غربالگري هموكاريون ها و توليدنژاد دورگ جديد قارچ دكمه‌اي سفيد (Agaricusbisporus) با استفاده از نشانگر ريزماهواره

Screening of the White Button Mushroom (Agaricusbisporus) Homokaryons and Producing New Hybrid Strain by SSR Markers

قارچ خوراكی دكمه¬ای سفید (Agaricusbisporus) رایج¬ترین قارچ خوراكی در ایران و دنیا است، اما بهدلایلی از جمله استفاده از نژادهای كم محصول وپسروی نژادی، عملكرد این قارچ دركشور كمتر از متوسط عملكرد آن در دنیا می¬باشد. یكی از برنامه¬های به نژادی،تولید نژاد F1 حاصل از آمیزش هموكاریون¬ها است كه مستلزم در اختیار داشتن جدایه¬های هموكاریون می¬باشد. در این مطالعه ابتدا 160جدایه تك¬اسپور كند رشد از نژاد A15 جداسازی شد و سپس براساس ویژگی¬های مورفولوژیك، از بین آنها 18 نمونه بررسی و انتخاب شدند. در مرحله¬ی بعد، از نشانگر هم¬بارز ریزماهواره (SSR) جهت شناسایی هموكاریون¬ها استفاده شد. ده آغازگر كه در نمونه¬های شاهد مادری دارای چندشكلی بودند برای نمونه¬های مورد آزمایش نیز استفاده شدند. جدایه¬ها براساس حضور و عدم حضور باندهای چندشكل در دو گروه كلی هتروآللیك و هموآللیك قرار گرفتند و هفت جدایه كه در تمامی جایگاه¬های بررسی شده الگوی باندی تك باند نشان دادند به عنوان جدایه¬ی هموكاریون در نظر گرفته شدند. به منظور محاسبه سطح تنوع ژنتیكی بین 7 جدایه هموكاریون به دست آمده، ماتریس فاصله ژنتیكی با استفاده از نرم¬افزار NTSYSpcمحاسبه شد و دندروگرام مربوطه ترسیم گردید. تشابه ژنتیكی جفت نمونه¬ها بین 17/0 تا 67/0متغیربود. در مرحله¬ی بعد نمونه¬های شماره¬ 4 و 8 با بیشترین فاصله ژنتیكی نسبت به سایر نمونه¬ها جهت تشكیل هیبرید در تلاقی شركت داده شدند. نتیجه این تلاقی، تولید هیبرید N1 بود كه با افزایش ناگهانی در رشد و تولید میسلیوم هوایی در محل الحاق همراه بود كه در مقایسه با والدهای هموكاریون، سرعت رشد بیشتری داشت. در ادامه، جهت تأیید مولكولی هیبرید حاصل، واكنش PCR-SSR با استفاده از یك پرایمر (AbSSR 45) انجام گرفت. همانطور كه انتظار می¬رفت در هیبرید N1 همانند نمونه¬ی شاهد هتروكاریون، دو باند قابل امتیازدهی حاصل شد كه نشان¬دهنده¬ی وجود دو هسته¬ی غیرخواهری در هر واحد سلولی آن می¬باشد. نتایج نشان دادند كه با استفاده از نشانگر SSR می¬توان با احتمالی بیش از 8/99 درصد نسبت به قطعیت هموكاریونی پس از اجرای آزمایش با 10 آغازگر دست یافت و به این ترتیب از آنها در برنامه¬های اصلاحی جهت تولید هیبرید بهره برد.

Introduction: Edible white button mushroom (Agaricusbisporus) is the most common edible mushroom in Iran and the world. The yield of this mushroom is less than the average of yield in the world because of strain degeneration and using strains with low yield. Most of the current hybrids are either identical or very similar to the first hybrids. Ongoing breeding programs are exploiting the variability in Agaricus germplasm to produce new varieties with better traits including higher yield and resistance to biotic and abiotic stresses. One of the breeding programs is F1 production from parental homokaryons crossing. These homokaryonsis were isolated among germinated basidiospores on the culture media. During the last decades, various molecular markers based on nucleic acid polymorphisms (such as Restriction Fragment Length Polymorphism, Random Amplification of Polymorphic DNA, Amplified fragment of Length Polymorphism, Inter Simple Sequence Repeat, Simple Sequence Repeat markers) have been used to differentiate homokaryons and heterokaryons. Microsatellites consist of short tandem repeat motifs distributed throughout the genome. Microsatellites are usually highly polymorphic due to a high degree of variation in the number of repeats among individuals. Microsatellite markers are multiallelic and co-dominant and thus tend to be more informative than other marker systems. Microsatellite markers have been widely developed in animals and plants and more recently in fungal species. The presence of microsatellites in the genome of A. bisporus was previously reported. Materials and Methods: In this research, 160 germinated basidiospores were collected from commercially cultivated strain A15 and they were grown on compost extract agar (CEA). The mycelial growth rate of these160 isolates was evaluated at 25°C on CEA medium. 18 isolates with slow growing rate were selected from 160 isolates. In the next step, co-dominant SSR markers were used to homokaryons detection. Ten SSR primers showed polymorphism in parental control samples that were used to this experiment. The isolates were divided into two general homoallelic and heteroallelic groups and seven isolates from homoallellic group, which showed one-band pattern, characterized as putative homokaryon. Genetic similarity was calculated by NTSYSpc software version 2.02 e using UPGMA method. In the next step of experiment, the isolates (4 and 8) had minimum genetic similarity that was crossed to produce hybrid. In order to confirm the hybrid formation, PCR-SSR reaction with a primer (AbSSR 45) was performed. Results and Discussions: Basidiospores were collected and allowed to germinate on CEA medium. Putative homokaryons were different in colony morphology and growth rate compared to the original heterokaryons. Mycelium samples showed different colony morphology including tomentose, apprised and strandy mycelium. Different growth rate can be affected by genetic factors in nucleus and mitoconderia. After four weeks, mycelium browning was appeared in liquid compost extract medium and created a disturbance in DNA extraction. To solve this problem, DNA was extracted from three-week old mycelium. Mycelium browning may cause by phenolic compounds produced by mycelium and enzymes that catalyze melanin biosynthesis reactions. Ten primers were used to homokaryon isolation. These primers were situated on the 9 linkage groups of 13 haploid chromosomes. Seven isolates were distinguished as putative homokaryon that showed one-band in all primers on the gel electrophoresis. The results of genetic similarity calculation showed that this index was variable between 0.17 to 0.67in 7 homokaryon isolates and the minimum genetic similarity (0.17) was observed between isolates 4 and 8. These two isolates were crossed and the result of this crossing was N1 hybrid. Also, other homokaryon isolates were crossed and mating incompatibility was observed in some of them. According to these observations, it is suggested that in future studies, in addition to genetic similarity, sexual incompatibility should also be considered. Hybrid N1 produced aerial mycelium and had higher growth rate in comparison to parental homokaryons and similar to heterokaryon control, had two-bands pattern. This two bands pattern indicates the presence of two non-sister nucleuse in each cells. Finally, the results showed that SSR marker can result to accurate detection of homokaryons. Conclusions: The aim of the present study was screening homokaryon isolates of A.bisporus using SSR markers to obtain hybrid. Results showed that growth rate of homokaryon isolates were lower than the heterokaryons. Since, SSR markers were able to show high polymorphism in the isolates, thus it can be said that these markers are suitable to homokaryon screening. Final result of this study is N1 hybrid that can compare to commercially cultivated strains.