بررسی مقاومت ژنوتیپ‌های گوجه‌فرنگی نسبت به پژمردگی ورتیسیلیومی و فوزاریومی با استفاده از نشانگرهای مولکولی

محورهای موضوعی : قارچ شناسی

بهار مرید ¹ , شهاب حاج منصور ²

1 - استادیار، گروه گیاه پزشکی، دانشگاه آزاد اسلامی، واحد تاکستان
2 - کارشناس ارشد، گروه بیماری شناسی گیاهی، دانشگاه آزاد اسلامی، واحد علوم و تحقیقات، تهران

تاریخ دریافت : 1395/10/11 تاریخ پذیرش : 1395/11/06 تاریخ انتشار : 1396/03/01

کلید واژه: پژمردگی ورتیسیلیومی, پژمردگی فوزاریومی, ژن I-2, ژن Ve, نشانگر مولکولی,

چکیده مقاله :

سابقه و هدف: بیماری‌ های پژمردگی ورتیسلیومی و فوزاریومی دو بیماری مهم در گوجه‌ فرنگی می‌باشند و تولید گوجه‌ فرنگی را در دنیا محدود می‌کنند. کاشت ارقام مقاوم گوجه‌ فرنگی بهترین روش برای کنترل این بیماری ها می‌ باشد. نشانگرهای مولکولی که به ژن‌ های مقاومت متصل می‌شوند برای توسعه برنامه‌ های اصلاحی مفید هستند. این مطالعه با هدف شناسایی ژنوتیپ‌ های مقاوم به پژمردگی ورتیسلیومی با استفاده از نشانگرهای اختصاصی آلل و شناسایی ژنوتیپ‌ های مقاوم به پژمردگی فوزاریومی به وسیله نشانگر CAPS انجام شد. مواد و روش ها: این پژوهش به صورت مقطعی-توصیفی بر روی 35 هیبرید و رقم تجاری گوجه‌ فرنگی تهیه شده از شرکت فلات انجام شد. DNA نمونه ها با روش CTAB استخراج شدند. سپس واکنش زنجیره‌ ای پلی مراز با استفاده از نشانگرهای مولکولی انجام گردید. به منظور شناسایی ژنوتیپ های مقاوم به پژمردگی فوزاریومی، از روش PCR-RFLP با استفاده از آنزیم‌ های برشی RsaI و FokI استفاده شد. درنهایت نتایج با آزمون بیماریزایی تایید شدند. یافته ها: از مجموع 35 ژنوتیپ مورد بررسی، 83 درصد ژنوتیپ ها نسبت به پژمردگی ورتیسیلیومی مقاوم و 17 درصد حساس بودند. از طرفی 46 درصد از ژنوتیپ ها نسبت به پژمردگی فوزاریومی مقاوم و 54 درصد حساس بودند. ژنوتیپ هایی که تولید باند اختصاصی مقاومت به بیماری پژمردگی ورتیسیلیومی کردند، دارای ژن مقاومت Ve-1 بودند. همچنین ژنوتیپ هایی دارای باندهای اختصاصی مقاومت به بیماری پژمردگی فوزاریومی، ژن مقاومت I-2 را داشتند. نتیجه گیری: با کاشت ژنوتیپ های مقاوم یافت شده در این تحقیق در مناطق آلوده می توان این دو بیماری را بدون استفاده از قارچ کش ها کنترل نمود.

چکیده انگلیسی:

Background & Objectives: Fusarium and Verticillium wilts are two major fungal wilt diseases of tomato that have restricted its production worldwide. Culturing resistant tomato cultivars is the best way to control such diseases. Molecular markers linked to resistance genes would be useful for improving tomato breeding programs. In this study allele, specific markers and cleaved amplified polymorphic sequences (CAPS) markers were used to identify tomato genotypes that are resistant to Verticillium wilt and Fusarium wilt, respectively. Materials & Methods: This cross-sectional study was carried out on 32 tomato hybrids and commercial varieties provided from Falat company. DNA was extracted using cetyl-trimethyl ammonium bromide (CTAB) method. Then, polymerase chain reaction (PCR) was performed using molecular markers. To detect Fusarium wilt resistant varieties, PCR-RFLP was down using RsaI and FokI restriction enzymes. The results were confirmed by pathogenicity test. Results: Out of 35 tomato genotypes, 83% were resistant to Verticillium wilt, while 17% were sensitive. Also, 46% of genotypes were resistant to Fusarium wilt, while 54% were sensitive. Genotypes that showed resistance to Verticillium and Fusarium wilts possessed Ve1 and I-2 genes, respectively. Conclusion: Planting resistant genotypes in infected areas can control fungal diseases such as Verticillium and Fusarium wilts, without using any fungicides.

منابع و مأخذ:

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2. FAOSTAT Database, 2014.
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4. Staniazsek M, Kozik EU, Marczewski W. A CAPS marker TAO1902 diagnostic for the I-2 gene conferring resistance to Fusarium oxysporum f. sp. lycopersici race 2 in tomato. Plant Breeding. 2007; 126(3): 331-333.
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6. Kawchuk LM, Hachey J, Lynch DR, Kulcsar F, van Rooijen G, Waterer DR, Robertson A, Kokko E, Byers R, Howard RJ, Fischer R, Prufer D. Tomato Ve disease resistance genes encode cell surface-like receptors. PNAS. 2001; 98: 6511-6515.
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9. Kuklev MY, Fesenko IA, Karlov GI. Development of a CAPS marker for the Verticillium wilt resistance in tomatoes. Russ J Genet. 2009; 45: 575-579.
10. Fradin EF, Zhang Z, Ayala JCJ, Castroverde CDM, Nazar RN, Robb J, Liu C,  Thomma BPHJ. Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1. Plant Physiol. 2009; 150: 320-332.
11. Shi A, Vierling R, Grazzini R, Chen P, Caton H, Weng Y. Development of single nucleotide polymorphism (SNP) markers for selection of Ve gene of tomato Verticillium wilt resistance. Int Res J Plant Sci. 2010; 1(2): 34-42.
12. Labate JA, Baldo, AM. Tomato SNP discovery by EST mining and resequencing. Mol Breeding. 2005; 16: 343-349.
13. Yang W, Bai X, Kabelka E, Eaton C, Kamoun S, van der Knaap E, Francis D. Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags. Mol Breeding. 2004; 14: 21-34.
14. Foolad MR. Genome mapping and molecular breeding of tomato. Int J Plant Genomics. 2007; 2007: 64358.
15. Jones E, Chu W, Ayele M, Ho J, Bruggeman E, Yourstone K, Rafalski A, Smith OS, McMullen MD, Bezawada C, Warren J, Babayew J, Basu S, Smith S. Development of single nucleotide polymorphism (SNP) markers for use in commercial maize (Zea mays L.) germplasm. Mol Breeding. 2009; 24: 165-176.
16. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current protocols in molecular biology. John Wiley and Sons Inc. USA; 1994.
17. Naraghi L, Heydari A, Rezaee S, Razavi M, Jahanifar H, Mahmoodi Khaledi E. Biological control of tomato Verticillim wilt disease by Talaromyces flavus. J Plant Prot Res. 2010; 50 (3): 360-365.
18. Acciarri N, Sabatini E, Ciriaci T, Rotino LG, Valentino D, Tamietti G. The presence of genes for resistance against Verticillium dahliae in Italian tomato landraces. Eur J Hortic Sci. 2010; 75(1): 8-14.
19. Kim JT, Park IH, Lee HB, Hahm YI, Yu SH. Identification of Verticillium dahliae and Verticillium albo-atrum causing wilt of tomato in Korea. Plant Pathol J. 2001; 17(4): 222-226.
20. Ganal MW, Altmann T, Roder MS. SNP identification in crop plants. Curr Opin Plant Biol. 2009; 12: 211-217.
21. Collard BCY, Mackill DJ. Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philos Trans R Soc Lond B Biol Sci. 2008; 363(1491): 557-572.
22. Wang Y, Tang X, Cheng Z, Mueller L, Giovannoni J, Tanksley SD. Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome. Genetics. 2006; 172: 2529-2540.
23. Shirasawa K, Asamizu E, Fukuoka H. An interspecific linkage map of SSR and intronic polymorphism markers in tomato. Theor Appl Genet. 2010; 121: 731-739.
24. Jimenez-Gomez JM, Maloof JN. Sequence diversity in three tomato species: SNPs, markers, and molecular evolution. BMC Plant Biol. 2009; 9: 85.
25. Hillier LW, Miller RD, Baird SE, Chinwalla A, Fulton LA, Koboldt DC, Waterston RH. Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny. PLoS Biol. 2007; 5: 167.
26. Eberle MA, Ng PC, Kuhn K, Zhou L, Peiffer DA, Galver L, Viaud-Martinez KA, Lawley CT, Gunderson KL, Shen R, Murray SS. Power to detect risk alleles using genome-wide tag SNP panels. PLoS Genet. 2009; 3: 1827-1837.
27. Sedlacek T, Marik P, Chrpova J. Development of CAPS marker for identification of rym4 and rym5 alleles conferring resistance to the Barley Yellow Mosaic Virus complex in barley. Czech J Genet Plant. 2010; 46(4): 159-163.
28. Akkale C, Tanyolac B. Screening of resistance genes to Fusarium root rot and Fusarium wilt diseases in F3 family lines of tomato using RAPD and CAPS markers. Int J Nat Eng Sci. 2009; 3(3): 144 -148.
29. Popoola AR, Ercolano M R, Kaledzi PD, Ferriello F, Ganiyu SA, Dapaah HK, Ojo DK, Adegbite DA, Falana Y, Adedibu OB. Molecular and phenotypic screening of tomato genotypes for resistance to Fusarium wilt. Ghana J Hort. 2012; 10: 61-67.
30. El Mohtar CA, Atamian HS, Dagher RB, Abou-Jawdah Y, Salus MS, Maxwell DP.
Marker-assisted selection of tomato genotypes with the I-2 gene for resistance to Fusarium oxysporum f. sp. lycopersici race 2. Plant Dis. 2007; 91: 758-762.
31. Popoola AR, Ercolano MR, Feriello F, Kaledzi PD, Kwoseh C, Ganiyu SA, OjoDK, Adegbite DA, Falana Y. CAPS markers TAO1 and TG105 in the identification of I-2 resistant gene in Nigerian accessions of tomato, Solanum lycopersicum L. Niger J Biotech. 2014; 28: 43-51.
32. Hansona P, Lua S, Wanga J, Chena W, Kenyona L, Tana C, Teeb K, Wanga Y, Hsua Y, Schafleitnera R, Ledesmaa D, Yangaa R. Conventional and molecular marker-assisted selection and pyramiding of genes for multiple disease resistance in tomato. Sci Hortic. 2016; 201: 346-354.

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1. Bal U, Abak K. Haploidy in tomato (Lycopersicon esculen­tum Mill.): a critical review. J Euphytica. 2007; 158 (1-2): 1-9.
2. FAOSTAT Database, 2014.
3. Aminaee MM, Mansoori B, Ershad D. 2006. A study on Verticillium wilt of tomato in Kerman province. Proceeding of the 17th Iranian Plant Protection Congress. 2-5 Sep, Tehran, Iran. 163.
4. Staniazsek M, Kozik EU, Marczewski W. A CAPS marker TAO1902 diagnostic for the I-2 gene conferring resistance to Fusarium oxysporum f. sp. lycopersici race 2 in tomato. Plant Breeding. 2007; 126(3): 331-333.
5. Acciarri N, Rotino GL, Tamietti G, Valentino D, Voltattorni S, Sabatini E. Molecular markers for Ve1and Ve2 Verticillium resistance genes from Italian tomato germplasm. Plant Breeding. 2007; 126: 617-621.
6. Kawchuk LM, Hachey J, Lynch DR, Kulcsar F, van Rooijen G, Waterer DR, Robertson A, Kokko E, Byers R, Howard RJ, Fischer R, Prufer D. Tomato Ve disease resistance genes encode cell surface-like receptors. PNAS. 2001; 98: 6511-6515.
7. Kawchuk LM, Lynch DR, Hachey J, Bains PS, Kulcsar F. Identification of a co-dominant amplified polymorphic DNA marker linked to the Verticillium wilt resistance gene in tomato. Theor Appl Genet. 1994; 89: 661-664.
8. Kawchuk LM, Hachey J, Lynch DR. Development of sequence characterized DNA markers linked to a dominant Verticillium wilt resistance gene in tomato. Genome. 1998; 41: 91-95.
9. Kuklev MY, Fesenko IA, Karlov GI. Development of a CAPS marker for the Verticillium wilt resistance in tomatoes. Russ J Genet. 2009; 45: 575-579.
10. Fradin EF, Zhang Z, Ayala JCJ, Castroverde CDM, Nazar RN, Robb J, Liu C,  Thomma BPHJ. Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1. Plant Physiol. 2009; 150: 320-332.
11. Shi A, Vierling R, Grazzini R, Chen P, Caton H, Weng Y. Development of single nucleotide polymorphism (SNP) markers for selection of Ve gene of tomato Verticillium wilt resistance. Int Res J Plant Sci. 2010; 1(2): 34-42.
12. Labate JA, Baldo, AM. Tomato SNP discovery by EST mining and resequencing. Mol Breeding. 2005; 16: 343-349.
13. Yang W, Bai X, Kabelka E, Eaton C, Kamoun S, van der Knaap E, Francis D. Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags. Mol Breeding. 2004; 14: 21-34.
14. Foolad MR. Genome mapping and molecular breeding of tomato. Int J Plant Genomics. 2007; 2007: 64358.
15. Jones E, Chu W, Ayele M, Ho J, Bruggeman E, Yourstone K, Rafalski A, Smith OS, McMullen MD, Bezawada C, Warren J, Babayew J, Basu S, Smith S. Development of single nucleotide polymorphism (SNP) markers for use in commercial maize (Zea mays L.) germplasm. Mol Breeding. 2009; 24: 165-176.
16. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current protocols in molecular biology. John Wiley and Sons Inc. USA; 1994.
17. Naraghi L, Heydari A, Rezaee S, Razavi M, Jahanifar H, Mahmoodi Khaledi E. Biological control of tomato Verticillim wilt disease by Talaromyces flavus. J Plant Prot Res. 2010; 50 (3): 360-365.
18. Acciarri N, Sabatini E, Ciriaci T, Rotino LG, Valentino D, Tamietti G. The presence of genes for resistance against Verticillium dahliae in Italian tomato landraces. Eur J Hortic Sci. 2010; 75(1): 8-14.
19. Kim JT, Park IH, Lee HB, Hahm YI, Yu SH. Identification of Verticillium dahliae and Verticillium albo-atrum causing wilt of tomato in Korea. Plant Pathol J. 2001; 17(4): 222-226.
20. Ganal MW, Altmann T, Roder MS. SNP identification in crop plants. Curr Opin Plant Biol. 2009; 12: 211-217.
21. Collard BCY, Mackill DJ. Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philos Trans R Soc Lond B Biol Sci. 2008; 363(1491): 557-572.
22. Wang Y, Tang X, Cheng Z, Mueller L, Giovannoni J, Tanksley SD. Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome. Genetics. 2006; 172: 2529-2540.
23. Shirasawa K, Asamizu E, Fukuoka H. An interspecific linkage map of SSR and intronic polymorphism markers in tomato. Theor Appl Genet. 2010; 121: 731-739.
24. Jimenez-Gomez JM, Maloof JN. Sequence diversity in three tomato species: SNPs, markers, and molecular evolution. BMC Plant Biol. 2009; 9: 85.
25. Hillier LW, Miller RD, Baird SE, Chinwalla A, Fulton LA, Koboldt DC, Waterston RH. Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny. PLoS Biol. 2007; 5: 167.
26. Eberle MA, Ng PC, Kuhn K, Zhou L, Peiffer DA, Galver L, Viaud-Martinez KA, Lawley CT, Gunderson KL, Shen R, Murray SS. Power to detect risk alleles using genome-wide tag SNP panels. PLoS Genet. 2009; 3: 1827-1837.
27. Sedlacek T, Marik P, Chrpova J. Development of CAPS marker for identification of rym4 and rym5 alleles conferring resistance to the Barley Yellow Mosaic Virus complex in barley. Czech J Genet Plant. 2010; 46(4): 159-163.
28. Akkale C, Tanyolac B. Screening of resistance genes to Fusarium root rot and Fusarium wilt diseases in F3 family lines of tomato using RAPD and CAPS markers. Int J Nat Eng Sci. 2009; 3(3): 144 -148.
29. Popoola AR, Ercolano M R, Kaledzi PD, Ferriello F, Ganiyu SA, Dapaah HK, Ojo DK, Adegbite DA, Falana Y, Adedibu OB. Molecular and phenotypic screening of tomato genotypes for resistance to Fusarium wilt. Ghana J Hort. 2012; 10: 61-67.
30. El Mohtar CA, Atamian HS, Dagher RB, Abou-Jawdah Y, Salus MS, Maxwell DP.
Marker-assisted selection of tomato genotypes with the I-2 gene for resistance to Fusarium oxysporum f. sp. lycopersici race 2. Plant Dis. 2007; 91: 758-762.
31. Popoola AR, Ercolano MR, Feriello F, Kaledzi PD, Kwoseh C, Ganiyu SA, OjoDK, Adegbite DA, Falana Y. CAPS markers TAO1 and TG105 in the identification of I-2 resistant gene in Nigerian accessions of tomato, Solanum lycopersicum L. Niger J Biotech. 2014; 28: 43-51.
32. Hansona P, Lua S, Wanga J, Chena W, Kenyona L, Tana C, Teeb K, Wanga Y, Hsua Y, Schafleitnera R, Ledesmaa D, Yangaa R. Conventional and molecular marker-assisted selection and pyramiding of genes for multiple disease resistance in tomato. Sci Hortic. 2016; 201: 346-354.