مطالعه وجود snpها در ژن‌های sunو ovateدخیل در شکل میوه گوجه‌فرنگی

شکل میوه از صفات بسیار مهم و موثر در کیفیت گوجه فرنگی (solanum lycopersicum l.) بوده و ژن های sunو ovate از ژن های کلیدی کنترل کننده طول میوه می باشند. به منظور شناسایی snpها (single nucleotide polymorphism) در این ژن ها، قطعاتی از نواحی کد کننده آنها در 96 ژنوتیپ گوجه فرنگی تکثیر و با آنزیم های محدود کننده trui وpsti  هضم شد. هضم قطعات تکثیری این دو ژن در ژنوتیپ ها چندشکلی تولید نکرد بنابراین چهار ژنوتیپ از جمعیت های مختلف انتخاب و قطعه تکثیری ژن ها در این ژنوتیپ ها توالی یابی شد. همردیفی توالی ها برای شناسایی snpها با استفاده از نرم افزار clustal omega انجام گرفت. توالی یابی قطعه تکثیری ژن sun منجر به شناسایی یک ناحیه اینترونی به طول 369 جفت باز شد. بر اساس نتایج، در ژن sun ده snp شناسایی شد. از کل snpهای شناسایی شده در این ژن، 80 درصد آنها از نوع همجنس و 20 درصد آنها از نوع غیرهمجنس بود. در ژن ovate پنج snp شناسایی شد که 80 درصد آنها از نوع همجنس و 20 درصد آنها از نوع غیرهمجنس بود. میانگین تعداد snpها به ازای هر 100 جفت باز در نواحی اگزونی و اینترونی ژن sun به ترتیب 0.9 و 1.62 بود. در ژن ovate به ازای هر 100 جفت باز نیمsnp  در ناحیه اگزونی شناسایی شد. با توجه به نقش مهم کیفیت میوه بخصوص شکل آن در بازارپسندی گوجه فرنگی، snpهای شناسایی شده در این تحقیق می تواند در برنامه های اصلاحی گوجه فرنگی برای مطالعه تنوع ژنتیکی، تهیه نقشه های ژنتیکی و شناسایی نشانگرهای عملکردی مرتبط با شکل میوه مورد استفاده قرار گیرد.

Studying SNPs in SUN and OVATE Genes Responsible for Fruit Shape in Tomato

Introduction: Commercial tomato (Solanum lycopersicum L.), one of the most widely grown vegetable crops worldwide, belongs to the Solanaceae family. The marketability of the commercial tomato mostly depends on the fruit quality. Tomato fruit quality is determined mainly by color, texture, shape and flavor. Fruit shape, one of the important traits affecting the quality of tomato fruit, is controlled by multiple minor genes and quantitatively inherited. Two important genes, involved in fruit shape, are SUN and OVATE genes. The SUN gene, which is a member of the IQD (IQdomain) gene family and the Calmodulin binding protein, controls fruit length. The more expression of both SUN and OVATE genes leads to increased fruit length. Moreover, the increased expression of OVATE gene reduces the size of flower and leaf components. Due to the important role of these genes in tomato fruit shape, identification of single nucleotide polymorphisms (SNPs) as a new generation of robust, frequent and reliable biallelic markers, in the coding regions of these genes might be necessary for generating functional markers associated with fruit shape. Materials and Methods: Seeds of 96 tomato genotypes from 12 populations were grown in the research greenhouse of Faculty of Agriculture and Natural Resources of Urmia University. The genotypes had been collected from different regions of West Azerbaijan of Iran and Turkey (Iğdır). The young and green plant leaves were used for genomic DNA extraction. The quality and quantity of the extracted DNA was assessed using spectrophotometry and agarose gel electrophoresis. To identify SNPs in SUN and OVATE genes, specific primers were designed by using FastPCR and Gene Runner software for amplifying fragments from coding regions of these genes in 96 tomato genotypes. Then, the amplified fragments of both genes were digested by using restriction enzymes TruI and PstI. Due to the lack of polymorphism in the digested patterns obtained by the used enzymes, four individuals from populations with close geographical distance were selected and amplified. The amplified bands were then purified by a purification kit (Kiagen, USA) and sequenced (Bioneer, South Korea). Sequencing was performed from both ends of the PCR fragments using both the forward and reverse primers used in the PCR reactions. The exon and intron regions of the sequenced fragments were identified by Softberry software. Following the retrieval of the sequenced fragments of each gene using FastPCR and Softberry software, multiple sequence alignment using Clustal Omega was used to identify SNPs in the exon and intron of the genes. Results and Discussion: Digestion of the amplified fragments of the genes using TruI and PstI restriction enzymes produced no polymorphism in the studied genotypes. Thus, four individuals were selected from geographically different populations and gene fragments were amplified, purified and sequenced in these genotypes. Sequencing of the amplified fragment of SUN gene revealed an intron region with a size of 369 bp. Out of the 10 SNPs detected in the SUN gene, four was found in the exon region, while the number of SNPs in intron was six. Of the total SNPs found in the SUN gene, the percentage of transition and transversion substitutions was 80 (50% T/C and 30% A/G) and 20 (T/G), respectively. In the OVATE gene, five SNPs were identified. The percentage of transition (40% G/A and 40% C/T) and transversion (20% G/T) substitutions in this genes were the same as SUN. The ratio of transition to transversion substitutions was 1:4 for both genes. The average number of SNPs in a 100 bp fragment in exonic and itronic region of SUN was 0.9 and 1.62, respectively, while it was 0.5 for exonic region of OVATE gene. Conclusion: The results of the current study revealed low polymorphisms and point mutations in the exon regions of SUN and OVATE genes, suggesting that the coding regions of these genes were conserved during the tomato evolution. Also, the number of SNPs in intron was more than those of exon. Considering the important role of fruit quality, especially fruit shape, in tomato market, the SNPs found in the current study may be used in genetic diversity studies, genetic map preparation, and saturation and identification of the functional markers associated with tomato fruit shape. These markers could accelerate tomato breeding programs aimed fruit shape improvement.