غربالگري برخي ارقام تجاري زردآلوي ايران با استفاده از نشانگرهاي ريزماهواره با هدف شناسايي هم‌نامي‌ها

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

در ايران، بسياري از ارقام محلي زردآلو توسط باغ­داران و نهال­كاران با هدف بهره‌برداري از ارقام جديد در ميان استان­ها جابجا شده و متعاقباً در برخي موارد نام آن­ها در مقصد تغيير يافته است. بنابراين براي مديريت بهينه ژرم­پلاسم زردآلوي كشور و شناسايي ارقامي كه از نظر ژنتيكي از يكديگر متفاوت هستند، غربالگري ژرم­پلاسم زردآلو در ايران ضروري به نظر مي­رسد. بنابراين هدف از انجام اين مطالعه، غربالگري تعدادي از ارقام تجاري زردآلوي ايران با استفاده از نشانگرهاي ريزماهواره، جهت بررسي وضعيت نام­هاي اطلاق شده به ارقام، شناسايي هم­نامي­ها و درك بهتر از تفاوت­هاي ژنتيكي ميان ارقام بود. در اين مطالعه 29 رقم مختلف زردآلو با پنج تكرار از 14 نهالستان از شش استان كشور جمع­آوري شدند. براي ارزيابي وضعيت حفظ اصالت ژنتيكي در نهالستان­هاي كشور، برخي از ارقام، دو بار از نهالستان­هاي مختلف جمع­آوري شدند. همچنين DNA 10 رقم بومي زردآلو كه قبلا ثبت و در فهرست ملي ارقام وارد شده بودند نيز به عنوان شاهد در مطالعه گنجانده شدند. علاوه بر آن زردآلوي رقم Orange Red (با نام محلي پرتقالي) به عنوان نمونه Outgroup در اين بررسي­ها گنجانده شد. براي انجام آزمايشات، استخراج DNA به روش CTAB انجام و از 13 جفت آغازگر ريزماهواره كه در مطالعات مختلف بيشترين ميزان تنوع را آشكار كرده بودند، براي انجام واكنش­هاي زنجيره­اي پليمراز استفاده شد. سپس محصولات اين واكنش­ها بر روي ژل پلي­اكريلاميد ده درصد الكتروفورز شد. نتايج نشان داد كه شاخص­هاي­ تنوع ژني ني و شانون به ترتيب 0/32 و 0/48 بودند. بيشترين فاصله ژنتيكي (0/74) ميان ارقام عسگرآبادي و زودرس و همچنين ارقام محلي گوشتي زودرس و نصيري مشاهده شد. در اين مطالعه، برخي از ارقام علي­رغم داشتن اسامي متفاوت، داراي زمينه ژنتيكي مشابهي بودند. همچنين نتايج نشان داد كه ارقام نصيري، طبرزه و شاهرودي كه هر كدام از دو نهالستان از استان­هاي متفاوت جمع­آوري شده بودند، علي­رغم نام يكسان داراي زمينه ژنتيكي متفاوتي بودند. در نهايت مي­توان گفت كه يكي از چالش­هاي جدي در مديريت ژرم­پلاسم و نهالستان­هاي كشور، عدم وجود باغات مادري استاندارد از ارقام اصيل بومي زردآلو جهت تامين پيوندك­هاي سالم و اصيل براي نهالستان­ها مي­باشد.

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 eco-geographical groups including: Central Asian, East Chinese, North Chinese, Dzhungar-Zailij, Irano-Caucasian and European. Iranian genotypes which belong to the Irano-Caucasian group are mostly self-incompatible 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 off-types 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 off-types. For identification of synonyms the off-type 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 UDP98-021, UDP98-409 and UDP98-411 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 off-types 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.