شناسایی مولکولی و تحلیل تنوع ژنتیکی Ocimum basilicum L. با استفاده از توالی‌های کلروپلاستی matK و نشانگرهای SNP در سطح کل ژنوم

مقالات آماده انتشار

نوع مقاله : مقاله پژوهشی

نویسندگان

دانشکده علوم، دانشگاه المستنصریه، بغداد، عراق.

10.22103/jab.2026.27125.1888

چکیده

هدف: ریحان شیرین (Ocimum basilicum L.) یکی از گیاهان معطر و اقتصادی مهم است که برای بهینه‌سازی برنامه‌های اصلاح نژادی و حفاظت از ذخایر ژرم‌پلاسم، نیازمند مطالعات ژنتیکی جامع می‌باشد. هدف این پژوهش، بررسی تنوع ژنتیکی و ساختار جمعیتی ریحان‌های کشت‌شده با استفاده از ترکیب روش بارکدگذاری DNA کلروپلاستی (matK) و نشانگرهای تک‌نوکلئوتیدی گسترده در سراسر ژنوم (SNP) بود.
مواد و روش‌ها: در این مطالعه، 30 نمونه ژنتیکی (اکسشن) مختلف از O. basilicum مورد بررسی قرار گرفتند (10 اکسشن از هر یک از سه منطقه جغرافیایی متفاوت در بغداد، عراق). ژن matK از تمامی نمونه‌ها تکثیر و با استفاده از روش تعیین توالی سانگر (Sanger Sequencing) توالی‌یابی شد. همچنین، نشانگرهای SNP ژنومی با استفاده از روش تعیین ژنوتیپ مبتنی بر توالی‌یابی (GBS؛ Genotyping-by-Sequencing) تولید شدند. پس از کنترل کیفیت داده‌ها، تعداد 6234 نشانگر SNP با کیفیت بالا برای تحلیل‌های بعدی تأیید گردید. تنوع ژنتیکی، ساختار جمعیت و میزان تمایز ژنتیکی با استفاده از تحلیل هاپلوتیپی، نرم‌افزار STRUCTURE، تحلیل مختصات اصلی (PCA)، تحلیل واریانس مولکولی (AMOVA) و شاخص‌های Fst مورد ارزیابی قرار گرفت.
نتایج: توالی‌یابی ژن matK وجود تنوع ژنتیکی متوسط را در قالب هشت هاپلوتیپ نشان داد (Hd = 0.685 ± 0.045؛ π = 0.0076 ± 0.0012). در مقابل، تحلیل SNPها کمبود قابل توجه هتروزیگوت‌ها را آشکار ساخت (Ho = 0.092 ± 0.038؛ He = 0.251 ± 0.072؛ Fis = 0.634 ± 0.089؛ P < 0.001). خوشه‌بندی بیزی سه جمعیت ژنتیکی متمایز (K = 3) را شناسایی کرد. نتایج تحلیل AMOVA نشان داد که 3/24 درصد از کل تنوع ژنتیکی به تفاوت میان جمعیت‌ها مربوط می‌شود (Φst = 0.243؛ P < 0.001). به‌طور کلی، نشانگرهای SNP نسبت به ژن matK قدرت تفکیک بیشتری داشتند و همبستگی متوسطی میان روابط ژنتیکی حاصل از این دو روش مشاهده شد (Mantel r = 0.523؛ P = 0.002).
نتیجه‌گیری: نتایج این مطالعه نشان می‌دهد که سه جمعیت ژنتیکی متمایز با جریان ژنی محدود وجود دارند که برای حفظ تنوع ژنتیکی، نیازمند برنامه‌های حفاظتی مستقل هستند. کمبود چشمگیر هتروزیگوت‌ها بیانگر آن است که تلاقی بین جمعیت‌های ژنتیکی متمایز می‌تواند موجب افزایش هتروزیس (برتری دورگه‌ای) در برنامه‌های اصلاح نباتات شود. یافته‌های این پژوهش بینش‌های جدیدی درباره ساختار ژنتیکی ریحان کشت‌شده فراهم می‌کند؛ از جمله وجود سه خوشه ژنتیکی مشخص، کمبود ساختارمند هتروزیگوت‌ها و همخوانی نسبی میان ژنوم کلروپلاستی و ژنوم هسته‌ای. این نتایج می‌توانند در درک بهتر جریان ژنی، اثرات اهلی‌سازی، تمایز جمعیت‌ها و تدوین راهبردهای آینده برای اصلاح و حفاظت ژنتیکی ریحان مؤثر باشند.

کلیدواژه‌ها

عنوان مقاله [English]

Molecular identification and genetic diversity analysis of Ocimum basilicum L. using chloroplast matK sequences and Genome-wide SNP markers

نویسندگان [English]

  • Ola Hatif Hazim
  • Maysam Ihsan Ali Al-Hassany
  • Zinah Ameer Abbood
College of Science, Mustansiriyah University, Baghdad, Iraq.
چکیده [English]

Objective
The economically significant aromatic crop sweet basil (Ocimum basilicum L.) needs thorough genetic analysis for breeding optimization and germplasm conservation. To clarify genetic diversity and population organization in farmed basil, we combined genome-wide SNP markers with chloroplast DNA barcoding (matK).
Materials and methods
A total of thirty different cultivated O. basilicum accessions were analyzed (10 accessions from three distinct geographic locations) in Baghdad, Iraq. The matK gene from each accession was amplified and sequenced using the Sanger sequencing. Genomic SNPs were generated using the genotyping-by-sequencing (GBS) method. After passing a quality control assessment, 6234 of the SNPs were considered valid high-quality SNPs. Genetic diversity, population structure, and differentiation were analyzed using haplotype analysis, STRUCTURE analysis, principal coordinates analysis (PCA), and statistical evaluation methods, including analysis of molecular variance (AMOVA) and Fst pairwise statistics.
Results
MatK sequencing revealed moderate genetic diversity among eight haplotypes was observed (Hd = 0.685 ± 0.045), whereas an excess of heterozygotes was identified in the SNP analysis (Ho = 0.092 ± 0.038; He = 0.251 ± 0.072; Fis = 0.634 ± 0.089; P < 0.001). Bayesian clustering suggests three genetically distinct populations (K = 3), while AMOVA results indicate 24.3% of the overall genetic variation is attributed to the population (Φst = 0.243, P < 0.001). Collectively, SNP markers exhibit relatively greater resolution than matK and demonstrate a moderate degree of correlation with matK-based genetic relationships (Mantel r = 0.523, P = 0.002).
Conclusions
Independent conservation is required for three genetically distinct populations with limited gene flow. The pronounced heterozygote deficit suggests that crosses between genetically distinct populations may enhance heterosis in breeding programs. Our results provide new biological insights into the genetic architecture of cultivated basil. These insights include the presence of three well-defined genetic clusters, a strong but structured lack of heterozygosity, and partial concordance between the chloroplast and nuclear genomes. These findings could improve our understanding of gene flow, domestication effects, and population differentiation in O. basilicum. Furthermore, they could provide a genomic framework for future breeding and conservation strategies.

کلیدواژه‌ها [English]

  • conservation genomics
  • DNA barcoding
  • heterozygote deficiency
  • population genetics
  • SNP genotyping

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