مجله بیوتکنولوژی کشاورزی
سال چهاردهم شماره 1 (پیاپی 46، بهار 1401)

Root structure modification is associated with the efficient water uptake and the nutrient utilization. It also provides structural support for the anchoring in soil. Genetic engineering for the improvement of plant root structure may help to maintain higher yields under drought conditions. The aim of this study was to modify the root structure of rice in order to improve drought tolerance and the efficiency of nutrient uptake. For this purpose, simultaneous transformation of Deeper Rooting1 or OsDRO1 gene, which is involved in the regulation of growth angle of the root in order to adapt to drought conditions, and Phosphorus-Starvation Tolerance1 or OsPSTOL1 gene, which is effective in increasing phosphorus uptake and improving root structure, were considered for rice root structure modification. 

The OsDRO1 and OsPSTOL1 genes derived from the wild rice cultivars were cloned together in a single construct under the control of the root specific and the ubiquitin promoters, respectively. The resulting construct, pUhrDroPstol is transformed into the Agrobacterium tumefactions strain EHA105 and used for the gene transformation into Hashemi cultivar. Putative transgenic plants, survived on 50 mg/L Hygromycin during tissue culture steps, are transplanted into the Yoshida solution and then into the pots until they set seeds. Construct specific and gene specific PCR analysis are used to confirm the transgenic plants. 

In this study, 12 putative transgenic rice events were obtained, of which 10 showed the presence of both OsDRO1 and OsPSTOL1 genes in the PCR analysis. Transgenic plants show stronger root structure compared to the non-transgenic ones. Molecular analysis in the T1 and T2 generations determined the homozygous events. 

In this study, two candidate genes affecting root structure, nutrient uptake and drought tolerance were transferred to the Hashemi rice using genetic engineering. So far, simultaneous transfer of these two candidate genes have not been reported. Transgenic plants present better root system compared to the control plants. The mentioned construct can be used for the transformation of other crops to improve their root structure, nutrient uptake and their drought tolerance. It is hoped that the production of the transgenic rice with modified root structure and efficient phosphorus uptake increases its drought tolerance and reduce water consumption in rice cultivation.

During the last decade, about 20-30% of the Zagros forests have faced a problem called oak decline. The aim of present study was to investigate the bacterial diversity in the healthy and declined forest areas by comparing two methods culture-dependent and culture-independent methods.

Sampling was done from different tissue and soil of oak trees in the forests of Ilam province. In the culture-based method, using microbiological and molecular methods the bacteria in the samples were isolated and identified to the genus and species level. In order to study metagenomics in culture-independent method, total microbial DNA was extracted directly from the samples and prepared metagenomic library using conventional and index primer, the diversity of bacterial was examined using Illumina's Miseq platform and then the results of the two methods were compared.

Results

Based on the culture-based method, only 3 phylum were identified, while in the metagenomics method, 9416 OTUs were obtained, that were divided into 12 phylum. In both methods, 3 families of Bacillaceae, Enterobacteriacea and Xanthomonadaceae were observed as the dominant families. The species diversity within a sample (Alpha diversity) was higher in the rhizosphere sample and in the Eyvan and Gale jar areas than other samples. The species diversity between samples (Beta diversity) was in Eyvan, Gale jar, Chavar, Tangedalab and Arghavan appear more similar to each other than the sites samples. Bacterial community of stems and leaves were more similar and also bulk and rhizosphere were more similar in bacterial species composition.

In the metagenomics method, very few species are identified, while in the culture method, the probability of this is low. Also, in the culture-independent method, alpha and beta diversity can be studied more comprehensively. In the culture-based method, the bacterial population can be identified up to the species level, while in the culture-independent method, it is usually identified up to the genus level. Therefore, it is suggested to use a combination of two methods simultaneously in studies of microbial communities.

In recent years, the use of biological methods to control the activity of molds with the help of lactic acid bacteria and their metabolites has received much attention. The aim of this study was to isolate and molecularly identify of lactic acid bacteria with antifungal ability from corn, sorghum and barley sour doughs. 

The anti-fungal properties of 68 cell bacterial isolates, bacterial supernatant and autoclaved cells were investigated on Aspergillus flavus, Penicillium expansum and Rhizopus coriniformis molds. Also, the anti-mold properties of the supernatant treated with pepsin and proteinase K enzymes were tested. 

The results showed that the percentage of inhibition of live bacterial cells on the molds of A. flavus, P. expansum and R. coriniformis was between 0-16, -0.33 and -0.56 percent, respectively. The lowest inhibitory effect of live bacterial cells was observed on R. coriniformis and the highest inhibitory effect was observed on P. expansum. The supernatants of most isolates had an inhibitory effect on R. coriniformis in the range of 0-30 percent, while this inhibition of growth on P. expansum mold was in the range of 100-90 percent, indicating that this mold is sensitive to the supernatant of most isolates. Regarding the inhibitory effect on P. expansum and R. coriniformis, the highest frequency of isolates was in the range of 91-100 percent, which indicated that these two molds were more sensitive to the heated cell of the most bacterial isolates. Based on the results, finally three isolates were selected as superior isolates, all of which were identified as Lactobacillus plantarum. The supernatants from these three bacterial isolates when treated with pepsin, inhibited the growth of A. flavus only and had no inhibitory effect on the other two molds. 

The results of this study showed that it is possible to use L. plantarum and its supernatant for control of mold growth.

Salinity stress is considered as one of the most important abiotic stresses that significantly decreased crop production in the world. The development of salt-tolerant varieties is one of the most strategies against decreasing crop performance in saline conditions. Hence, assessment of expression patterns of genes responsible for the biosynthesis pathways can be very effective in creating tolerant genotypes. The main objective of this study was to compression of expression patterns of HvSOS1, HvSOS3, HvNHX3, and HvHKT3 genes in some advanced barley genotypes under salinity stress conditions.

In the present study, the effect of salt stress on relative changes expression of HvSOS1, HvSOS3, HvNHX3, and HvHKT3 genes in the six advanced barley genotypes along with Mehr cultivar (as a check) was investigated under the control (no NaCl) and salt (200 mM NaCl) treatments. All tested genotypes were planted under controlled greenhouse conditions using a hydroponic system. After salt treatment duration, all seedling plants were subjected to sampling and the relative expression of selected genes was estimated.

Based on obtained results, there are significant differences between treatments as well as among tested genotypes. Salt treatment significantly increased expression of HvSOS1, HvSOS3, HvNHX3, and HvHKT3 genes by 14.76-, 4.65-, 4-, and 4-fold compared to the control treatment, respectively. Comparison of the expression patterns of studied genes showed that two genotypes G3 and G6 had the highest transcription rate of mentioned genes and it seems that they have an acceptable tolerance to the high levels of salinity stress. A positive association between HvHKT3 and HvSOS genes was another obtained result so that two identified genotypes indicated the highest expression of these genes under salt treatment.  

It is inferred that increasing expression of studied genes is an important part of the salt tolerance mechanism because each of them has a key role in the signaling pathway and translocation of Na ions in the plant cells. Two identified genotypes, G3 and G6, with having this potential can be appreciated candidates for future research and use as salt-tolerant cultivars.

The objective of present study was to evaluate the genetic diversity within 23 genotypes of Freesia hybrida including 6 parents and their 17 F1 hybrids, using 10 ISSR primers in order to utilize such diversity in breeding program of this plant. 

After extraction of genomic DNA from fresh leaves and amplification of marker regions by the PCR, followed by the electrophoresis, a matrix of binary data was created based on scoring of electrophoretic bands. Marker parameters including number of polymorphic loci, polymorphism information content, effective multiple ratio, resolution power index, and marker index as well as genetic variation indices including observed number of alleles, effective number of alleles, Nei's gene diversity index, and Shannon's information index were calculated by using the ISSR marker data. Principle components analysis based on the Jaccard similarity coefficient with UPGMA algorithm were done followed by the cluster analysis using Dice coefficient and based on Ward’s grouping method. 

Out of 110 amplified loci of the 10 ISSR markers, the mean of polymorphism percentage 94.7% within the used genotypes was estimated. Maker IS-HB11 showed maximum number of polymorphic loci (12), effective multiple ratio (7.93), resolution power index (7.83), and marker index (2.67) as well. Cluster analysis grouped genotypes into four clusters, which was confirmed by the result of principle component analysis. 

Relative high value of the polymorphism information content (0.368), showed that the ISSR makers utilized in present study were genetically well informative regarding to the number of identified alleles and their distribution in the genome of Freesia. Based on the gene diversity indices, there was no significant difference between the parental genotypes and the F1 hybrids in terms of genetic variation. However, the level of this variation was acceptable and is capable to be utilized for breeding program in this plant.

Reproductive traits (especially litter size at birth) is one of the most important economic traits in sheep breeding systems. The present study aimed to conduct a genome wide association studies based on Gene-set enrichment analysis for identifying the genomic region, candidate genes and biological pathways associated with Litter size in Zandi sheep breed. 

Prolific ewes with at least one twining record and others with only singleton records were genotyped using the medium-density Illumina Ovine SNP50 array. The gene set analysis consists basically in three different steps: the assignment of SNPs to genes, the assignment of genes to functional categories, and finally the association analysis between each functional category and the phenotype of interest. Genome-wide association study for litter size evaluated using Plink software method case-control. Using the biomaRt2 R package the SNP were assigned to genes. Subsequently, gene enrichment analysis was performed with the goseq R package and bioinformatics analysis was implemented to identify the biological pathways performed in GO, KEEG, DAVID and PANTHER databases. 

We identified different sets of candidate genes related to litter size: AFP, FOXO3, ESR1, ESR2, RBP4, AURKA, DLG1 and ZGLP1 in Zandi sheep. According to pathway analysis, 11 pathways were associated with the litter size trait. Among biological pathways, the Oocyte differentiation, Ovulation from ovarian follicle, Estrogen signaling pathway and Positive regulation of peptide hormone secretion pathways have significant association with ovulation rate and ovarian steroidogenesis traits. 

Considering, this study supported previous results from GWAS of  litter size, also revealed additional regions in the sheep genome associated with these economically important traits, presented here should be contribute to a better understanding of the genetic control of litter size in sheep and using these findings can accelerate the genetic progress in sheep breeding programs.

Early heading as an adaptation mechanism for end season heat and drought stress is an important goal in wheat breeding programs. Photoperiod (Ppd) and vernalization (Vrn) are the most important genes controlling early heading in bread wheat. The aim of the present study was to investigate the effect of Ppd and Vrn genes on early heading, grain yield and important agronomic traits in BC4F2 generation of Roshan (recurrent parent) and Excalibur backcross.

A BC4F2 population resulted from backcross of Roshan and Excalibur was generated in Shahid Bahonar University of Kerman. In this breeding program, early heading was transferred from Excalibur cultivar to Roshan (recurrent parent). This population, including 175 BC4F2 progenies, and their parents were cultivated in the research field of Shahid Bahonar University of Kerman in the 2019-2020 growing season. The progenies were genotyping using specific markers Vrn-B1, Vrn-D1 and Ppd-D1. In this population, early heading, grain yield and important agronomic traits were evaluated.

Excalibur and Roshan had Ppd-D1a, and Ppd-D1b alleles, respectively. The frequency of Ppd-D1a/Ppd-D1a and heterozygous genotypes in the evaluated population were 27.33% and 415%, respectively. In this population, Ppd-D1a/Ppd-D1a, which is photoperiod insensitive genotype, was 6 and 4 days earlier heading than Ppd-D1b/Ppd-D1b and Ppd-D1a/Ppd-D1b genotypes, respectively. There was no allelic diversity between progeny for Vrn-1 locus. Early heading had a significant negative correlation with grain yield, 1000-grain weight, spikes number and grains number per spike.

The results confirm the importance of Ppd-D1a gene in the early heading, grain yield and yield components. Selection for early heading increased grain yield, 1000-grain weight, spikes number, biological yield, stover biomass at harvest and spikes dry weight. Due to the large and significant effect of Ppd-D1a gene on grain yield and yield components, we recommend marker assisted selection of this gene in wheat breeding programs. Phenotypic selection was performed before BC4F2 generation and Vrn gene diversity was established in the population.

Date palm is one of the most important trees in arid and tropical regions with very valuable fruit as a good food source. Therefore, the aim of this study was to investigate allelic diversity, the population structure and genetic diversity of important date palm genotypes using ISSR molecular marker for applying in genomic studies. 

Genomic DNA was extracted from leaf tissue of 68 date palm genotypes by Zhang method. The quantitative and qualitative evaluation of the extracted DNA was performed by using a spectrophotometer. For qualitative evaluation, DNA samples were visualized on 2% agarose gel. Polymerase chain reaction was performed using 10 ISSR primers. 

In this study, 74 polymorphic bands were created that showed an average of 65.78% polymorphism. Based on the results of this experiment, (AG)8YC', (TC)8C and (GA)8C are suitable markers in the study of genetic diversity of date palm. Structural analysis divided the study population into three sub-structures. The results of analysis of molecular variance between and within the three subpopulations showed that the share of variance within and between subpopulations was 79 and 21% of the total variance, respectively. The principle component analysis based on the Euclidean square distance matrix showed that the first ten components explained 54.57% of the total changes and diagram of the principal component analysis based on the first two principal components divided population into four groups. In addition, cluster analysis based on complete linkage clustering method and the Euclidean distance divided 68 date palm genotypes into four groups. 

The ISSR marker can be useful as a suitable marker for studying the differentiation and structure of the date palm population. High genetic diversity within and between subpopulations makes it possible to select parents for suitable traits in future breeding programs of date palm.

Plants are often exposed to a variety of environmental stresses such as drought and salinity, which leads to osmotic stress in the plant and ultimately reduces crop growth and productivity. Identification of effective genes at different treatments of osmotic stress can be very helpful in finding genes that are effective in tolerating plant stresses. Therefore, the aim of this study was to identify the hub genes of Arabidopsis model plant in osmotic stress and to introduce them for crop breeding under environmental stresses .

In this study, Arabidopsis microarray data that were exposed to mannitol-induced osmotic stress for 1.5, 3, 12 and 24 hours were analyzed separately by GEO2R online tool. Genes with significant expression were identified and the most important genes at each stress level were identified using bioinformatics tools. Then, the hub genes in osmotic stress were identified and their protein interactions and biological processes were studied and discussed .

The result showed that 26, 79, 138 and 184 genes had significant expression at 1.5, 3, 12 and 24 hours after stress, respectively. Based on protein network analysis and biological processes, SDA1, CRK11, CYP81F2, EDA39, PLA2A, T1K7_24, F6N7_24, AT2G25735 and MRH10_18 genes were reported as hub genes at different levels of osmotic stress. The results of molecular function analysis of hub genes showed that these genes involved in oxidative stress, response to hypoxia, regulation stomata movement, hypersensitive response, response to chitin, induced systemic resistance, indole glucosinolate biosynthetic process, defense response by callose deposition in cell wall, response to cadmium ion, phytochelatin biosynthetic process, arsenite transport, defense response to bacteria, insects, fungi, viruses and other environmental stress responsive biological process. 

It seems that the key genes introduced in this study can be used to breed crops under environmental stresses that cause osmotic stress in plants.

The omp (Outer membrane protein) protein located at outer surface of the bacterial membrane body agent citrus greening disease (Candidatus Liberibacter asiaticus). The mentioned-protein can be used as antigen for generating polyclonal and monoclonal antibodies. Therefore, the purification of membrane proteins is exposed to many problems due to presence of hydrophilic structure. The purpose of this study was optimizing recovery and purification of the recombinant omp protein.

The pET28a bacterial expression construct containing omp gene was used to recombinant production of the mentioned-protein. The omp gene expression was conducted in E. coli strain Rosetta-Gami2. With considering to be hydrophilic of omp protein, its purification was investigated under denature condition. To attain it, the different concentrations of NaCL, urea and guanidine at the different acidity pH condition was applied. To obtain the pure omp recombinant protein, the protein recovery from polyacrylamide gel was performed by methods based on elution by diffusion, sonication, and electroelution. The evaluation of quality and quantity purified protein was analyzed by SDS-PAGE and the protein concentration was calculated by ImageJ software. Also, to validate the purified proteins, Western blot was applied by specific antibody.

Primary results showed that using the 500 mM NaCL was detected as an essential share in purification buffers. Also, using urea as denature agent had twice the efficiency on the quantity of purified protein more than guanidine hydrocholoride and thus, when 8 m urea supplemented in purification buffer, the highest purified protein was attained (450 µl/ml). Furthermore, the results related to recovery the single band of protein from gel indicated that the electroelution has highest efficient on recovery of the target protein from gel matrix compared to sonication and elution by diffusion methods. The results of SDS-PAGE confirmed to appear a protein with 34 kDa related to molecular weight of target protein. Western blot validated the recombinant protein in membrane. 

The current study showed that using urea in 8 M, NaCL in 500 mM concentration in purification buffers following adjusting the elution buffer on pH (4) in elution purification step can be apply as an optimized condition to attain the highest target protein concentration. Furthermore, electroelution method introduced as efficient method in order to recovery band of the target protein from polyacrylamide gel matrix.

Awareness of genetic diversity and population structure is very effective in conserving germplasm and preventing the weakening of the genetic base and provides the opportunity to harness the potential of desirable genes in the genetic repository in breeding programs. The aim of this study was to investigate the genetic diversity in the germplasm of Rosa damascena collected from different regions of Iran and to identify the genetic relationships of different populations for use in breeding and Conservation of genetic resources programs.

Genetic diversity and population structure were evaluated in a collection of Rosa damascena Mill germplasm, including 40 accessions collected from five geographical regions of Iran using inter-simple sequence repeats (ISSR) markers.

Twelve ISSR primers replicated 202 multiform genomic fragments, the number of these bands ranged from 15 to 18 in different primers and the average was 16.83. The average Polymorphism Information Content (PIC) and marker index (MI) for the primers used ranged from 0.35 to 0.46 and 5.25 to 8.28, respectively. Analysis of molecular variance (AMOVA) showed that intra-population diversity accounts for a greater share (93%) of total molecular diversity. The dendrogram obtained from cluster analysis based on method neighbor joining categorized the accessions into 3 main groups, which were confirmed by principal coordinate analysis (PCoA). Based on Jacard coefficient, the highest genetic distance (0.837) was observed between Hormozgan and Barzok 3 accessions, and the lowest genetic distance between Semnan accessions (0.141). The results of population structure using STRUCTURE software showed no strong relationship with the geographical distribution of accessions.

Cluster analysis and principal coordinate analysis were consistent with the genetic relationships derived by STRUCTURE analysis in many cases. The results showed that the grouping of accessions based on molecular data is not strong related to their geographical origin, thus strengthening the probability of gene flow between populations. Genetic diversity obtained by ISSR marker indicates the ability to identify interspecies and intraspecies differences of this marker.