Rasheed A., Wen W., Gao F., Zhai S., Jin H., Liu J., Guo Q., Zhang Y., Dreisigacker S., Xia X. & He Z. Development and validation of KASP assays for genes underpinning key economic traits in bread wheat // Theoretical and Applied Genetics. – 2016. – Vol. 129. – № 10. – Pр. 1843–1860.Rasheed A., Wen W., Gao F., Zhai S., Jin H., Liu J., Guo Q., Zhang Y., Dreisigacker S., Xia X., He Z. Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theoretical and Applied Genetics. 2016; 129 (10): 1843–1860.Rogers S.O., Bendich A.J. Extraction of total cellular DNA from plants, algae and fungi // Plant Molecular Biology Manual. – Dordrecht: Springer Netherlands, 1994. – Pр. 183–190.Rogers S.O., Bendich A.J. Extraction of total cellular DNA from plants, algae and fungi. Plant Molecular Biology Manual. Dordrecht: Springer Netherlands, 1994: 183–190.Shavrukov Y. Comparison of SNP and CAPS markers application in genetic research in wheat and barley // BMC Plant Biology. – 2016. – Vol. 16. – № S1. – P. 11.Shavrukov Y. Comparison of SNP and CAPS markers application in genetic research in wheat and barley. BMC Plant Biology. 2016; 16 (S1): 11.Tan C., Assanga S., Zhang G., Rudd J.C., Haley S.D., Xue Q., Ibrahim A., Bai G., Zhang X., Byrne P., Fuentealba M.P., Liu S.. Development and Validation of KASP Markers for Wheat Streak Mosaic Virus Resistance Gene Wsm2 // Crop Science. – 2017. – Vol. 57. – № 1. – Pр. 340–349.Tan C., Assanga S., Zhang G., Rudd J.C., Haley S.D., Xue Q., Ibrahim A., Bai G., Zhang X., Byrne P., Fuentealba M.P., Liu S. Development and Validation of KASP Markers for Wheat Streak Mosaic Virus Resistance Gene Wsm2. Crop Science. 2017; 57 (1): 340–349.Tariq H. A novel and high throughput wheat (Triticum aestivum L.) genotyping using Kompetitive Allele Specific PCR assay for genes underpinning major economic attributes // Pakistan Journal of Agricultural Sciences. – 2021. – Vol. 58. – № 06. – Pр. 1799–1807.Tariq H. A novel and high throughput wheat (Triticum aestivum L.) genotyping using Kompetitive Allele Specific PCR assay for genes underpinning major economic attributes. Pakistan Journal of Agricultural Sciences. 2021; 58 (06): 1799–1807.Terracciano I., Maccaferri M., Bassi F., Mantovani P., Sanguineti M.C., Salvi S., Šimková H., Doležel J., Massi A., Ammar K., Kolmer J. & Tuberosa R. Development of COS-SNP and HRM markers for high-throughput and reliable haplotype-based detection of Lr14a in durum wheat (Triticum durum Desf.) // Theoretical and Applied Genetics. – 2013. – Vol. 126. – № 4. – Pр. 1077–1101.Terracciano I., Maccaferri M., Bassi F., Mantovani P., Sanguineti M.C., Salvi S., Šimková H., Doležel J., Massi A., Ammar K., Kolmer J., Tuberosa R. Development of COS-SNP and HRM markers for high-throughput and reliable haplotype-based detection of Lr14a in durum wheat (Triticum durum Desf.). Theoretical and Applied Genetics. 2013; 126 (4): 1077–1101.Walkowiak S., Gao L., Monat C. et al. Multiple wheat genomes reveal global variation in modern breeding // Nature. – 2020. – Vol. 588. – № 7837. – Pр. 277–283.Walkowiak S., Gao L., Monat C. et al. Multiple wheat genomes reveal global variation in modern breeding. Nature. 2020; 588 (7837): 277–283.Wei B., Jing R., Wang C., Chen J., Mao X., Chang X. & Jia J. Dreb1 genes in wheat (Triticum aestivum L.): development of functional markers and gene mapping based on SNPs // Molecular Breeding. – 2009. – Vol. 23. – № 1. – Pр. 13–22.Wei B., Jing R., Wang C., Chen J., Mao X., Chang X., Jia J. Dreb1 genes in wheat (Triticum aestivum L.): development of functional markers and gene mapping based on SNPs. Molecular Breeding. 2009; 23 (1): 13–22.Winfield M.O., Allen A.M., Burridge A.J. et al. High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool // Plant Biotechnology Journal. – 2016. – Vol. 14. – № 5. – Pр. 1195–1206.Winfield M.O., Allen A.M., Burridge A.J. et al. High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool. Plant Biotechnology Journal. 2016; 14 (5): 1195–1206.Yang Y., Zhao X.L., Xia L.Q., Chen X.M., Xia X.C., Yu Z., Z.H. He & Röder M. Development and validation of a Viviparous-1 STS marker for pre-harvest sprouting tolerance in Chinese wheats // Theoretical and Applied Genetics. – 2007. – Vol. 115. – № 7. – Pр. 971–980.Yang Y., Zhao X.L., Xia L.Q., Chen X.M., Xia X.C., Yu Z., He Z.H., Röder M. Development and validation of a Viviparous-1 STS marker for pre-harvest sprouting tolerance in Chinese wheats. Theoretical and Applied Genetics. 2007; 115 (7): 971–980.

The authors declare that there are no conflicts of interest present.