كاربرد سيستم الكتروفورز ژل عمودي پلي‌اكريل‌آميد بهينه و مقرون‌به‌صرفه با رنگ‌آميزي اتيديوم برومايد در بررسي تنوع ژنتيكي ژنوتيپ‌هاي آفتابگردان با استفاده از نشانگر مولكولي TRAP

Application of a High-throughput Polyacrylamide Electrophoresis Gel System with Ethidium Bromide Staining in Genetic Diversity Estimation of Sunflower Genotypes Using TRAP Markers

در اﯾﻦ ﭘﮋوﻫﺶ ﮐﺎرﺑﺮد روش ﺟﺪﯾﺪ اﻟﮑﺘﺮوﻓﻮرز ژل ﭘﻠﯽاﮐﺮﯾﻞآﻣﯿﺪ ﺑﺎ اﺳﺘﻔﺎده از رﻧﮓآﻣﯿﺰي اﺗﯿﺪﯾﻮم ﺑﺮوﻣﺎﯾﺪ ﺑﺮاي ﺑﺮرﺳﯽ ﺗﻨﻮع ژﻧﺘﯿﮑﯽ ژﻧﻮﺗﯿﭗﻫﺎي آﻓﺘﺎﺑﮕﺮدان ﺑﺎ اﺳﺘﻔﺎده از ﻧﺸﺎﻧﮕﺮ ﻣﻮﻟﮑﻮﻟﯽ ﻧﻮﯾﻦ TRAP اراﺋﻪﺷﺪه ﮐﻪ ﻧﺴﺒﺖ ﺑﻪ روشﻫﺎي ﻣﺘﺪاول رﻧﮓآﻣﯿﺰي ﻧﻘﺮه و ﻧﺸﺎندار ﮐﺮدن آﻏﺎزﮔﺮﻫﺎ ﺑﺎ ﻓﻠﻮرﺳﻨﺖ، داراي ﻣﺰﯾﺖﻫﺎﯾﯽ ﻫﻤﭽﻮن ﻣﻘﺮونﺑﻪﺻﺮﻓﻪ ﺑﻮدن، ﺳﺮﻋﺖ ﺑﺎﻻي ﺗﺠﺰﯾﻪ، ﺗﺠﺰﯾﻪ ﺗﻌﺪاد زﯾﺎدي ﻧﻤﻮﻧﻪ در واﺣﺪ زﻣﺎن )196 × 2( در ﻫﺮ آزﻣﺎﯾﺶ و ﻫﻢﭼﻨﯿﻦ اﺳﺘﻔﺎده ﻣﺠﺪد از ژلﻫﺎ ﻣﯽﺑﺎﺷﺪ. 6 آﻏﺎزﮔﺮ ﺛﺎﺑﺖ و 6 آﻏﺎزﮔﺮ ﺗﺼﺎدﻓﯽ ﻧﺸﺎﻧﮕﺮ ﻣﻮﻟﮑﻮﻟﯽ TRAP ﺑﻪﮐﺎررﻓﺘﻪ ﺗﻮﺳﻂ اﯾﻦ روش اﻟﮑﺘﺮوﻓﻮرز 19 ﺗﺮﮐﯿﺐ ﻧﺸﺎﻧﮕﺮي اﯾﺠﺎد ﮐﺮدﻧﺪ ﮐﻪ از ﻣﯿﺎن 116 ﻧﻮار اﯾﺠﺎدﺷﺪه ﺗﻮﺳﻂ ﻧﺸﺎﻧﮕﺮ TRAP، 109 ﺗﺎي آنﻫﺎ ﭼﻨﺪﺷﮑﻞ ﺑﻮدﻧﺪ. ﻣﯿﺎﻧﮕﯿﻦ ﺗﻌﺪاد ﻧﻮارﻫﺎي ﭼﻨﺪﺷﮑﻞ 5/79 ﺑﻮد. اﻧﺪازه ﻗﻄﻌﺎت آﺷﮑﺎرﺷﺪه ﺑﯿﻦ 920-25 ﺟﻔﺖ ﺑﺎز ﺑﻮد. ﻣﺤﺘﻮاي اﻃﻼﻋﺎت ﭼﻨﺪﺷﮑﻞ )PIC( اﯾﻦ ﻧﺸﺎﻧﮕﺮ ﺑﯿﻦ 0/03 و 0/50 ﺑﺎ ﻣﯿﺎﻧﮕﯿﻦ 0/33 ﺑﻮد. ﺗﺮﮐﯿﺐ ﻧﺸﺎﻧﮕﺮي )MAX3BR و Sa12( ﺑﺎ ﻣﺤﺘﻮاي اﻃﻼﻋﺎت ﭼﻨﺪﺷﮑﻞ 0/49 ﭼﻨﺪﺷﮑﻞﺗﺮﯾﻦ ﺟﺎﯾﮕﺎه ﺑﻮد. ﻧﺘﺎﯾﺞ آزﻣﺎﯾﺶ ﻧﺸﺎن داد ﮐﻪ اﯾﻦ روش ﻧﻪﺗﻨﻬﺎ در آﺷﮑﺎرﺳﺎزي ﻣﺤﺼﻮﻻت ﻧﺸﺎﻧﮕﺮﻫﺎي رﯾﺰﻣﺎﻫﻮاره ﺑﻠﮑﻪ در ﺳﺎﯾﺮ ﻧﺸﺎﻧﮕﺮﻫﺎ ﻣﺎﻧﻨﺪ TRAP ﮐﻪ ﺗﻌﺪاد زﯾﺎدي ﻧﻮار ﭼﻨﺪﺷﮑﻞ ﺗﻮﻟﯿﺪ ﻣﯽﮐﻨﻨﺪ ﻣﯽﺗﻮاﻧﺪ ﺑﺴﯿﺎر ﻣﻔﯿﺪ واﻗﻊ ﺷﻮد

In this research, the application of new electrophoresis method of polyacrylamide gel using ethidium bromide staining for genetic variation of sunflower genotypes using TRAP novel marker has been proposed which has advantages such as low-cost, higher speed of analysis, analysis of a large number of samples per unit time (196×2) per experiment, as well as the reuse of gels in comparison with conventional methods of silver staining and fluorescent primer labeling. Six fixed and six arbitrary primers produced 19 TRAP markers so that from 116 bands, 109 of them were polymorphic. Mean number of polymorphic bands were 5.79. DNA fragments size were between 25-920 bp. Polymorphic Information Content (PIC) of this marker were between 0.03 and 0.50 (with the average of 0.33). Primer combination (MAX3BR and Sa12) with PIC value of 0.49 was the highest polymorphic locus. The results showed that this method could be very useful not only in detecting products of the microsatellite markers, but also in other markers, such as TRAP, which produces many polymorphic bands.