Detection of variation among clonal selections of grapevine (Vitis vinifera L.) ‘Kishmish Chernyi’ by AFLP analysis

Summary

Clonal selection is an important method for varietal improvement in grapevine. Ampelometric and morphological markers fail to differentiate clones from their parent genotype. Molecular markers offer the opportunity to identify the clonal material. In this study, five clones of the grapevine variety ‘Kishmish Chernyi’ were analysed using microsatellite (SSR) and AFLP markers. These clones differed significantly in their bunch characteristics including berry size, shape, and colour. Microsatellite (SSR) analysis using 24 primers could not distinguish between these clones. The allele profiles of the clones and the parent variety were identical. AFLP analysis using 13 primer pair combinations yielded 592 markers ranging in size from 50 – 500 bp. Of these, 79 markers (13%) were polymorphic. The majority of the polymorphic markers (75/79) were detected in the clone ‘Sharad Seedless’. Three AFLP primer combinations detected unique markers in three clones which could be useful for future identification.     

Influence of 1-methylcyclopropene on pectic enzyme activities,gene expression,and cell wall modification in papaya (Carica papaya cv. ‘Sunrise’) fruit

SUMMARY

‘Sunrise’ papaya fruit harvested at two stages of maturity [colour break (< 10% yellow peel colour) and 25% yellow peel colour] were treated with 100 nl l^–1 1-methylcyclopropene (1-MCP) to determine its effects on ripening, on the activities and levels of gene expression of polygalacturonase (PG), pectin methyl esterase (PME), and βgalactosidase ( βGal), and on the degradation of cell wall components. 1-MCP delayed ripening and the onset of the climacteric, although the peak in the respiration rate was almost the same as that in untreated control fruit. Colour-break fruit treated with 1-MCP exhibited a continuous increase in ethylene production, but at a lower rate than in control fruit. Consequently, 1-MCP-treated fruit ripened with a concomitant reduction in firmness, which was accompanied by an increase in PG and βGal enzyme activities and gene expression. On the other hand, fruit treated with 1-MCP at the 25% yellow stage exhibited lower levels of ethylene production and developed pulp with a rubbery texture at the ripe stage which was attributed to reduced PG, βGal, and PME enzyme activities and gene expression. This was consistent with the higher level of cell wall polysaccharides measured in 1-MCP-treated fruit. The above results indicated that ‘Sunrise’ papaya fruit can be treated with 1-MCP at the colour break stage since they have a greater capacity to recover from the effects of 1-MCP than fruit treated at the 25% yellow stage.     

Sequence analysis,prokaryotic expression and antiserum preparation of coat protein gene of Actinidia virus A in Sichuan province北大核心CSCD

【Objective】The purpose of the research was to study the occurrence and molecular diversity of Actinidia virus A (AcVA), so as to provide scientific basis for the rapid detection, scientific prevention and control of AcVA and the variety breeding of kiwifruit in China.【Methods】Total RNA was extracted from kiwifruit leaves by CTAB (Cetyl Trimethyl Ammonium Bromide). Nested- PCR was used to amplify AcVA CP gene sequence. The first round PCR amplification was performed by using primers AcVA- 1F (5’- ATGAATCGTTCGAGCA TAGGT- 3’) and AcVA- 1R (5’- TGCGAACATGGTCCCACACTTA-3’), and the pair of primers was designed according to the full length of AcVA sequence, and the amplified fragment was 888 bp that included the complete CP gene sequence. The reaction was conducted under conditions of initial 5 min denaturation at 94 ℃, 34 cycles of 94 ℃ for 60 s, 54 ℃ for 60 s, 72 ℃ for 60 s and extension for 10 min at 72 ℃. Then, 1 μL PCR product was used as template for the second round of PCR amplification with the primers AcVA- 2F (5’- ATGGCAAAGAATATCTCAAG-3’) and AcVA-2R (5’-CTATATTTCAACAGCCTGC-3’). PCR was performed using the following parameters: one cycle at 94 ℃ for 5 min, 34 cycles at 94 ℃ for 30 s, 54 ℃ for 30 s, and 72 ℃ for 40 s, and extension for 10 min at 72 ℃. The BLAST algorithm was used to search the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) databases for homologous sequences and ascertain the identity of target gene. DNAMAN was used to analyze the AcVA CP gene sequence, and MegAlign was used to analyze the sequence identity. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA 6.0. The restriction enzymes Bam HⅠ and Sal Ⅰ were added to the corresponding end of the AcVA CP gene sequence, respectively. The PCR purified products and pET28a vector were digested by Bam HⅠ and Sal Ⅰ, after that they were ligated with T4 DNA ligase (TaKaRa) and transferred into E. coli (Escherichia coli) strains DH5á (TaKaRa), and finally plated onto LB (Luria-Bertani) agar containing Kana (Kanamycin). The expression strain BL21 containing the recombinant plasmid was cultured at 37 ℃ overnight, and transferred to a new medium at a 10% inoculum on the second day, until OD600 reached 0.4-0.6, IPTG (Isopropyl β-D-Thiogalactoside) was added to a final concentration of 0.2, 0.4, 0.6, 0.8, 1.0 mM, and incubated at 16 ℃ overnight. The expressed protein was purified and then anti-AcVA-CP antibody was obtained from a rabbit. The optimal titer of the antiserum was tested by Western blot.【Results】The complete CP gene sequences of AcVA Sichuan isolates were cloned by nested-PCR and named as AcVA-DJY4, AcVA-HYZ1 and AcVA-WBS12, respectively. In the study the frequency of AcVA in Sichuan province was also counted. The full length of the CP gene sequence was 597 bp, encoding 198 amino acids. Based on the comparison of the nucleotide sequence and the deduced amino acid sequence of CP gene of the AcVA isolates, the nucleotide identity between the AcVA isolates from Sichuan province ranged from 86.9% to 88.3%, and the identity of the encoded amino acids ranged from 96.0 to 96.5%. The CP genes of three AcVA Sichuan isolates shared 87.9%-90.8% nucleotide identity and 94.9%-97.5% amino acid identity with the Actinidia virus A isolate TP7-93A (AcVA-TP7-93A) from New Zealand and the Actinidia virus A isolate Haenam (AcVA-Haenam) from South Korea. Phylogenetic analysis based on nucleotide sequence of AcVA CP gene showed that AcVA-HYZ1 was clustered with AcVA-Haenam (Group I), and AcVA-WBS12 was clustered with AcVA-TP7-93A (Group II), but AcVA-DJY4 was an independent branch. Phylogenetic analysis based on amino acid sequence of AcVA CP gene showed that AcVA-WBS12 was a single branch, AcVA-DJY4 and AcVA-HYZ1 were clustered into one group (Group I), and the AcVA-Haenam reported in South Korea and the AcVA-TP7-93A reported in New Zealand were clustered into another group (Group II). The results of the phylogenetic analyses indicated that there were significant differences among the AcVA isolates. The prokaryotic expression plasmid pET28a-AcVA-DJY4-CP was successfully constructed. The target fusion protein (27 kDa) was highly expressed in E. coli induced by 0.6 mmol · L- 1 IPTG at 16 ℃. The expressed protein was purified and retrieved and then used to immune rabbits and the corresponding specific antiserum was prepared. Western blot analysis confirmed that the antiserum reacted strongly and specifically with the CP of AcVA-DJY4, with the optimal titer of the antiserum being up to 1:5 000.【Conclusion】Three AcVA Sichuan isolates were obtained for the first time, enriching the sequence diversity of the AcVA CP gene sequences. In the study, the optimal conditions were explored for prokaryotic expression and specific antisera was prepared for detection of AcVA-DJY4-CP. Western blot analysis showed that the antiserum could be used for the detection of AcVA in the kiwifruit producing districts. These results can also provide a technical support for the rapid detection, scientific prevention and control of virus disease in kiwifruit plant and the breeding of kiwifruit varieties. © 2019 Journal of Fruit Science     

A molecular detection and identification method for papaya mealybug （Paracoccus marginatus）, the first recorded invasive pest in Fujian province of China北大核心CSCD

【Objective】Paracoccus marginatus Williams and Granara de Willink is an invasive pest with strong diffusion and fecundity. It has caused serious damage to the papaya industry in Central America, Florida (USA), Guam (USA) and India. Pa. marginatus was first discovered in Fujian Province (Fuzhou and Zhangzhou) in 2017, showing great potential risks to papaya and other fruit crops, as well as flower industry in Fujian province. Because of the small body size and similar morphological characteristics, the morphological identification of mealybugs was inefficient. Rapid molecular identification of different species could be achieved through the use of DNA barcoding technology. Therefore, a technology for rapid molecular identification of Pa. marginatus was established based on the species-specific PCR method. 【Methods】A species-specific PCR method based on ribosomal DNA-28S gene fragment (28S rDNA) was exploited to establish one technology for rapid detection and identification of Pa. marginatus. The additional 10 species of mealybugs (Phenacoccus solenopsis, Dysmicoccus boninsis, Nipaecoccus viridis, Phenacoccus solani, Pseudococcus comstocki, Pseudococcus cryptus, Planococcus lilacinus, Pseudococcus odermatti, Planococcus minor and Phenacoccus madeirensi) were collected in the fields as the contrast. In order to ensure the uniqueness of the source of DNA, the DNA templates were all extracted from one single female adult of these 11 species of mealybugs, respectively. 28S rDNA of the 11 species was amplified by a pair of universal primers (S3660/A335). The obtained partial fragments of 28S rDNA were sequenced. And the phylogenetic tree was established by using a Neighbor-joining (NJ) method. According to the obtained 28S rDNA gene partial sequence of the 11 species and 28S rDNA gene sequences of Paracoccus galzerae in GeneBank database, the sequence alignment and analysis were performed on DNAMAN. 28S rDNA species-specific primers (28S-ParF/ 28S-MarR) for Pa. marginatus were designed by selecting the sites with large differences in the sequence. And then, the specific effects, versatility and sensitivity of the specific primers were examined. 【Results】The comparative results showed that the similarity between Pa. marginatus and Paracoccus marginatus isolate S3-668, KP692333 in the GenBank database was 100%. It was also indicated that the mealybugs were identified as Pa. marginatus by molecular identification. Phylogenetic analysis indicated that Pa. marginatus from Fuzhou and Zhangzhou was clustered in a clade. And that combined with Paracoccus galzerae (inter-species genetic distance is 0.058) to form a clade of the genus Paracoccus. The results of specificity tests showed that all Pa. marginatus specimens could be detected positively and a 446 bp fragment of the 28S rDNA of Pa. marginatus was obtained by the species-specific primers, while there was no cross reactions with other 10 species of mealybugs. The species-specific primers not only had a stable amplification effect on female adults, but also were proved to be applicable for the 2nd instar nymphs and the 3rd instar nymphs. Pa. marginatus from three different regions (Fuzhou and Zhangzhou in Fujian province, Jinghong in Yunnan province) and six different host plants (Carica papaya, Solanum melongena, Plumeria rubra, Solanum tuberosum, Tithonia diversifolia and Duranta erecta) was also successfully detected by the species-specific primers.【Conclusion】Molecular identification of Pa. marginatus first reported in Fujian province was carried out based on 28S rDNA molecular markers. It was proved by experiments that the 28S rDNA species-specific primers had ideal and stable specificity for Pa. marginatus and could be used to identify Pa. marginatus accurately. A rapid molecular detection technique for Pa. marginatus was established based on the species-specific PCR method. The technology has the characteristics of accuracy, rapidity, sensitivity and simplicity. Our present results indicated that the rapid detection technique should be useful in quarantine at ports, in pest detection and in monitoring during transportation of papaya and other fruit tree seedlings, as well as flowers. However, in view of the fact that no other mealybugs of the genus Paracoccus has been reported in China, this study can provide a reference for the molecular identification for the closely related species of Pa. marginatus. © 2019 Journal of Fruit Science     

Determination of Pesticide Residues in Apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) Grown at Good Agricultural Practices (GAPs) by LC-MS/MS and GC-MS

Good Agricultural Practices (GAPs) includes agricultural techniques which environmentally-conscious, is not harmful to human and animal health, target protection of natural resources, provide traceability and food security. With these kinds of production techniques, it is aimed at agricultural production which is socially viable, economically profitable and sustainable. In this study conducted under good agricultural practices, pesticide residues in apricot fruit were investigated. The experiment was set up to have 3 replications and 15 trees in each replicate, according to randomized trial design. From the trees included in the experiment, necessary samples were taken at the harvest time and analyzes were carried out. In this study, pesticide residue levels were determined in fruit extracts with high-precision analytical instruments such as LC-MS/MS and GC-MS. A total of 385 pesticide active substances were analyzed in LC-MS/MS and 101 pesticide active substances in GC-MS in fruit extracts. In this research carried out in 2014 and 2015, samples of both years were not found to be detectable to the tolerance values of Turkish Food Codex (TFC).