Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ برآورد میزان تنوع ژنتیکی در ذخیره مولدین فیل‌‌ماهی (Huso huso Linnaeus, 1758) با استفاده از داده‌های حاصل از توالی یابی ژنوم Genomic-based investigation of genetic diversity in broodstock of beluga (Huso huso Linnaeus, 1758) ژنتيك و اصلاح نژاد ژنتيك و اصلاح نژاد پژوهشي Research فیل‌‌ماهی به عنوان اصلی‌ترین گونه پرورشی از ماهیان خاویاری در ایران مطرح بوده که ذخیره مولدین کنونی آن از تعداد محدودی مولد وحشی در دو گذشته ایجاد شده است. فقدان گونه‌‌های شناسنامه‌دار یکی از مهم‌ترین عوامل محدودکننده در توسعه صنعت آبزی‌پروری ماهیان خاویاری در ایران بوده است. از این‌رو، در پروژه حاضر و برای اولین بار از تکنیک توالی‌‌یابی مبتنی بر ژنوم<a href="#_ftn1" name="_ftnref1" title="">[1]</a> به منظور برآورد تنوع ژنتیکی و تهیه شناسنامه مولکولی مبتنی بر ژنوم از ذخیره مولدین فیل‌‌ماهی موجود در انستیتو ماهیان خاویاری، استفاده شد. توالی‌‌یابی ژنومی منجر به تولید GB294 داده حاصل از 261974459 خوانش ژنومی شد که در نهایت باعث شناسایی 1836 نشانگر SNP<a href="#_ftn2" name="_ftnref2" title="">[2]</a> در ژنوم فیل‌‌ماهی شد که بیشترین تعداد آن بر کروموزوم شماره 58 مشاهده شد. نتایج حاصل از تجزیه‌‌وتحلیل SNPs کشف‌‌شده نشان داد که میزان هتروزیگوسیتی موردمشاهده در ذخیره مولدین فیل‌‌ماهی موجود در انستیتو، 35/0 است. همچنین 105 آلل در ذخیره مولدین مورد بررسی یافت شد که بیانگر تنوع آللی مناسب در این گونه بود. نتایج حاصل از روابط هم‌‌تباری با استفاده از مارکرهای SNP شناسایی شده برای اولین بار نشان داد که ذخیره ژنتیکی مولدین فیل‌‌ماهی در انستیتو در دو خوشه ژنتیکی، قابل تقسیم بندی هستند. میزان همخونی در مولدین فیل‌‌ماهی مورد بررسی 02/0 برآورد گردید که این میزان پایین همخونی در کنار ساختار جمعیتی مختلط مورد مشاهده در آزمون PCA می‌تواند بیانگر تنوع بالا در مولدین وحشی ایجادکننده جمعیت حاضر باشد. با توجه به دسترسی بسیار محدود به مولدین وحشی برای غنی‌سازی خزانه ژنی گله‌های پرورشی، پیشنهاد می‌‌گردد تا با استفاده از روش‌‌های نوین مبتنی بر ژنومیکس در زمینه شناسایی خزانه‌های ژنی، حفظ ساختارهای ژنتیکی و تکثیر هدفمند بر اساس اطلاعات ژنتیکی در بازسازی ذخایر و آبزی‌پروری اقدام گردد. <div>  <hr align="left" size="1" width="33%" > <div id="ftn1"><a href="#_ftnref1" name="_ftn1" title="">[1]</a> Genotyping-by sequencing</div> <div id="ftn2"><a href="#_ftnref2" name="_ftn2" title="">[2]</a> Single nucleotide polymorphism (SNP)</div> </div> Introduction Sturgeons (Acipenseriformes) are of the most commercially and scientifically important fish species in the globe. These fish are mostly anadromous, spending most of the life cycle in the marine and brackish waters and migrating to the freshwater rivers to spawn (Kottelat and Freyhof, 2007). The Caspian Sea basin has been the main refuge for six species of sturgeons namely Huso huso, Acipenser persicus, Acipenser stellatus, Acipenser guldenstaedtii, Acipenser nudiventris and Acipenser ruthenus, all of which under critically endangered level of the IUCN red list (IUCN, 2025). Generally speaking, the number of sturgeon species has declined, and the beluga (H. huso) is currently in the most critical situation due to a decline in the number of adults leading to local extinctions of the wild populations (Dudu et al., 2014). It is worth noting that female beluga does not spawn every year and the optimal reproductive performance of these fish occurs at an age equivalent to twice the age of first sexual maturity, which can be considered as a limiting factor in the survival of this species considering the shrinking populations (Boscari et al., 2021). Several factors such as overfishing and poaching for trade of caviar and meat, dam construction, loss of nursery grounds, pollution, global warming, and reduced volume of freshwater flowing into the seas are considered to be the most important components in the decline of wild sturgeon populations. In order to compensate the loss of wild populations of H. huso and on the other hand providing the human society required caviar and protein, Iranian Fisheries Organization started the restocking activity since five decades ago and stablished sturgeon aquaculture from two decades ago. Due to the sharp decrease in wild mature beluga of the southern Caspian Sea during the last decades, nowadays most of the aquaculture and restocking activities are forced to be focused on the brood stock generated from few wild parents in the past. Therefore, it is necessary to use modern molecular methods and prepare a genomic-based certificate to protect genetic reserves and enhance the genetic diversity of the native beluga in the south of the Caspian Sea. Genetic diversity represents the variations in the number and type of alleles available in chromosomal loci, which is also considered the main basis for the adaptation of species and populations against changing environmental conditions. While several molecular markers such as SSR, AFLP, and mtDNA, have been widely used during the four decades in fisheries studies (Robledo et al., 2018), these days due to the revolution in the genome sequencing technologies (NGS) more robust genotyping methods such as Genotyping-by Sequencing (GBS) can provide more diverse, sensitive and accurate estimations by screening thousands of variant markers throughout the whole genome of organisms even in species without reference genome (Sonah et al., 2013; Andrews et al., 2016; Barría et al., 2018; Liu et al., 2018). Therefore, during the present study for the first we used a genome-based technique known as GBS in order to estimate the amount of genetic diversity and identify possible genetic nuclei in H. huso brood stock available at International Sturgeon Research Institute (ISRI). Methodology The feeding was stopped a week before sampling and each fish specimen was PIT tagged for later traceability purposes. Caudal fin tissues of 23 Huso huso was sampled and preserved in absolute ethanol for later molecular experiments. DNA extraction was done from the fin tissues through Phenol-Chloroform method with minor modifications (Jafari et al., 2022) and their quality and quantity in terms of DNA integrity, purity and concentration were determined using 0.8% agarose gel and Nano Drop instrument (ND 1000). After DNA samples were qualitatively approved, DNA pellets were sent to Novogene for 150bp PE sequencing on a lane of Illumina Novaseq 6000. Quality of the raw reads generated by sequencing were assessed through Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and only window reads with mean quality of 20 were kept in the final dataset. The clean data set then mapped to the genome reference of Huso huso using Bowtie2 (Langmead and Salzberg, 2012) and the SNP calling was performed based on Stacks. After conducting SNP calling, SNPs were filtered based on parameters such as MAF of 0.05, LD and Hardy-Weinberg equilibrium. Dispersal of fish individuals were visualized using Principal Component Analysis (PCA) based on the two first components in R (Team, 2013). Furthermore, FineRADstructure was used to illustrate the probable genetic clusters based on coancestry information through SNPs (Malinsky et al., 2018). Results The sequence quality analysis showed that the quality of the sequences was high with an average of 92.55. In total, 294 Gb of DNA data obtained from GBS libraries contained an average of 41% guanine (G) and cytosine (C) bases in the genome of Huso huso. PE genomic sequencing produced 261,974,459 reads, with an average alignment of 90% to the reference genome (Table 1). The average number of reads obtained from DNA sequencing in each specimen was about 11 million reads. The results of variant calling showed that a total of 1836 SNP markers with a minimum allele frequency of 0.05 were called from genome sequencing in H. huso with the maximum number of SNPs detected on chromosome 58 (Fig. 1). Based on the results obtained from SNPs obtained from genomic sequencing, the amount of genetic diversity observed in the H. huso broodstock was 0.35. Also, the inbreeding and nucleotide diversity were calculated to be 0.02 and 0.28, respectively (Table 2). The distribution of genetic diversity in the H. huso broodstock using the two first components of PCA is depicted in Figure 2. Based on the results of the PCA, the first two components accounted for 31.89% of the variance. The results of the phylogenetic relationships based on coancecstry relationship also showed that the studied broodstock of H. huso can be considered in two main genetic sub-groups (Fig. 3). Discussion and conclusion Identifying genetic reserves and maintenance of genetic diversity are among the most important measures in managing broodstock either in a sustainable aquaculture activity or in a restocking program. In sturgeons’ aquaculture, there is a particular concern about the reduction of genetic diversity in intensive beluga farming. Preservation of an appropriate level of genetic diversity within the population is of great importance in diminishing negative effects of inbreeding and genetic drift. In the present study, and to the best of our knowledge for the first time in Iran, the identification of genetic groups in the broodstock of Huso huso available at the International Sturgeon Research Institute was carried out using GBS method. Accordingly, creation of a genetic certificate using 1836 SNP markers obtained from GBS was successfully implemented on 23 H. huso. Based on the obtained results from 1836 SNP marker, the observed heterozygosity was 0.36 in the investigated broodstock of H. huso. Observed heterozygosity is the most common indicator of genetic diversity used in determining the genetic health of fish broodstocks. Alongside with the observed heterozygosity, allelic richness should also be taken into consideration in assessment of genetic diversity. Results of the SNP markers showed a considerable number of alleles (An= 105) in the studied broodstock of H. huso, indicating a satisfactory level of heterogeneity in this broodstock. This was also supported by the small value of inbreeding (Fis= 0.02). The UPGMA tree based on coancestry information revealed an admixture pattern of genetic clustering in H. huso. It seems that wild parents from different populations had been used in generating the broodstock of H. huso at ISRI during the past decades. However, based on the genetic clustering heatmap it is suggested to consider two genetic nuclei in the investigated broodstock of H. huso in order to keep the genetic diversity for future sustainable aquaculture purposes. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment Hereby we wish to appreciate Iranian Fisheries Science Research Institute and Iranian Fisheries Organization for their financial support and also providing laboratory required equipment to conduct the current project. چندشکلی تک‌نوکلئوتیدی, خزانه ژنی, درون‌آمیزی, ژنومیکس, ماهیان خاویاری Gene pool, Genomics, Inbreeding, SNP, Sturgeons 19 28 http://isfj.ir/browse.php?a_code=A-10-2919-2&slc_lang=fa&sid=1 Omid Jafari امید جعفری jaafari.omi@yahoo.com 100319475328460040940 100319475328460040940 Yes Agricultural Research, Education and Extension Organization انستیتو تحقیقات بین المللی ماهیان خاویاری، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، رشت، ایران Naser Karamirad ناصر کرمی راد Naserkaramiradddd@yahoo.com 100319475328460040941 100319475328460040941 No سازمان شیلات ایران Mehdi Golshan مهدی گلشن Mahdigolshan@yahoo.com 100319475328460040942 100319475328460040942 No مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران Alinaghi Sarpanah علی نقی سرپناه Alinaghisarpanahhh@yahoo.com 100319475328460040943 100319475328460040943 No انستیتو تحقیقات بین المللی ماهیان خاویاری، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، رشت، ایران Mehrshad Zeinolabedini مهرشاد زین العابدینی Mzeinalabediniii@yahoo.com 100319475328460040944 100319475328460040944 No پژوهشگاه بیوتکنولوژی کشاورزی ایران، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران Esmaeil Abdollahzadeh اسماعیل عبداله زاده abdolahzadehhhh@rocketmail.com 100319475328460040945 100319475328460040945 No انستیتو تحقیقات بین المللی ماهیان خاویاری، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، رشت، ایران Mohammad Hassanzadeh Saber محمد حسن زاده صابر meraj.saberrrr@yahoo.com 100319475328460040946 100319475328460040946 No انستیتو تحقیقات بین المللی ماهیان خاویاری، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، رشت، ایران Maryam Nasrolahpourmoghadam مریم نصراله پورمقدم Pourmoghadamm@yahoo.com 100319475328460040947 100319475328460040947 No Iranian Fisheries Organization سازمان شیلات ایران Mehdi Shakoori مهدی شکوری mshakouriiiii@yahoo.com 100319475328460040948 100319475328460040948 No سازمان شیلات ایران Mahmood Mohseni محمود محسنی mohseniiii@yahoo.com 100319475328460040949 100319475328460040949 No انستیتو تحقیقات بین المللی ماهیان خاویاری، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، رشت، ایران