غربالگری برخی ارقام تجاری زردآلوی ایران با استفاده از نشانگرهای ریزماهواره با هدف شناسایی هم‌نامی‌ها

در ایران، بسیاری از ارقام محلی زردآلو توسط باغ داران و نهال کاران با هدف بهره‌برداری از ارقام جدید در میان استان ها جابجا شده و متعاقباً در برخی موارد نام آن ها در مقصد تغییر یافته است. بنابراین برای مدیریت بهینه ژرم پلاسم زردآلوی کشور و شناسایی ارقامی که از نظر ژنتیکی از یکدیگر متفاوت هستند، غربالگری ژرم پلاسم زردآلو در ایران ضروری به نظر می رسد. بنابراین هدف از انجام این مطالعه، غربالگری تعدادی از ارقام تجاری زردآلوی ایران با استفاده از نشانگرهای ریزماهواره، جهت بررسی وضعیت نام های اطلاق شده به ارقام، شناسایی هم نامی ها و درک بهتر از تفاوت های ژنتیکی میان ارقام بود. در این مطالعه 29 رقم مختلف زردآلو با پنج تکرار از 14 نهالستان از شش استان کشور جمع آوری شدند. برای ارزیابی وضعیت حفظ اصالت ژنتیکی در نهالستان های کشور، برخی از ارقام، دو بار از نهالستان های مختلف جمع آوری شدند. همچنین dna 10 رقم بومی زردآلو که قبلا ثبت و در فهرست ملی ارقام وارد شده بودند نیز به عنوان شاهد در مطالعه گنجانده شدند. علاوه بر آن زردآلوی رقم orange red (با نام محلی پرتقالی) به عنوان نمونه outgroup در این بررسی ها گنجانده شد. برای انجام آزمایشات، استخراج dna به روش ctab انجام و از 13 جفت آغازگر ریزماهواره که در مطالعات مختلف بیشترین میزان تنوع را آشکار کرده بودند، برای انجام واکنش های زنجیره ای پلیمراز استفاده شد. سپس محصولات این واکنش ها بر روی ژل پلی اکریلامید ده درصد الکتروفورز شد. نتایج نشان داد که شاخص های تنوع ژنی نی و شانون به ترتیب 0/32 و 0/48 بودند. بیشترین فاصله ژنتیکی (0/74) میان ارقام عسگرآبادی و زودرس و همچنین ارقام محلی گوشتی زودرس و نصیری مشاهده شد. در این مطالعه، برخی از ارقام علی رغم داشتن اسامی متفاوت، دارای زمینه ژنتیکی مشابهی بودند. همچنین نتایج نشان داد که ارقام نصیری، طبرزه و شاهرودی که هر کدام از دو نهالستان از استان های متفاوت جمع آوری شده بودند، علی رغم نام یکسان دارای زمینه ژنتیکی متفاوتی بودند. در نهایت می توان گفت که یکی از چالش های جدی در مدیریت ژرم پلاسم و نهالستان های کشور، عدم وجود باغات مادری استاندارد از ارقام اصیل بومی زردآلو جهت تامین پیوندک های سالم و اصیل برای نهالستان ها می باشد.

Screening of some Iranian Commercial Apricot Cultivars by SSR Markers for Identification of Synonyms

Introduction: Apricot (Prunus armeniaca L.) as an important fruit crop belongs to the Amygdaloideae in the Rosacea family and is grown in regions with Mediterranean climates in the world. Apricot species were classified into six ecogeographical groups including: Central Asian, East Chinese, North Chinese, DzhungarZailij, IranoCaucasian and European. Iranian genotypes which belong to the IranoCaucasian group are mostly selfincompatible with low chill requirement. The high level of genetic diversity in Iranian apricots is due to sexual reproduction by seeds during the years. In Iran, many of apricot local varieties have been relocated between provinces and subsequently, in some cases their names, have been changed over the years. Hence, to determine the genetically different cultivars and detection of synonyms, screening of apricot germplasm seems necessary in Iran.Materials and Methods: Thirty eight commercial genotypes of apricot with five biological replications were collected from 14 nurseries in West Azarbaijan, East Azarbaijan, Esfahan, Semnan, Alborz, and Tehran Provinces in Iran. Additionally Orang Red apricot (Porteghali) included in the study as an outgroup sample. Also DNA sample of previously registered apricots in national list were used in this study. Young and healthy leaves of each cultivar were sampled and stored at 70 °C. Samples were powdered using mortar and pestle in presence of liquid nitrogen. CTAB extraction buffer was used for nucleic acid extraction. Quantity and quality of extracted DNA were measured by spectrophotometry and agarose gel electrophoresis.Thermal cycles were done in Eppendorf thermocycler and the cycling program were set on one cycle of 94°C for 4 minute, 30 cycles of  94°C for 30 seconds, annealing temperature of each primer for 30 seconds and 72°C for 30 seconds followed by one cycle of 72°C for 5 minutes. PCR products were resolved on 10% polyacrylamide gels in 1x TBE buffer. GelRed (Biotium) was applied for gel staining and amplified bands were revealed by UV (300 nm). Eight SSR markers which showed more diversity were selected and scored. Polymorphic alleles were scored as one for presence and zero for absence. For detection of off types, samples were classified by Paired Group method and Euclidean algorithm in PAST software. Then the data of offtypes were removed from the dataset and samples were reclassified by the method. Principal Coordinate Analysis (PCoA) was carried out using PAST software. Genetic diversity indices were evaluated in Popgene 32 software.   Results and Discussion: Eight SSR loci produced 124 alleles with the average 15.5 allele per locus. Nei’s gene diversity and Shannon’s information index were 0.32 and 0.48, respectively which showed high level of diversity in this collection. Distance matrix based on Nei’s gene diversity showed that the most genetic distance (0.74) was between Askar Abadi and Zodras, Mahali Goushti Zodras and Nasiri cultivars. Clustering of samples indicated that some samples including 19 (Shahroudi), 59 (Nakhjavan), 107 (Shahroudi), 127 (Soltani), 137 (Ghavami), 144 (Tabraze) and 156 (Daneshkade) were offtypes.For identification of synonyms the offtype samples were disregarded. Cluster analysis illustrated that some local cultivars with different names had same genetic backgrounds. Thus, the names of these samples should be unified in the germplasm. Depicted graph based on first and second coordinates in PCoA demonstrated that the 38 collected groups of apricots are genetically 26 distinct cultivars and there are some duplicates in the germplasm. Results showed that three loci including UDP98021, UDP98409 and UDP98411 were able to distinguish all 26 genotypes. To check the genetic identity of saplings with the same name, some cultivars including Jahangiri, Askar Abadi, Shamlou, Saltanati, Shahroudi, Shams, Tabarze and Rajab Ali were collected from two different nurseries. Surprisingly, the results showed that Nasiri, Tabraze and Shahroudi which were sampled twice from distinct nurseries and provinces, despite of identical names, had different genetic backgrounds.Conclusion: Detected offtypes among five biological replications of local varieties propagated asexually by nurseries showed that there was not sufficient attention in the selection of propagating material in the nurseries. In this context, establishment of foundation blocks by public sector and mother orchards by private sector of economy from Iranian apricot local varieties can be an effective solution so that nursery operators can provide certified propagating material for production of certified nursery stocks. Moreover, seed and plant certification and registration institute should made inspection and testing procedures in mother orchards and nurseries to ensure that propagated trees are healthy, genetically uniform and original.