شناسایی نشانگرهای EST-SSR عدس (Lens culinaris) تحت تنش سرما

نوع مقاله: علمی پژوهشی

نویسندگان

1 دانشجوی دکتری گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

2 دانشیار گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

3 استاد گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

4 استادیار گروه زیست شناسی، دانشکده علوم پایه، دانشگاه رازی، کرمانشاه، ایران

چکیده

توسعه برنامه‌های به‌نژادی عدس برای مقابله با عوامل نامساعد محیطی نسبت به سایر حبوبات، به علت کمبود منابع ژنتیکی با چالش‌های بیشتری روبرو می‌باشد. نشانگرهای EST-SSR به‌دلیل ایجاد چندشکلی در نواحی کدکننده ژن‌ها، یکی از پرکاربردترین نشانگرهای مولکولی در بسیاری از برنامه‌های اصلاحی به‌شمار می‌روند. از این‌رو در تحقیق حاضر به‌منظور توسعه نشانگرهای EST-SSR در مقیاس بالا، از فرآیند سرهم‌بندی مجموعه خوانش‌های کوتاه حاصل از فناوری توالی‌یابی RNA عدس تحت تنش سرما و حالت طبیعی استفاده شد. به‌منظور اعمال تنش سرما، گیاهچه‌های 21 روزه عدس به‌مدت 48 ساعت در معرض دمای 4 درجه سانتی‌گراد قرار گرفتند. از نمونه‌های گیاهی تهیه شده، RNA کل استخراج شد و مورد توالی‌یابی قرار گرفتند. تعداد 8905 مکان ریزماهواره در 7211 یونی‎ژن حاصل از داده‌های RNA-seq عدس شناسایی شد که 1293 یونی‌ژن شامل بیش از یک مکان نشانگر SSR بود. فراوان‌ترین نوع نشانگر EST-SSR یافت شده از نوع تک نوکلئوتیدی بود. در این مطالعه موتیف‌های A/T، AG/CT و AAG/CTT به‌ترتیب بیشترین فراوانی را در بین موتیف‌های تک، دو و سه نوکلئوتیدی به خود اختصاص دادند. نتایج بلاست یونی‌ژن‌های حاوی SSR، نشان داد که 80 درصد از یونی‌ژن‌ها دارای رکورد مشابه در پایگاه پروتئین‌های غیرتکراری بودند. تفسیر کارکردی ژن‌ها، حضور یونی‌ژن‌های حاوی ریزماهواره را در زیر‌گروه‌های مهم پاسخ به تنش سرما نظیر اتصال، سلول و اجزای سلول و متابولیک را نشان داد. همچنین با توجه به نتایج می‌توان چنین اظهار نمود که اغلب نشانگرهای شناسایی شده در ژن‌هایی قرار داشتند که نقش مهمی در پاسخ به تنش‌ سرما دارند و جایگاه احتمالی اغلب آن‌ها نواحی UTR می‌باشد. از این‌رو بررسی بیشتر این نواحی در رونوشت‌ ژن‌های کاندید پاسخ‌دهنده به تنش سرما از اهمیت بیشتری برخوردار می‌باشد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Discovery of EST-SSRs Markers in Lentil (Lens culinaris) under Cold Stress

نویسندگان [English]

  • Seyed Sajad Sohrabi 1
  • Ahmad Ismaili 2
  • Farhad N azarian Firouz-Abadi 3
  • Hossein Fallahi 4
1 Ph.D. Student, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
2 Associate Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
3 Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
4 Assistant Professor, Department of Biology, Faculty of Basic Sciences, Razi University, Kermanshah, Iran
چکیده [English]

Development and improvement of lentil breeding programs to deal with adverse environmental factors in comparison to other legumes has more challenges due to poor pool of genetic resources. EST-SSR (EST-Simple Sequence Repeats) markers are one of the most commonly used molecular markers in many plant breeding programs due to polymorphism in genes coding regions. Hence, in the present study, for the development of EST-SSR markers, assembly process of RNA sequences of lentil under cold stress and normal condition was used. In order to apply cold stress, lentil plants treated at 4 °C condition. Total RNA was extracted from plant samples and was sequenced. 8905 microsatellite locations in 7211 unigene derived from lentil RNA sequences data was identified that 1293 unigene of them contained more than one SSR marker location. The most abundant type of EST-SSR marker was found to be of single nucleotide type. In this study, A/T, AG/CT and AAG/CTT motifs had the highest frequency among the one, two and three nucleotide motifs, respectively. The results of blast of unigene containing SSR showed that 80% of the unigenes had a similar record in the non-redundant protein database. The functional annotation of the unigenes showed that unigene containing SSR marker are subordinate to the critical stress-responsive terms such as binding, cell, cell parts and metabolic. Also, according to the results of this study, it can be stated that most of the identified EST-SSR markers were found in genes that play an important role in responding to cold stress, and UTR regions is often possible position. Hence, more analysis of these areas in candidate gene transcripts in response to cold stress is more important.

کلیدواژه‌ها [English]

  • Cold stress
  • Lentil
  • Tri-nucleotide repeat motifs
  • EST-SSR markers
Alisoltani A, Ebrahimi S, Azarian S, Hematyar M, Shiran B, Jahanbazi H, Fallahi H, Mousavi-Fard S, Rafiei F (2016) Parallel consideration of SSRs and differentially expressed genes under abiotic stress for targeted development of functional markers in almond and related Prunus species. Sci. Hortic. 198: 462-472.

Al-Quraan NA, Ghunaim AI, Alkhatib RQ (2015) The influence of chlorsulfuron herbicide on GABA metabolism and oxidative damage in lentil (Lens culinaris Medik) and wheat (Triticum aestivum L.) seedlings. Acta Physiol. Plant. 37: 227.

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman, DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.

Alves‐Carvalho S, Aubert G, Carrère S, Cruaud C, Brochot AL, Jacquin F, Klein A, Martin C, Boucherot K, Kreplak J (2015) Full‐length de novo assembly of RNA‐seq data in pea (Pisum sativum L.) provides a gene expression atlas and gives insights into root nodulation in this species. Plant J. 84: 1-19.

Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 9: 208-218.

Barrios A, Caminero C, García P, Krezdorn N, Hoffmeier K, Winter P, Pérez de la Vega M (2017) Deep Super-SAGE transcriptomic analysis of cold acclimation in lentil (Lens culinaris Medik.). BMC Plant Biol. 17: 111.

Buchwaldt L, Anderson KL, Morrall RAA, Gossen BD, Bernier CC (2004) Identification of Lentil Germ Plasm Resistant to Colletotrichum truncatum and Characterization of Two Pathogen Races. Phytopathology. 94: 236-243.

Chen J, Li R, Xia Y, Bai G, Guo P, Wang Z, Zhang H, Siddique KHM (2017) Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies. PLoS One. 12(9): e0184736.

Dai Y, Su W, Yang C, Song B, Li Y, Fu Y (2017) Development of Novel Polymorphic EST-SSR Markers in Bailinggu (Pleurotus tuoliensis) for Crossbreeding. Genes, 8: 325.

Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res, 38: 64-70.

Erskine W (2009) The Lentil: Botany, Production and Uses, CABI, Wallingford, Oxfordshire.

Gupta DS, Cheng P, Sablok G, Thavarajah P, Coyne CJ, Kumar S, Baum M, McGee RJ (2016) Development of a panel of unigene-derived polymorphic EST–SSR markers in lentil using public database information. Crop. J, 4: 425-433.

Hamwieh A, Udupa SM, Sarker A, Jung C, Baum M (2009) Development of new microsatellite markers and their application in the analysis of genetic diversity in lentils. Breed. Sci, 59: 77-86.

Kaplan F, Kopka J, Sung DY, Zhao W, Popp M, Porat R, Guy CL (2007) Transcript and metabolite profiling during cold acclimation of Arabidopsis reveals an intricate relationship of cold‐regulated gene expression with modifications in metabolite content. Plant J. 50: 967-981.

Kaur S, Cogan NOI, Pembleton LW, Shinozuka M, Savin KW, Materne M, Forster JW (2011) Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery. BMC Genomics. 12:265

Khazaei H, Caron CT, Fedoruk M, Diapari M, Vandenberg A, Coyne CJ, McGee R, Bett KE (2016) Genetic diversity of cultivated lentil (Lens culinaris Medik.) and its relation to the world's agro-ecological zones. Front. Plant Sci. 7:1093.

Kumar S, Barpete S, Kumar J, Gupta P, Sarker A (2013) Global lentil production: constraints and strategies. SATSA Mukhapatra–Annu Tech. 17: 1-13.

Kumar S, Hamwieh A, Manickavelu A, Kumar J, Sharma T, Baum M (2014) Advances in lentil genomics. In: Gupta S, Nadarajan N, Gupta D S (eds) Legumes in the Omic Era, Springer, New York. pp 111-130.

Long Y, Zhang J, Tian X, Wu S, Zhang Q, Zhang J, Dang Z, Pei XW (2014) De novo assembly of the desert tree Haloxylon ammodendron (C. A. Mey.) based on RNA-Seq data provides insight into drought response, gene discovery and marker identification. BMC Genomics. 15:1111.

Luro FL, Costantino G, Terol J, Argout X, Allario T, Wincker P, Talon M, Ollitrault P, Morillon R (2008) Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other Citrus species and their effectiveness for genetic mapping. BMC Genomics. 9:287.

Majnoon Hosseini N (2008) Legumes in Iran. Jihad Daneshgahi Press, Tehran.

Pérez-de-Castro AM, Vilanova S, Cañizares J, Pascual L, Blanca JM, Díez MJ, Prohens J, Picó B (2012) Application of genomic tools in plant breeding. Curr. Genomics. 13: 179-195.

Powell W, Machray GC, Provan J (1996) Polymorphism revealed by simple sequence repeats. Trends Plant Sci. 1: 215-222.

Primmer CR (2009) From conservation genetics to conservation genomics. Ann. Ny. Acad, Sci., 1162: 357-368.

Sabaghpour S, Seyedi F, Mahmoodi A, Safikhani M, Pezeshkpour P, Rostemi B, Kamel M, Ferayedi Y, Alahyari N, Poursiabidi M (2013) Cultivar release: kimiya, a new high yielding lentil cultivar for moderate cold and semi warm climate of iran. Seed. Palt. Improve J. 29: 397-399.

Saha GC, Vandemark GJ (2013) Stability of Expression of Reference Genes Among Different Lentil (Lens culinaris) Genotypes Subjected to Cold Stress, White Mold Disease, and Aphanomyces Root Rot. Plant Mol. Biol. Report., 31: 1109-1115.

Singh D, Singh CK, Taunk J, Tomar RSS, Chaturvedi AK, Gaikwad K, Pal M (2017) Transcriptome analysis of lentil (Lens culinaris Medikus) in response to seedling drought stress. BMC Genomics. 18:206.

Srivastava R, Vasishtha H (2012) Saponins and lectins of Indian chickpeas (Cicer arietinum) and lentils (Lens culinaris). Indian J. Agric. Biochem. 25: 44-47.

Sudheesh S, Verma P, Forster JW, Cogan NOI, Kaur S (2016) Generation and characterisation of a reference transcriptome for lentil (Lens culinaris Medik.). Int. J. Mol. Sci. 17(11): 1887.

Sultana R, Choudhary AK, Pal AK, Saxena KB, Prasad BD, Singh R (2014) Abiotic Stresses in Major Pulses: Current Status and Strategies. Springer, New Delhi.

Thiel T, Michalek W, Varshney R, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor. Appl. Gent. 106: 411-422.

Tian DQ, Pan XY, Yu YM, Wang WY, Zhang F, Ge YY, Shen XL, Shen FQ, Liu XJ (2013) De novo characterization of the Anthurium transcriptome and analysis of its digital gene expression under cold stress. BMC Genomics. 14(1): 827.

Varshney RK, Graner A, Sorrells ME (2005) Genic microsatellite markers in plants: features and applications. Trends Biotechnol. 23: 48-55.

Varshney RK, Tuberosa R (2007) Genomics-Assisted Crop Improvement. Springer, Netherlands.

Verma P, Shah N, Bhatia S (2013) Development of an expressed gene catalogue and molecular markers from the de novo assembly of short sequence reads of the lentil (Lens culinaris Medik.) transcriptome. Plant Biotechnol. J. 11: 894-905.

Wang P, Yang L, Zhang E, Qin Z, Wang H, Liao Y, Wang X, Gao L (2017a) Characterization and development of EST-SSR markers from a cold-stressed transcriptome of centipedegrass by Illumina paired-end sequencing. Plant Mol. Biol. Rep. 35: 215-223.

Wang S, Zhou G, Huang X, Hu J, Wang B, Lin C, Li X, Jia Y, Wang A (2017b) Transcriptome analysis of non-heading Chinese cabbage under heat stress by RNA-seq and marker identification. Euphytica. 213:109.

Wang Z, Li, J Luo Z, Huang L, Chen X, Fang B, Li Y, Chen J, Zhang X (2011) Characterization and development of EST-derived SSR markers in cultivated sweetpotato (Ipomoea batatas). BMC Plant Biol. 11:139.

Wu R, Wang T, Warren BAW, Allan AC, Macknight RC, Varkonyi-Gasic E (2017) Kiwifruit SVP2 gene prevents premature budbreak during dormancy. J. Exp. Bot. 68: 1071-1082.

Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell. 14: 165-183.

Xu X, Zhou C, Zhang Y, Zhang W, Gan X, Zhang H, Guo Y, Gan S (2018) A novel set of 223 EST-SSR markers in Casuarina L. ex Adans. polymorphisms, cross-species transferability, and utility for commercial clone genotyping. Tree Genet. Genomes. 14: 30.

Yilmaz Temel H, Göl D, Kaya Akkale HB, Kahriman A, Tanyolaç MB (2015) Single nucleotide polymorphism discovery through illumina-based transcriptome sequencing and mapping in lentil. Turk. J. Agric. For. 39: 470-488.

Zheng X, Pan C, Diao Y, You Y, Yang C, Hu Z (2013) Development of microsatellite markers by transcriptome sequencing in two species of Amorphophallus (Araceae). BMC Genomics. 14:490.