بررسی بیان ژن‌های دخیل در مقاومت درخت‌ سیب به شانکر اروپایی با استفاده از آنالیز داده‌های ترانسکریپتوم

نوع مقاله : مقاله پژوهشی

نویسندگان

1 نویسنده مسئول: استادیار، گروه تنوع زیستی، پژوهشگاه علوم و تکنولوژی پیشرفته و علوم محیطی، دانشگاه تحصیلات تکمیلی صنعتی و فناوری پیشرفته، کرمان، ایران

2 استاد، گروه اصلاح نباتات، دانشگاه علوم کشاورزی سوئد، کیخانستاد، سوئد

چکیده

چکیده
هدف: یکی از مخرب ترین بیماری‌های درختان میوه، بیماری شانکر اروپایی با عامل قارچ بیماری زای Neonectria ditissima، در کشورهای تولید کننده سیب با هوای خنک می‌باشد. از آنجایی که کنترل این بیماری به دلیل حضور قارچ بیماری زا در طول کل سال دشوار است، این تحقیق به منظور بررسی الگوی بیان ژن‌های پاسخ دهنده به این عامل بیماری زا در سیب انجام شد که می‌تواند درک بهتر از تعامل بین میزبان و پاتوژن در جهت بهبود استراتژی‌های مدیریتی فراهم کند.
مواد و روش ها: بدین منظور رقم نیمه مقاوم سیب ("جاناتان"، منشا نیویورک) با سوسپانسیون قارچ تلقیح داده شد و نمونه‌های شاهد و تلقیح داده شده برای استخراج RNA و توالی یابی در سه نقطه زمانی 5، 15 و 30 روز پس از تلقیح برداشت شدند. پس از کنترل کیفی و کمی RNA کل استخراج شده، توالی‌یابی کل ژنوم به صورت دو سویه توسط شرکت ایلومینا و دستگاه‌ توالی‌یاب Hiseq2000 انجام شد. کنترل کیفیت داده‌ها توسط نرم افزارFastQC صورت پذیرفت. سپس خوانش‌ها با استفاده از نرم افزارTopHat2 با ژنوم مرجع سیب نقشه‌یابی شدند. نرمال‌سازی و تجزیه و تحلیل ژن‌های با بیان متفاوت با نرم افزار DESeq2 و آنالیز غنی‌سازی مسیرهای DEGs با نرم افزار KEGG انجام شد.
یافته ها: تجزیه و تحلیل‌های غنی‌سازی GO و KEGG در ژن‌های دارای بیان افتراقی (Differentially expressed genes, DEGs) ، تعدادی ژن مرتبط با پاسخ دفاعی را شناسایی نمود. در رقم سیب تلقیح ‌شده با N. ditissima، تغییرات قابل‌توجهی در ژن‌های مرتبط با دفاع و ژن‌های دخیل در سم‌زدایی، پراکسیداز و متابولیسم فنیل پروپانوئید مشاهده شد. بالاترین سطح بیان ژن‌های مرتبط با دفاع، 30 روز پس از تلقیح با N. ditissima دیده شد. این موضوع می‌تواند بیانگر این باشد که پاتوژن برای ایجاد آلودگی نیاز به زمان دارد و به سرعت نمی‌تواند در بافت گیاه گسترش پیدا کند.
نتیجه گیری: ژن‌های شناسایی شده درگیر در بیماری ‌زایی N. ditissima دخیل در لیگنین شدن، سم‌زدایی، فسفوریلاسیون و دفاع پاتوژن بوده و منبعی ارزشمند در تحقیقات ژنتیکی هستند و به ما این امکان را می‌دهد تا تعامل پاتوژن با گیاه میزبان را بهتر درک کنیم و می توانند در برنامه‌های اصلاحی کنترل این بیماری مورد استفاده قرار گیرند.

کلیدواژه‌ها

عنوان مقاله [English]

Identification of genes associated with tolerance to apple canker by genome-wide transcriptome analysis

نویسندگان [English]

  • Marjan Ghasemkhanni 1
  • Hilde Nybom 2
1 Corresponding author. Assistant Professor, Department of Biodiversity, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
2 Professor, Department of Plant Breeding, Balsgård, Swedish University of Agricultural Sciences, Kristianstad, Sweden
چکیده [English]

Objective
Neonectria ditissima, the causal agent of fruit tree canker, is one of most destructive diseases of apple trees in producing countries with cool weather. Since it is difficult to control this disease due to the presence of the pathogenic fungus throughout the year, for better understanding of this host–pathogen interaction in order to improve management strategies, a transcriptional analysis of apple gene expression in response to N. ditissima was conducted.
Materials and methods
In the present study, we evaluated transcriptome responses to N. ditissima by selecting the partially resistant cultivar “Jonathan”. The leaf scars of apple trees were artificially inoculated with suspension of N. ditissima and water as control. Then the samples were taken for RNA extraction at three different time points, 5, 15, and 30 days after inoculation. The quality and quantity of the extracted RNA were checked and total RNA was sequenced using Illumina paired-end sequencing and Hiseq2000 sequencer. The quality of the sequence data was done by FastQC software. Then the reads were mapped to apple reference genome by TopHat2 software. Normalization and differential expression analysis of genes were performed with DESeq2. Enrichment analysis of DEGs pathways was done through KEGG software.
Results
Based on GO enrichment and KEGG pathway analyses, it was found that some of the defense response genes were differentially expressed between control and treatment groups. The data provides evidence that apple cultivars inoculated with N. ditissima exhibit significant upregulation of defense-related genes and genes involved in detoxification, peroxidase related reactions, phenylpropanoid metabolism. The highest expression level of genes related to defense was observed 30 days after inoculation. It shows that the pathogen needs time to cause infection and cannot spread quickly in the plant tissue.
Conclusion
Identification of candidate genes involved in pathogenicity of N. ditissima are involved in lignification, detoxification, phosphorylation and pathogen defense. They are a valuable resource in genetic research and allow us to better understand interaction of fungus and the apple defense system, and may assists in apple canker breeding programs.

کلیدواژه‌ها [English]

  • Defense response
  • Next-generation sequencing (NGS)
  • Malus × domestica
  • RNA-Seq

مراجع

جعفری احمدآبادی سید علی اصغر، عسکری­همت حشمت­اله، محمدآبادی محمدرضا (1402) تاثیر شاهدانه بر بیان ژن DLK1 در بافت‌ قلب بره‌های کرمانی. مجله بیوتکنولوژی کشاورزی، 15(1)، 217-234.
شکری سمیرا، خضری امین، محمدآبادی محمدرضا، خیرالدین حمید (1402) بررسی بیان ژن MYH7  در بافت‌های ران، دست و راسته بره‌های پرواری نژاد کرمانی. مجله بیوتکنولوژی کشاورزی، 15(2)، 217-236.
محمدآبادی محمدرضا، گلکار افروز، عسکری حصنی مجید (1402) اثر رازیانه (Foeniculum vulgare) بر بیان ژن فاکتور 1 رشد شبه انسولین در بافت شکمبه گوسفند کرمانی. مجله بیوتکنولوژی کشاورزی، 15(4)، 239-256.
References
Atzema, JL (1998) Durability of MLO resistance in barley against powdery mildew caused by Erysiphe graminis f. sp. hordei. ETH Zurich.
Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11, R106.
Anders S, Pyl PT, Huber W (2014) HTSeq–A Python framework to work with high-throughput sequencing data. Bioinform btu638.
Andrews S (2010) FastQC: A quality control tool for high throughput sequence data. Reference Source.
Barazandeh A, Mohammadabadi MR, Ghaderi-Zefrehei M, Nezamabadipour H (2016a) Predicting CpG Islands and Their Relationship with Genomic Feature in Cattle by Hidden Markov Model Algorithm. Iran J Appl Anim Sci 6 (3), 571-579.
Barazandeh A, Mohammadabadi MR, Ghaderi-Zefrehei M, Nezamabadipour H (2016b) Genome-wide analysis of CpG islands in some livestock genomes and their relationship with genomic features. Czech J Anim Sci 61, 487.
Beno-Moualem D, Prusky D (2000) Early events during quiescent infection development by Colletotrichum gloeosporioides in unripe avocado fruits. Phytopathology 90, 553-9.
Beresford RM, Kim KS (2011) Identification of regional climatic conditions favorable for development of European canker of apple. Phytopathology 101, 135-46.
Bolger A, Giorgi F (2014) Trimmomatic: A Flexible Read Trimming Tool for Illumina NGS Data. URL http://www.usadellab.org/cms/index.php.
Bordbar F, Mohammadabadi M, Jensen J, et al. (2022) Identification of candidate genes regulating carcass depth and hind leg circumference in simmental beef cattle using Illumina Bovine Beadchip and next-generation sequencing. Animals 12 (9), e1103.
Bus VGM, Scheper RWA, Walter MR et al. (2019) Genetic mapping of the European canker (Neonectria ditissima) resistance locus Rnd1 from Malus ‘Robusta 5’. Tree Genet 15, 1-13.
Carneiro, Benedito A. Wafik S. El-Deiry (2020) "Targeting apoptosis in cancer therapy." Nat Rev Clin Oncol 17.7, 395-417.
Carstens E, Van Niekerk JM, Smit WA, Fourie PH (2010) Resolving the status of Neonectria ditissima in South Africa. Australas. Plant Pathol 39, 43-5.
Castlebury LA, Rossman AY, Hyten AS (2006) Phylogenetic relationships of Neonectria/Cylindrocarpon on Fagus in North America. Can J Bot 84, 1417-33.
Chevalier M, Parisi L, Gueye B et al. (2008) Specific activation of PR-10 pathogenesis-related genes in apple by an incompatible race of Venturia inaequalis. Biol Plant 52, 718-22.
Coleman JJ, Rounsley SD, Rodriguez-Carres M et al. (2009) The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion. PLoS genet 5, e1000618.
Dixon RA, Achnine L, Kota P et al. (2002) The phenylpropanoid pathway and plant defence—a genomics perspective. Mol Plant Pathol 3, 371-90.
Du Z, Zhou X, Ling Y et al. (2010) AgriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res pp.64-70.
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol pathol 35, 495-516.
Garkava-Gustavsson L, Ghasemkhani M, Zborowska A, Englund JE, Lateur M et al (2014). Approaches for evaluation of resistance to European canker (Neonectria ditissima) in apple. 29th International Horticultural Congress (IHC2014), Aug. 17-22, 2014. Brisbane, Australia, pp. 75-82.
Garkava-Gustavsson L, Zborowska A, Sehic J et al. (2013) Screening of apple cultivars for resistance to european canker, Neonectria ditissima. Acta Hortic 976, 529-36.
Gayoso C, Pomar F, Novo-Uzal E, Merino F, De Ilárduya ÓM (2010) The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H2O2, peroxidase and lignins and drives PAL gene expression. Bmc Plant Biol 10, 232.
Ghasemkhani M, Liljeroth E, Sehic J et al. (2015) Cut-off shoots method for estimation of partial resistance in apple cultivars to fruit tree canker caused by Neonectria ditissima. Acta Agric Scand B Soil Plant Sci 1-10.
Gusberti M, Gessler C, Broggini GA (2013) RNA-Seq analysis reveals candidate genes for ontogenic resistance in Malus-Venturia pathosystem. PloS one 8, e78457.
Jafari Ahmadabadi SAA, Askari-Hemmat H, Mohammadabadi M, et al. (2023) The effect of Cannabis seed on DLK1 gene expression in heart tissue of Kermani lambs. Agric Biotechnol J 15 (1), 217-234 (In Persian).
Katile SO, Perumal R, Rooney WL et al. (2010) Expression of pathogenesis‐related protein PR‐10 in sorghum floral tissues in response to inoculation with Fusarium thapsinum and Curvularia lunata. Mol Plant Pathol 11, 93-103.
Kemmerling B, Schwedt A, Rodriguez P et al. (2007) Mü ssig C, Thomma BPHJ, Albrecht C, de Vries S, Hirt H, Nü rnberger T: The BRI1- associated kinase1, BAK1, has a brassinolideindependent role in plant cell-death control. Curr Biol 17, 1116-1122.
Kędzierski Ł, Montgomery J, Curtis J. Handman E (2004) Leucine-rich repeats in host-pathogen interactions. AITE 52, 104-112.
Kim KS, Beresford RM (2012) Use of a climatic rule and fuzzy sets to model geographic distribution of climatic risk for European eanker (Neonectria galligena) of apple. Phytopathology 102, 147-57.
Komjanc M, Festi S, Rizzotti L et al. (1999) A leucine-rich repeat receptor-like protein kinase (LRPKm1) gene is induced in Malus× domestica by Venturia inaequalis infection and salicylic acid treatment. Plant Mol Biol 40, 945-57.
Linhard Pedersen H, Vittrup Christensen J, Hansen P (1994) Susceptibility of 15 apple cultivars to apple scab, powdery mildew, canker and mites. Fruit Var J 48, 97-100.
Love M, Anders S, Huber W (2013) Differential analysis of count data–the DESeq2 package. In., 1-48.
Masoudzadeh, S.H., Mohammadabadi, M.R., Khezri, A., et al. (2020) Dlk1 gene expression in different Tissues of lamb. Iran J Appl Anim Sci 10, 669-677.
McCracken AR, Berrie A, Barbara DJ et al. (2003) Relative significance of nursery infections and orchard inoculum in the development and spread of apple canker (Nectria galligena) in young orchards. Plant Pathol 52, 553-66.
McLeod LC, Poole PR (1994) Changes in enzymic activities after harvest and in early stages of Botrytis cinerea infection of kiwifruit. J. Sci. Food Agric. J SCI FOOD AGR 64(1), 95-100.
Mohamadipoor L, Mohammadabadi M, Amiri Z, et al. (2021) Signature selection analysis reveals candidate genes associated with production traits in Iranian sheep breeds. BMC Vet Res 17 (1), 1-9.
Mohammadabadi M, Masoudzadeh SH, Khezri A, et al. (2021) Fennel (Foeniculum vulgare) seed powder increases Delta-Like Non-Canonical Notch Ligand 1 gene expression in testis, liver, and humeral muscle tissues of growing lambs. Heliyon 7 (12), e08542.
Mohammadinejad F, Mohammadabadi M, Roudbari Z, Sadkowski T (2022) Identification of Key Genes and Biological Pathways Associated with Skeletal Muscle Maturation and Hypertrophy in Bos taurus, Ovis aries, and Sus scrofa. Animals 12 (24), 3471.
Mohammadabadi M, Golkar A, Askari Hesni M (2023) The effect of fennel (Foeniculum vulgare) on insulin-like growth factor 1 gene expression in the rumen tissue of Kermani sheep. Agric Biotechnol J 15 (4), 239-256 (In Persian).
Morant M, Bak S, Møller BL, Werck-Reichhart D (2003) Plant cytochromes P450: tools for pharmacology, plant protection and phytoremediation. Curr Opin Biotechnol 14, 151-62.
Nekrasov V, Wang C, Win J et al. (2017) Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci Rep 7(1), 482.
Nybom H, Cervin-Hoberg C, Andersson M (2013) Oral challenges with four apple cultivars result in significant differences in oral allergy symptoms. Int Arch Allergy Appl Immunol 161, 258-64.
Petkovsek MM, Slatnar A, Stampar F, Veberic R (2011) Phenolic compounds in apple leaves after infection with apple scab. Biol Plant 55, 725-30.
Pourcel L, Routaboul J-M, Kerhoas L et al. (2005) TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat. Plant Cell 17, 2966-80.
Puehringer HM, Zinoecker I, Marzban G et al. (2003) MdAP, a novel protein in apple, is associated with the major allergen Mal d 1. Gene 321, 173-83.
Robert N, Ferran J, Breda C et al. (2001) Molecular characterization of the incompatible interaction of Vitis vinifera leaves with Pseudomonas syringae pv. pisi: Expression of genes coding for stilbene synthase and class 10 PR protein. Eur J Plant Pathol 107, 249-61.
Safaei SMH, Dadpasand M, Mohammadabadi M, et al. (2022) An Origanum majorana Leaf Diet Influences Myogenin Gene Expression, Performance, and Carcass Characteristics in Lambs. Animals 13 (1), e14.
Sasnauskas A, Gelvonauskiene D, Gelvonauskis B et al. (2006) Resistance to fungal diseases of apple cultivars and hybrids in Lithuania. Agron Res 4, 349-52.
Singh V, Singh A, Chand R. Singh B (2012) Genome wide analysis of disease resistance mlo gene family in sorghum. Journal Plant Genom 2, 18-27.
Schenk MF, Cordewener JH, America AH et al. (2009) Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen. BMC plant biol 9, 1-15.
Schovankova J, Opatova H (2011) Changes in phenols composition and activity of phenylalanine-ammonia lyase in apples after fungal infections. Hort-Science 38, 1-10.
Shahsavari M, Mohammadabadi M, Khezri A, et al. (2022) Effect of Fennel (Foeniculum Vulgare) Seed Powder Consumption on Insulin-like Growth Factor 1 Gene Expression in the Liver Tissue of Growing Lambs. Gene Express 21 (2), 21-26.
Shokri S, Khezri A, Mohammadabadi M, Kheyrodin H (2023). The expression of MYH7 gene in femur, humeral muscle and back muscle tissues of fattening lambs of the Kermani breed. Agric Biotechnol J 15 (2), 217-236 (In Persian).
Simons TJ, Ross A (1970) Enhanced peroxidase activity associated with induction of resistance to Tobacco mosaic virus in hypersensitive Tobacco. Phytopathology 60, 383-4.
Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinform 25, 1105-11.
Trognitz F, Manosalva P, Gysin R et al. (2002) Plant defense genes associated with quantitative resistance to potato late blight in Solanum phureja× dihaploid S. tuberosum hybrids. MPMI 15(6), 587-597.
Torii KU (2004) Leucine-rich repeat receptor kinases in plants: structure, function, and signal    transduction pathways. Int rev cyt 234, 1-46.
Veitch NC (2004) Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry 65, 249-59.
Velasco R, Zharkikh A, Affourtit J et al. (2010) The genome of the domesticated apple (Malus ×  domestica Borkh.). Nat Genet 42, 833-9.
Venisse J-S, Malnoy M, Faize M et al. (2002) Modulation of defense responses of Malus spp. during compatible and incompatible interactions with Erwinia amylovora. MPMI 15, 1204-12.
Wan J, Zhang XC, Neece D et al. (2008) A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis. Plant Cell 20(2), 471-81.
Wang X, Gao Y, Yan Q, Chen W (2016) Salicylic acid promotes autophagy via NPR3 and NPR4 in Arabidopsis senescence and innate immune response. Acta Physiol. Plant 38, 1-12.
Wang J, Tian W, Tao F et al. (2020) TaRPM1 positively regulates wheat high-temperature seedling-plant resistance to Puccinia striiformis f. sp. tritici. Front Plant Sci 10, 1679.
Zagaja SW, Millikan DF, Kaminski W, Myszka T (1971) Field resistance to Nectria canker in apple. Plant Dis Rep 55, 445-7.
Zhu L, Ni W, Liu S et al. (2017) Transcriptomics analysis of apple leaves in response to Alternaria alternata apple pathotype infection. Front. Plant Sci 8, 22.
مجله بیوتکنولوژی کشاورزی
دوره 16، شماره 2
خرداد 1403
صفحه 131-152

فایل ها

هم رسانی

ارجاع به این مقاله

آمار