| The apple ring rot disease is one of the most serious diseases in China apple production and the apple industry suffered huge economic losses in recent year.The study was aimed to screening the highly resistant varieties by combine artificial inoculation of Botryosphaeria berengriana with field investigation.Then we used SA(salicylic acid)to treatment two different resistance varieties of apples,to determine the changes of some enzyme activity associated with resistance before and after the induction.And we further clarified genes associated with resistance of differentially express in different treatment by high-throughput sequencing technology and validated some of these genes.The results are as follows: 1.Screening germplasms resistant to apple ring rot.The study filtered 190 malus germplasms in the malus germplasm repository.(1)All malus germplasms were found that have infected of apple ring rot by artificial inoculation.There were significant difference in the resistance among varieties and no immune to apple ring rot of malus germplasm tested was found.By cluster analysis there were 15 highly resistant varieties,is 7.89 percent of in inoculated varieties;77 medium resistant varieties,40.53 percent of the total;80 medium susceptible varieties,42.11 percent of the total and 18 highly susceptible varieties,9.47 percent of the total.(2)The survey of field natural morbidity for 190 malus germplasms in 2012 and 2013 two consecutive years shows that there were majority of highly resistant varieties in 2012,there was only one medium susceptible variety named Liquanduanfu.In these 190 varieties of the survey,there were 164 varieties whose disease index are less than 30,is about 86.32 percent of survey total.But the disease index of Liquanduanfu was more than 50.The overall resistance trends in 2013 consistented with it in 2012,early maturing varieties accounted for a higher proportion of resistant varieties and susceptible varieties are mostly Fuji varieties,and only three resistant varieties include the Kitanosach,1996-1-1 and Qinguan.There was no highly susceptible variety in the findings of 2012 and 2013.Combining the two-year investigation,the variety Kitanosach which was performed stabilized and high resistance and the variety Liquanduanfu which was susceptible.2.We used resistant variety Kitanosach and susceptible variety Liquanduanfu for materials to test resistance enzyme activity of apple leaves by treated with different concentrations of SA.the test demonstrated that PAL,POD,?-1,3-glucanase and PPO enzyme activity of Kitanosach and Liquanduanfu improved in a short time and significantly higher than the control.PAL activity of resistant variety Kitanosach by SA treatment increased by 1.36-2.73 times more than the control;POD activity increased by 4.74-6.19 times;?-1,3-glucanase activity increased by 1.71-2.04 times;PPO activity reached the maximum,1.88-2.32 times more than that of the control at late 11 th day.PAL activity of the susceptible variety Liquanduanfu having SA treatment increased by 1.32-1.70 times.It increased more slowly and reached the maximum at 11 th day if having high concentration treatment;POD activity increased slower than it of Kitanosachs and reached the maximum value at 11 th day,higher than the control by 4.11-5.68 times;?-1,3-glucanase enzyme increased 1.53-1.82 times and PPO activity reached the maximum at early 7th day and increased by 3.13-4.00 times than the control.It could increased the activity of pathogenesis-related proteins enzyme and enhanced apple's resistance to disease to apply SA exogenously.Resistances of different varieties were different.From the results of PPO activity determined that there were some differences in PPO activity itself,having no SA in different apple varieties.PPO activity of disease-resistant varieties was higher than susceptible varieties.The disease-resistant varieties may be resist the invasion of pathogens.3.We used resistant variety Kitanosach and susceptible variety Liquanduanfu for materials to do RNA-Seq before and after SA treatment.Andthe different expression genes and different gene types were analyzed after treated by SA,the digital expression spectrum was built when by having SA treatment in different resistant varieties.The results were as follows:(1)The Illumina sequencing was made for four samples: Kitanosach and Liquanduanfu before and after SA treatment,and have gotten 35.30 M reads.(2)All reads which obtained by sequencing compared with Unigene libraries and got 2,590 differentially expressed genes.There 1382 were the maximum differentially expressed genes,150 the minimum differentially expressed genes and 9 co-expressed genes are: MDP0000039357,MDP0000158474,MDP0000223968,MDP0000225361,MDP0000232908,MDP0000329063,MDP0000362305,MDP0000838695 and MDP0000916403.In co-expressed genes,MDP0000223968,MDP0000329063 and MDP0000362305 were involved in defense and response stimuli.(3)In the treatment group and the control group of resistant variety Kitanosach,there were 257 differentially expressed genes,include 213 upregulated genes and 44 downregulated genes.In the treatment group and the control group of susceptible variety Liquanduanfu,there were 150 differentially expressed genes,include 110 upregulated genes and 40 downregulated genes.In the treatment groups resistant variety Kitanosach and susceptible variety Liquanduanfu,there were 828 differentially expressed genes,include 356 upregulated genes and 472 downregulated genes.In the untreated groups resistant variety Kitanosach and susceptible variety Liquanduanfu,there were 1382 differentially expressed genes,include 540 upregulated genes and 842 downregulated genes.As can be seen from the results,in the same variety,the number of upregulated genes expression was more than it of downregulated genes expression after SA treatment.And the number of differentially expressed genes between different varieties is the most and more than the same variety of genes after treatment.(4)A total of 2,202 results of Unigene annotations by bioinformatics annotations,the majority of differentially expressed genes were general function prediction only,and the functional annotations related to the defense were mainly defense mechanisms,signal transduction mechanisms,energy production and conversion and inorganic ion transport and metabolism,but the genes involved defense mechanisms in susceptible variety Liquanduanfu were not annotated.(5)By GO classifications most are metabolic process,response to stimulus,biological regulation,immune system process and antioxidant activity.Significant classification types GO of susceptible variety Liquanduanfu are fewer than resistant variety Kitanosach,and the number of differentially expressed genes was less than Kitanosach.(6)The analysis for KEGG pathways shows that metabolic pathways in varieties are also more than it in species,and the number of differentially expressed genes decreased after induced by SA among different varieties,but significant functions GO classification and KEGG pathway involving the quantity changed little and covered roughly the same.Mainly related to or involved in disease resistance related metabolic pathways include: Peroxisome,Phenylpropanoid biosynthesis,Terpenoid backbone biosynthesis,Plant-pathogen interaction,Flavonoid biosynthesis,Plant hormone signal transduction,Phenylalanine metabolism and the Ribosome.The difference among or in varieties was that there was ribosome but no flavonoid biosynthetic among varieties,and there was flavonoid biosynthetic but no ribosomes in varieties.The KEGG pathway of susceptible variety Liquanduanfu was significantly less than it of disease-resistant variety Kitanosach and the disease resistance related KEGG pathways were less.After SA induced treatment,the number of differential genes of different resistance cultivars was significantly different.Resistance varieties had a more number of genes and susceptible varieties had a relatively less of it.And the KEGG pathways involved in were less,which associated with the resistance of the varieties itself and also suggests that resistance-related genes of disease-resistant varieties were induced easily by SA.(7)In these 2,590 differences gene have filtered,there were 304 chitin enzyme related genes,include 170 genes participate defense mechanism and 120 genes related to salicylic acid response;there were 39 glucanase related genes which include 2 genes participate defense mechanism,2 genes participate phenylpropanoid metabolism and 9 genes participate oxidation restore;there were 2 NPR1 related genes,one was annotated to the participation in the defense mechanism;there were 16 WRYK transcription factor-related genes,of which 5 genes involved in the defense,4 genes responsed stimulus-related;3 PR1 genes are associated with defense mechanisms and there was one TGA gene.(8)The KEGG pathways related to plant disease resistance: Phenylpropanoid metabolism pathway and salicylic acid signal pathways were detected the presence of the genes.By treating of SA,the genes of phenylalanine ammonia-lyase,cinnamic acid-4-hydroxylase(C4H)and 4-coumaric acid-CoA ligase(4CL)exist not only differential expression in the resistant varieties,but also exist differential expression in susceptible varieties.To intermediate products of flavonoids,lignin and alkaloid with antibacterial activity,the differences between resistant and susceptible varieties were also expressed.In Salicylic acid signal pathway,susceptible varieties Liquanduanfu differences genes expression were lower than it in the resistant varieties Kitanosach.It showed that exogenous application of SA are more likely to induce genes expression related salicylic acid signaling pathway in resistance was raised except the inherent differences between varieties.(9)We took Q-PCR verify for 6 co-expressed differential genes,one glucanase transporter genes and 2 chitin enzyme genes.The rusult showed that it consisted with the RNA-Seq sequencing,there was a certain bias only in expression multiples,but the trend of upregulated and downregulated were basically the same. |
PDF Full Text Request