ارزیابی تنوع ژنتیکی Lotus corniculatus L. در خوزستان با استفاده از نشانگرهای مولکولی و DNA بارکد

نوع مقاله : مقاله پژوهشی

نویسندگان

گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه شهید چمران اهواز، اهواز، ایران

10.22103/jab.2025.24998.1681

چکیده

چکیده
هدف: گونه .Lotus corniculatus L یک لگوم چندساله، تتراپلوئید و با پراکنش گسترده است که به‌دلیل سازگاری با شرایط متنوع محیطی و کاربردهای آن در تولید علوفه، بهبود خاک و مصارف دارویی، ارزش بالایی دارد. ارزیابی تنوع ژنتیکی این گونه با استفاده از نشانگرهای مولکولی، نقش کلیدی در تدوین راهبردهای اصلاح نژاد و حفاظت ژرم‌پلاسم ایفا می‌کند. این مطالعه، نخستین بررسی مولکولی این گونه را در استان خوزستان، جنوب‌غرب ایران، ارائه می‌دهد.
مواد و روش‌ها: در این مطالعه، ۲۱ اکسشن L. corniculatus از هشت منطقه جغرافیایی متفاوت در خوزستان جمع‌آوری شد. برای بررسی تنوع ژنتیکی، از ۱۲ آغازگر SCoT و ۶ آغازگر ISSR استفاده گردید. روابط و ساختار ژنتیکی با بهره‌گیری از تحلیل خوشه‌ای، تحلیل مختصات اصلی (PCoA) و نرم‌افزار STRUCTURE بررسی گردید. به منظور تکمیل تحلیل مبتنی بر نشانگر، سه اکسشن دارای بیش‌ترین فاصله ژنتیکی برای توالی‌یابی دو ناحیه بارکدی ITS و trnH-psbA انتخاب شدند. جایگاه فیلوژنتیکی توالی‌های به‌دست‌آمده با BLAST و تحلیل Maximum Likelihood بررسی شد.
نتایج: نشانگرهای SCoT و ISSR به ترتیب 87/96 درصد و 8/85 درصد چندشکلی را نشان دادند که بیانگر تنوع ژنتیکی قابل توجه در میان اکسشن‌ها بود. تحلیل خوشه‌ای و PCoA دو گروه ژنتیکی اصلی را شناسایی کردند به طوری‌که اکسشن جزیره مینو یک خوشه متمایز را تشکیل داد. تحلیل STRUCTURE وجود سه گروه ژنتیکی را تأیید کرد که بیانگر هم‌آمیختگی ژنتیکی و گروه‌بندی‌های مجزا در میان اکسشن‌ها بود. توالی‌یابی بارکدهای DNA نشان داد که هر سه اکشن منتخب (شامل اکسشن جزیره مینو) دارای توالی یکسان در نواحی ITS و trnH-psbA بودند که به‌عنوان هاپلوتیپ ایرانی تعریف شد. توالی ITS شباهت 71/99 درصدی با L. tenuis و ناحیه trnH-psbA تطابق کامل (۱۰۰ درصد) با L. japonicus را نشان داد. با این حال، این نواحی حفاظت شده توان تفکیک مناسبی در سطح درون‌گونه‌ای نداشتند.
نتیجه‌گیری: هر دو نشانگر عملکرد مناسبی داشتند؛ نشانگرهای SCoT مقادیر بالاتری از درصد چندشکلی و میانگین محتوای اطلاعات چندشکلی را نشان دادند؛ در حالی که نشانگرهای ISSR قدرت تفکیک و شاخص نشانگر بیش‌تری داشتند. تمایز ژنتیکی آشکار اکسشن‌‌ جزیره مینو نشان‌دهنده‌ی سازگاری محلی و ضرورت حفاظت منطقه‌ای است. در مقابل، عدم تنوع در توالی‌های بارکدی، محدودیت نواحی محافظت‌شده مانند ITS و trnH-psbA را در شناسایی ساختار ژنتیکی ظریف نشان می‌دهد. استفاده از ابزارهای مکمل مانند شمارش کروموزومی و نشانگرهای ژنومی (تعیین ژنوتیپ SNP، توالی‌یابی RAD و توالی‌یابی کل ژنوم) برای بهبود تفکیک تاکسونومیکی پیشنهاد می‌شود.

کلیدواژه‌ها

عنوان مقاله [English]

Genetic Diversity Assessment of Lotus corniculatus L. in Khuzestan Using Molecular Markers and DNA Barcoding

نویسندگان [English]

  • Zahra Sadat Mousavi
  • Fatemeh Nasernakhaei
Department of Plant Production and Genetics, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran
چکیده [English]

Abstract
Objective
Lotus corniculatus L. is a widely distributed, tetraploid perennial legume valued for its adaptability to diverse environments and its applications in forage production, soil improvement, and medicine. It is essential to assess its genetic diversity using molecular markers for conservation and breeding strategies. This study presents the first molecular characterization of L. corniculatus accessions from Khuzestan province in southwestern Iran.
Materials and methods
Twenty-one L. corniculatus accessions were collected from eight geographically distinct sites. Genetic variation was assessed using 12 SCoT and 6 ISSR primers. Genetic structure and relationships among accessions were analyzed using cluster analysis, principal coordinate analysis (PCoA), and the STRUCTURE software. To complement these analyses, three genetically divergent accessions were selected for sequencing of two DNA barcode regions: the internal transcribed spacer (ITS) of nuclear ribosomal DNA and the trnH-psbA intergenic spacer of the chloroplast genome. Barcoded sequences were analyzed using BLAST and Maximum Likelihood phylogenetic reconstruction.
Results
SCoT and ISSR markers revealed high polymorphism (96.87% and 85.8%, respectively), indicating substantial genetic diversity among the accessions. Cluster and PCoA consistently grouped the accessions into two main clusters, with the Minoo Island accession forming a distinct and divergent group. STRUCTURE analysis supported the existence of three genetic clusters, reflecting both admixture and clear genetic differentiation. DNA barcoding revealed that all three sequenced accessions, including the genetically distinct Minoo Island accession, shared identical sequences in both the ITS and trnH-psbA regions, forming a single haplotype, here designated as the Iranian haplotype. The ITS sequence exhibited 99.71% identity with L. tenuis, while the trnH-psbA region showed 100% identity with L. japonicus. However, both barcode regions lacked sufficient resolution to distinguish intraspecific variation.
Conclusions
Both marker systems were effective in detecting genetic diversity, with SCoT markers showing higher average polymorphism and mean PIC values, and ISSR markers exhibiting stronger marker index and resolving power. The distinct clustering of the Minoo Island accession highlights the potential for local adaptation and underlines the importance of conserving regional germplasm. In contrast, the lack of variation in the barcode sequences emphasizes the limited ability of conserved regions such as ITS and trnH-psbA to resolve fine-scale genetic structure. Complementary tools such as chromosome counting and genome-wide markers (e.g., SNP genotyping, RAD-seq, WGS) are recommended to improve taxonomic resolution.

کلیدواژه‌ها [English]

  • Bird's-foot trefoil
  • Fingerprinting
  • Genetic differentiation
  • Genetic resource management
  • Phylogenetic relationships

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مجله بیوتکنولوژی کشاورزی
دوره 17، شماره 3
شهریور 1404
صفحه 113-134

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