نشانگرهای ریزماهواره‌ای و کاربرد آن در تجزیه و تحلیل تنوع ژنتیکی جمعیت‌های بالتوری سبز (neuroptera:chrysopidae) (stephens, 1836) chrysoperla carnea

مطالعات کمی روی تنوع و ساختار ژنتیکی دشمنان طبیعی آفت‌ها که به عنوان عوامل کنترل زیستی نیز شناخته می‌شوند، در کشور انجام شده است. این پژوهش با هدف مقایسه تنوع­ژنتیکی جمعیت­های بالتوری­سبز chrysoperla carnea انجام شد. جمعیت­های مختلف از چهارده استان کشور شامل استان­های آذربایجان­شرقی، آذربایجان­غربی، اصفهان، کرمان، کرمانشاه، لرستان، مازندران، گیلان، هرمزگان، تهران، کردستان، شیراز، زنجان و همدان جمع­آوری شدند. این جمعیت‌ها با جمعیت­های هلندی با استفاده از 10 نشانگر مولکولی issr مقایسه شدند. بیشترین درصد چند­شکلی مربوط به جایگاه ژنی ubc809 به میزان 88/88 درصد و کمترین آن متعلق به جایگاه ژنی ubc886 به میزان 33/33 درصد بود. کمترین فاصله ژنتیکی جمعیت هلندی با جمعیت­های مورد مطالعه، مربوط به جمعیت استان گیلان و بیشترین فاصله ژنتیکی با جمعیت استان کرمان بود. میانگین تعداد آلل، برای جمعیت هلندی، گیلان و کرمان به‌ترتیب معادل، 6/9، 6/6، 4/1 و میانگین تعداد آلل موثر، 1/48، 1/41 و 1/19 بدست آمد. مطالعه حاضر نشان داد که نشانگرهای issr در مطالعه تنوع­ژنتیکی این حشره کارایی بالایی دارند.

Use of Microsatellite Markers for Analysis of Genetic Diversity between Populations of Green Lacewings Chrysoperla carnea (Neuroptera, Chrysopidae)

Introduction: Green lacewings are active predators of Aphids, Psylids, Mealy bugs. We used ISSR markers with 10 primers to evaluate the genetic polymorphism between populations of Chrysoperla carnea collected in 30 cities of 14 provinces of Iran with different climatic conditions and compared them with specimens of a population from Netherlands. Maximum and minimum of polymorphism were calculated successively of 88.88% for UBC809 and 33.33% for UBC886 primers. The Dutch specimens had the smallest genetic distance with populations of Guilan province and the largest genetic distance to Kerman province. The mean number of alleles for Dutch, Guilan and Kerman populations were successively 6.9, 6.6 and 4.1 and mean number of effective alleles were successively 1.48, 1.41 and 1.19.Materials and Methods: 2000 specimens of Chrysoperla carnea collected from alfa alfa plantations in each of 14 provinces of Iran, 27 specimens sampled from each province besides another 27 specimens of a Dutch population acquired from Hamedan center for Science and technology and DNA extracted using Topaz Inc. kit , ISSR marker with 10 primers used. BioRad® thermocycler was used for PCR and PCR end product was electrophoresed in 2% Agarose gel and stained with ethidium bromide. Bands photographed using Gel Doc 2000. Hardyweinberg’s chi square ( χ2)  equation was used for evaluate of equilibrium in the number of polymorphic alleles,Shannon and Nei indice for evaluation of heterozygosity, POPGENE for genetic distance , GenAlex 6  for cluster analysis, AMOVA for analysis of inter and intra populations diversity.   Results and Discussion: The total number of electrophoresis bands were 64 bands, 43 of them were polymorphic The range of changes of allelic sizes of primers were between 1501500 bp. Polymorphic information content(PIC) was between 0.3020.643 the maximum of it belonged to the primerUBC809 and the minimum belonged to the primer UBC812 (Table 3). In three populations of Dutch, Guilan and Kerman the maximum of alleles were seen in gene locus of UBC809 suggesting that it is highly polymorphic, similar findings was found by Barbosa and coworkers. As in Fig (2) the cluster analysis based on use of Jaccard similarity coefficient, the populations clustered in three groups, the Dutch population together with those of Guilan, Mazendaran, Lorestan, East and west Azerbaijan provinces were gathered in the first cluster and populations of Kerman, Sistan and Beluchestan, Isfahan and Fars province were gathered in the second cluster and the populations of Hamedan, Zanjan, Kermanshah, Tehran and Kudistan provinces were in the third cluster. Principal Component analysis was also done, Eigen values, variances percentage and cumulative variances percentages by independent components and the range of variations represented by these components was shown in Table (5). 100% of variations were represented by first ten independent components. The first, second and third components successively represented 27.68,20.57 and 13.01 percentages of all variations.These components independently from each other  represented  a percent of genetic diversity, not already  known. The maximum of Shannon index was seen in gene locus of UBC809was 0.53 in Guilan province and the minimum 0.04 in Kerman province.These data were conceivable because there were nine alleles in gene locus of UBC809 in population of Guilan province and two alleles in gene locus of UBC812 in populations of Kerman province, the same index was calculated 0.58 for Dutch population. The maximum and the minimum of Nei’s index (H) of gene diversity was calculated 0.35for gene locus of UBC809 in Guilan province and 0.009 for gene locus of UBC812 in Kerman province successively and for Dutch population the index was 0.39. The maximum and minimum of observed heterozygosity (Ho) was 0.49 in gene locus of UBC809 in Guilan and 0.031 in gene locus of UBC812 in Kerman province successively. The maximum and minimum of expected heterozygosity was calculated as 0.579 in gene locus of UBC809 in Mazendaran province and 0.034 in gene locus of UBC886 in Hormozgan province successively. Having nine alleles in its gene locus and getting of maximum observed and expected heterozygosity therefore we can consider the UBC809 primer as the best gene locus to express of gene diversity in all Chrysoperla carnea populations studied in our present study. We used χ2 of HardyWeinberg equilibrium for all alleles studied, and with regard to H0(Equilibrium in alleles frequency in studied populations) the  most well adapted status   to Hardyweinberg equilibrium was seen in gene loci of UBC809, some of populations and gene loci did have HardyWeinberg equilibrium but others did have significant biases from this equilibrium which should be attributed to inadequate  number of samples chosen (sampling errors) in the studied populations and or  existence of nonreproducing  alleles. Our study on population genetic diversity of Chrysoperla carnea showed that ISSRPCR markers are the best to study the polymorphism of populations of this species, did not study hitherto in Iran.The primers   used by us in this study for Chrysoperla carnea was equally used for other orders of insects such as Lepidoptera and Coleoptera (UBC809) and used for order of Hemiptera (UBC812, UBC819).Conclusion: More genetic diversity of alleles in populations of Chrysoperla carnea collected from nontreated alfa alfa plantations than those treated with insecticides. Similar results was obtained in Brazil concerning populations of Chrysoperla externa. Our present study on Chrysoperla carnea in Iran showed us the similarity between gene frequencies in the studied provinces, similar results was found for other species of Chrysoperla. Some authors believe that reduction in heterozygotes alleles in population was caused by a reduction in effective number of alleles in that population. The decrease of allele’s diversity could bring about a reduction of populations, even species, as well as the reproduction vigor of populations in a new environment. In insect breeding programs a reduction in genetic diversity has an impact of reduction of fitness or even the loss of fitness in such an environment in which those populations should be released.  We propose that our research on heterozygosity of populations of Chrysoperla carnea to be continued in more agroecosystems in Iran with a similar or different molecular markers to have more informations about populations genetic diversity and finally to consider a logical management for preserving genetic reservoirs of them.