Shadi Attar; Gholamhossein Davarynejad; Leila Samiee; Mohammad Moghadam
Abstract
Introduction: Persian walnut (Juglans regia L.), belonging to the Juglandaceae family, has its natural origin in the mountainous regions of central Asia and especially northern forests of Iran. Most walnut genotypes are seedling and sexually reproduced. Conducting studies on the genetic structure of ...
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Introduction: Persian walnut (Juglans regia L.), belonging to the Juglandaceae family, has its natural origin in the mountainous regions of central Asia and especially northern forests of Iran. Most walnut genotypes are seedling and sexually reproduced. Conducting studies on the genetic structure of these genotypes to identify, select and maintain their genetic resources is important. Identifying and collecting local varieties of fruit trees is considered as the first step on the path of breeding programs and lack of information regarding plants genetic characteristics causes the breeding work to be done slowly. Various methods have been used for studying genetic diversity and determining the genetic relationship between European and Asian varieties of walnut and identifying commercial walnut varieties, among which we can mention: Morphologic indices, Alozyme, Isozym, RFLP, RAPD, AFLP and ISSR markers. ISSR molecular marker was used in order to investigate genetic diversity of some genotypes of Persian walnut (Juglans regia L.) in Mashhad orchards. .
Materials and methods: To begin with, about 56 walnut trees from 4 orchards in Mashhad (Esteghlal (1), Golestan (2), Alandasht (3) and Emam Reza (4)) were selected and tagged from 2014 to 2016. In the spring of 2014 with the beginning of trees growth and opening of leaves, a number of leaves from each genotype were collected. After DNA extraction, the quality of samples by agarose gel (1 percentage) and electrophoresis method and quantity of them via spectrophotometer device at 260 and 280 nm wavelengths were determined. First, 24 primers of ISSR marker were prepared and after initial evaluation on 5 random genotypes, 9 primers with high polymorphism and repeatability were selected for further investigation. For PCR reaction, Amplicon kit (code 180 301, made in Denmark) was used. Gel electrophoresis images of primers that produced polymorphic bands with suitable resolution were analyzed manually. After scoring the bands, in which 0 used for showing the absence of a band and 1 showing the presence of a band in each sample, 1 and 0 numbers were changed to matrix by using NTedit software. Genetic similarities of samples were calculated by using NTSYSpc software, SIMQUAL method and DICE similarity coefficient. Dendrogram by cluster analysis was drawn by using UPGMA method. Principle coordinate analysis (PCO) was performed using the NTSYS software and grouping samples were evaluated in a two-dimensional plot.
Results and discussion: Results showed that from 9 primers in total, 118 bands amplified were in 300 to 3000 base pairs, while 29 bands were polymorphic. The number of amplified fragment for each primer was different so that UBC 844 (14 bands) had the highest and UBC 890 (8 bands) had the lowest amplified bands. The average amplified fragments for each primer was 9.83. The percentage of polymorphic in various primers was different. Maximum polymorphism (80%) of the primers was observed in UBC830. In general, according to the percentage of polymorphic bands, low adjustment to any changes in the environment was indicated. This can be used as an indicator to determine the value of diversity and genetic erosion. In genotypes cluster analysis, clustering was performed based on Dice similarity coefficient and UPGMA method, and 10 clusters were formed. ISSR molecular marker somewhat revealed genetic diversity among walnuts genotypes, whereas the genetic diversity was lower than expected. In general, by reviewing the findings in other parts of the world about walnut genetic diversity and comparing them with the results of this study, despite existing high genetic diversity among walnuts in many areas, some reports of low genetic diversity among walnuts populations have been published and unfortunately in recent years these reports has made more attention. Based on the results of several studies reporting low genetic diversity among walnuts, the following factors can be effective in this problem: natural disasters, human impacts such as deforestation and selection and propagation of superior genotypes, and sometimes walnuts self - pollination. In this respect, there is concern that if this trend of decreasing genetic diversity in the walnut population continues, this invaluable crop will be in danger of extinction. So we should think about a remedy. Finally, this investigation can be used as a start for conducting more researches in the region to maintain and manage this valuable crop germplasm and maximize genetic diversity for performing breeding programs in the future.
Shadab Faramarzi; Abbas Yadollahi; Ghasem Karimzadeh
Abstract
Introduction: Apple (Malus Miller) belongs to Rosacease family and the subfamily of Pomoideae. This fruit is at first place among fruits of temperate zones. The cultivated apple (Malus × domestica Borkh.) is a complex hybrid of the apple species. Chromosomal basis of this subfamily are x = 17 and the ...
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Introduction: Apple (Malus Miller) belongs to Rosacease family and the subfamily of Pomoideae. This fruit is at first place among fruits of temperate zones. The cultivated apple (Malus × domestica Borkh.) is a complex hybrid of the apple species. Chromosomal basis of this subfamily are x = 17 and the ploidy levels have been reported for diploid (2n=2x = 34), triploid (2n =3x= 51) and tetraploid (2n= 4x= 68). Since Iran is close to the apple diversity region (Central Asia), it has a good variation of apple varieties. Despite the high levels of variation in apple cultivars and species in Iran, there is not still a database of genome size. Classification of plants according to their genome size, especially at lower taxonomic levels is important for breeders. Over the past years, several methods for estimation of nuclear DNA content (genome size) was common, but recently, the use of flow cytometry (FCM) has been increasingly used. Flow cytometry is the best method to estimate DNA c-value and ploidy levels in apples. In this study, DNA c-Value and ploidy level of Iranian apple varieties has been estimated by flow cytometry and propidum iodide staining.
Materials and Methods: Fully expanded young leaves of all apple varieties were collected in the summer 2013. Nuclear extraction was performed using Partec kit as following: 1 cm2 apple leaf and 1 cm2 parsley leaf (as internal standard) were co-chopped with razor blade after adding 500 µl of nuclear extraction buffer. Then, the extract was filtered by two kind of filters (50 and 30 um). One ml of staining buffer, 4 µl of RNAase and 4 µl ofpropidium iodide was added for 15 min at room temperature. Finally, nuclei were counted using flow cytometry (BD FACSCanto II, USA) at Tarbiat Modarres University. The genome size was estimated according to bellow formula:
DNA 2C-value sample =
Also, given the high levels of phenolic compounds in apples, treating with PVP and PVP 1% were performed to evaluate the effect of phenolic compounds on estimation of genome size. Finally, Histogram analysis and DNA c-value estimation were done with Partec Flow Max software. The difference between means was obtained by SAS software ver. 9.2 and LSD tests.
Results and Discussion: The results showed that genome size obtained from Partec Flow Max software and ranged from 1.57 pg for ‘Golab- Bastam’ to 1.73 pg for ‘Golab- Kermanshah’. Histogram analysis was demonstrated that all studied cultivars are diploid. The average genome size in this study was 1.62 pg. Research conducted on foreign apple varieties have showed that the genome size of diploid species from was obtained 1.45 for M. fusca to1.68 pg for M. ransitoria. The genome size for triploid species was ranged from 2.37 to 2.57 pg. In this study, genome size was calculated in terms of mega base pairs and was different from 748 Mbp in ‘Golab- Bastam’ to 846 Mbp in ‘Golab- Kermanshah’. Thus, the size of the genome was closed to M. ransitoria (1.68 pg). This species is native to China, which is a Crab apple and used as an ornamental tree. It has been reported that Iranian apple are M. domestica Borkh. In another study, genome size was identified in the range from1.245 pg for diploid species of M. tschonoskii to 1.653 pg for M. florentina. M. florentina species is native to Balkans and Italy, that is an ornamental tree and its genome size is close to M. domestica Borkh. (1.653 pg).
Conclusion: Classification of plants according to their genome size seems to be important, especially at lower taxonomic. Genome size, even in very close species can also be different, for example, northern corn with more heterochromatin has larger genomes than those who are located in south (less heterochromatin). This study appears the variation of DNA 2C-value in Golab cultivars, even though Golab cultivars are known clones with low genetic diversity. Therefore, it is likely that Iranian apple varieties, with the same ploidy level, have been had difference in genome size. There are various ploidy level in apple, including diploid (2n = 34), triploid (2n = 51), tetraploid (2n = 68) and hexaploid (2n = 102). Thus, it is expected that current apple M. × domestica Borkh., have been contributed some several species such as M. prunifolia (Willd.) Borkh., M. baccata (L.) Borkh., M. sieboldii (Regel) Rehder, M. sylvestris, ،M. orientalis Uglitzk and M. sieversii.
