In order to explore the role of heat shock protein(HSP90)gene family members in heat tolerance of apple(Malus × domestica Borkh.),bioinformatics method was used to identify the apple HSP90 gene family members,and their physicochemical properties,gene structure,cis elements,phylogenetic relationship and expression characteristics of HSP90 gene family under high temperature stress as well as in different tissues were analyzed. The results showed that a total of 11 HSP90 family members were identified in apple genome,which were distributed on 10 chromosomes. The amino acid sequence size was ranged from 104 to 818 aa,and molecular weight of protein ranged from 11.81 to 93.58 kD. Transcriptome analysis showed that four genes(MdHSP90-1,MdHSP90-3,MdHSP90-5and MdHSP90-11)of the 11 members were significantly up-regulated under high temperature stress. Subcellular localization analysis showed that MdHSP90-1,MdHSP90-3 and MdHSP90-5 proteins were located in the cell cytoplasm and nucleus,whereas MdHSP90-11 was targeted to the nucleus. Subsequent qRT-PCR analysis showed that the expression trend of the four genes in the two kinds of apple rootstocks were similar,the expression level increased greatly in the early stage of high temperature stress,and decreased in the middle and late stage. Four genes were also detected in different apple tissues,and the expressions of fourHSP90 genes were induced by heat stress to varying degrees,which indicated that the above four HSP90 genes may play an important role in the heat tolerance of apple.
To explore the pathways and mechanism of response to powdery mildew in Malus hupehensis(Pamp.)Rehder,we used the BGISEQ-500 sequencing technology and de novo transcriptome assembly to gain a comprehensive overview of the transcriptome of the leaves of‘Qingzhen 1’, ‘Qingzhen 1’/Malus hupehensis and M. hupehensis after the outbreak of powdery mildew in the same period. The resistance to powdery mildew of three materials increased in turn. Gene Ontology(GO)enrichment showed significant enrichment of GO term associated with metabolic process and catalytic activity in Qingzhen 1,Qingzhen 1/Malus hupehensis and M. hupehensis. KEGG analysis showed linoleic acid metabolism,plant-pathogen interaction,inositol phosphate metabolism,brassinosteroid biosynthesis and phosphatidylinositol signaling system etc. were significantly enriched in Qingzhen 1/Malus hupehensis and resistance to powdery mildew,respectively. And the carbon metabolism pathway was only significantly enriched in M. hupehensis. Expression analysis of genes related to the resistance to powdery mildew showed that WRKY70,WRKY33,NPR3and WRKY40 were significantly up-regulated in Qingzhen 1/Malus hupehensis and M. hupehensis compared to Qingzhen 1. The accuracy of sequencing were confirmed by qRT-PCR experiment.
This work aims to establish the CRISPR/Cas9 gene editing system using European pear calli,and provides a technical platform for functional genomics study and molecular breeding of pear. We edited theGUS gene in proDAM3:GUS transgenic pear calli using a dual-cut strategy. Based on the sequence characteristics of the GUS gene,two sgRNAs were designed to target the second exon of the GUS gene. The corresponding GUS-pYLCRISPR/Cas9 plasmids were constructed and transformed into the proDAM3:GUS transgenic calli by Agrobacterium-mediated transformation. The results from sequencing data revealed that four out of six independent transgenic lines carried mutations at the target sites,with mutation rates of 66.7%. A total of 56 successfully sequenced clones from the gene edited lines were generated,which indicated that sgRNA1 target had the higher mutation frequency(71.4%)than that of sgRNA2 target(46.4%). We further analyzed the mutation types of the four GUS editing lines,and found that #1,#2,#3 lines showed deletion and insertion of nucleotides,while #5 lines showed deletion of large fragments between two target sites,whereas no nucleotide substitution was observed in any edited line. To check the phenotype,GUS staining showed that the control calli were blue in color,while the four gene editing lines were white,indicating that the CRISPR/Cas9 system was a powerful and precise method to induce targeted mutagenesis in pear calli.
In order to explore the effects of water stress on volatile compounds of grape berries,the 10-year-old table grape variety ‘Muscat Hamburg’ under three different water states were used as materials. Gas chromatography-mass spectrometry(GC-MS)was employed to analyze volatile composition,the expression of relevant genes of the berries at different development stages were determined by qRT-PCR. The results showed that water stress could reduce 100-berry weight and the content of soluble solids,there was no significant difference on titratable acid and total phenol of berries at harvest stage. In this study,41,43 and 33 volatile compounds were detected in control,light water stress and severe water stress,respectively,in which aldehydes were the main volatile compounds,terpenes were the second,the contents of phenols and esters were at a low level. The total content of volatile compounds in light water stress was 34.74% and 36.92% higher than that in control and severe water stress at ripening stage,respectively. The content of esters,aldehydes and phenols in light water stress was the highest,while the content of alcohols was the lowest. The content of acids and alcohols of severe water stress were significantly higher than that of the other two treatments. The content of terpenes with rose scent increased with the aggravation of water stress. Water stress upregulated the expression of VvTPS of berries,but was not conducive to the expression of VvRiLinNer and VvCCD1. The expression of VvGPPS in light water stress was significantly higher than that in control,but the severe water stress decreased the expression of this gene of berries. In a word,moderate water stress can improve berries quality by increasing the content of volatile compounds of‘Muscat Hamburg’grape.
In order to further understand the genetic diversity and variation of F1 population in intra-specific and inter-specific hybrids of chestnut,and the influence of genetic effects and configuration of different parents on F1 generation,the genome DNA of 235 F1 hybrids and their parents were amplified by PCR and analyzed by capillary electrophoresis with 32 pairs of SSR markers,and then the genetic parameters,molecular variance,UPGMA clustering and PCA analysis for the statistical data were performed. Two hundred and seventy-eight polymorphic loci were amplified by 32 pairs of primers in the 235 progenies;and the average number of alleles was 8.69;the average value of Shannon’s diversity index (I),genetic diversity index(Hs)and gene flow(Nm)were 1.3707,0.6574 and 1.5951,respectively;the genetic differentiation coefficient(Fst)showed that 85.18% of the variation of F1 generation existed in the cross combination. Shannon’s diversity index of different combinations ranged from 0.8816 to 1.1317,indicating that there were abundant genetic diversity in their offspring,and the genetic diversity level of those offspring were affected by the genetic effect of different parents and the configuration of parents. The results of molecular variance analysis showed that the genetic variation of F1 hybrid population mainly came from the combination(78.06%),which was consistent with the result of genetic differentiation coefficient. The genetic distance and UPGMA clustering of different hybrid combinations showed that C3 and C5,which had the same parents,had the closest genetic distance and got together first,followed by the combination with the same female parent or the same male parent,got together respectively. The PCA scatter plot of hybrid progenies further verified the result of cluster analysis of hybrid combination. Most progenies of the same hybrid combination or the combinations with relatively close genetic distance could gather together. At the same time,there was cross overlap phenomenon in different combinations,which indicated that there was more extensive gene exchange among them,and the phenomenon of offspring separation was obvious.
To study the potential activity of pri-miR319a encoding regulatory peptide of Dimocarpus longan,the pri-miR319a full-length sequences were cloned from seven longan varieties and the positions of potential miPEP(miRNA encode regulatory peptide)in longan pri-miR319a sequences were further analyzed. Then,the activity of miPEP319a was verified by genetic transformation and artificial synthesis of miPEP319a. The results showed that among the seven longan varieties,pri-miR319a had 10 nucleotide differences and three potential ORFs that could encode miPEPs,and one nucleotide mutation site resulted in the change of miPEP319a amino acid sequence. The biological activity of longan miPEP was further verified by thein plantatransformation technology and the synthetic miPEP319 treated embryogenic callus of longan. The results indicated that only the miPEP319a-2 of the three potential miPEPs was biological activity and promoted the expression of miR319a. Finally,the vectors were transiently expressed in tobacco leaves by Agrobacterium-mediated transformation method to verify the activity of longan miPEP319a in tobacco. The results showed that there was no significant difference in the expression of miR319a in tobacco leaves,which indicated that miPEP319a was specie-specific. The results suggested that longan miPEP319a-2 had biological activity and species-specific,and also suggested that longan miPEP319a-2 might participate in the growth and development of longan.
In order to understand the potential functions of DRB(dsRNA-binding proteins)in longan,the bioinformatics analysis was carried out to identify members of the DRB gene family,and then their expression patterns were analyzed. The results showed that the DlDRB gene family contained eight members with two or three DSRM(double-stranded RNA binding motif)domains and could be distributed into five subgroups. The numbers of introns varied from one to seven. DlDRBs contain six kinds of motifs. The promoters ofDlDRBscontain a large number of light response elements,hormone response elements and stress response elements. DlDRBswere expressed in all stages of longan somatic embryogenesis and different organs. Under ABA,SA and MeJA treatments,the expression ofDlDRBsis clearly inhibited. This study indicated that members of the DlDRBs were highly conserved,they may be involved in the growth and development of somatic embryogenesis and organs in longan,and may be related to the response processes of ABA,SA and MeJA.
SSR markers were used to analyze the genetic diversity of 23 spring cabbage and 29 autumn cabbage inbred lines. The results showed that the 23 spring cabbage inbred lines were classified into two major groups,namely the extremely early maturing group and the early maturing group at the genetic similarity coefficient of 0.61. Meanwhile,twenty-nine inbred lines of autumn cabbage were classified into three groups,namely middle-late maturing,middle-early maturing,and middle maturing group. Fifteen inbred lines were selected to make 105 hybrid combinations with complete diallel method to analyze their heterosis and explore the relationship between genetic distance and heterosis by SSR markers. At the genetic similarity coefficient of 0.58,the heterosis analysis of 105 hybrid combinations obtained from 15 parental materials meant that the offspring produced by crosses among different groups or between different subgroups of the same group had stronger heterosis. Coefficient among genetic distance based on polymorphism of SSR markers and gross weight mid-parental heterosis was significant at 0.01 level. Thus,the classification based on SSR markers had a certain value for heterosis prediction. In addition,this study also found that among 105 hybrid combinations,the 10 combinations with the strongest mid-parental heterosis among gross weight was spring cabbage × autumn cabbage,indicating that the combination of spring cabbage × autumn cabbage had a potential utilization on heterosis breeding.
The plant phosphatidyl ethanolamine-binding protein(PEBP)is divided into three subfamilies:MFT-like,FT-like,and TFL1-like. The FT and TFL1 subfamilies comprise the florigen-antiflorigen system of plants that regulate flowering time and plant architecture. Here,the PEBP gene family was identified by genome-wide identification of the whole genome data of the annual pepper (Capsicum annuum),and the structures of gene and protein,comparative evolution,collinearity and tissue expression profile were analyzed. The results showed that the C. annuum genome contains nine PEBP genes,including one MFT,four FT,and four TFL1 genes. All the nine PEBPgenes contain four exons and three introns,which are located on six chromosomes. Moreover,these nine PEBP proteins are located in the cytoplasm and nucleus. Comparative evolution analysis between C. annuum and Solanum lycopersicum showed that CaPEBP genes were subjected to purification selection during evolution. Among them,CaFT2 and CaFT4 were relatively stable during evolution;there were eight pairs of collinearity genes between pepper and tomato. The qRT-PCR expression profile ofCaPEBP genes were significantly differentially expressed in the tissues tested. For example,CaMFT/CaTFL1-1 was highly expressed in fruits,and CaFT1 had the highest relative expression level in leaves;the expression of CaFT3 was the highest in shoot apical meristem and flower,while CaTFL1-4 was specifically expressed in roots.
The biocontrol efficiency of a local isolate of Bjerkandera adusta‘M1’against the gray mold and growth promotion in tomato was studied in order to further understand its potential function in agriculture. The confrontation culture,fermentation broth inhibition and greenhouse pot experiments were conducted to evaluate the inhibitory effect ofBj. adusta‘M1’and its fermentation broth on theBotrytis cinerea Pers.,and their biocontrol efficiency against the tomato gray mold and plant growth promotion. The growth of Bo. cinerea was obviously suppressed by Bj. adusta‘M1’through the observations of their confrontation culture. Results from the optical and scanning electron microscopic observations showed that the hyphae ofBo. cinerea were interspersed and twined by Bj. adusta‘M1’,and resulted in hyphal swelling, atrophy and deformation,indicating a strong mycoparasitism. Results from the plate antagonism test indicated that the inhibition rate of theBj. adusta‘M1’fermentation broth againstBo. cinereareached up to 47.90%,which was equivalent to that of the chemical procymidone. Compared to the Bo. cinereaspore suspension application,the greenhouse pot experiment showed that the disease index of gray mold was significantly decrease,and 55.2% control effect was thus obtained;the activity of leaf peroxidase,superoxide dismutase and catalase was significantly increased by 29.57%,136.15% and 534.13%,respectively. Meanwhile,the malondialdehyde content and the relative electrolyte leakage rate were significantly reduced by 19.98% and 51.10%,indicating a reduction of the pathogen damage to plants. Plant height,stem thickness,aboveground biomass and leaf chlorophyll content in tomato plants were increased significantly by 63.92%,38.04%,87.08%,and 35.02%,respectively. These results demonstrate that Bj. adusta‘M1’,and its fermentation broth can inhibit theBo. cinerea growth and effectively control the gray mold,while promoting the tomato growth. This study concludes that Bj. adusta‘M1’has a great potential to control gray mold in the field.
The overall objective of this research was to explore the effects of exogenous 5-Aminolevulinic acid(ALA)on the flavor quality of tomato fruit. Tomato(Solanum lycopersicum‘Yuanwei 1’)fruits were applied with different concentration of ALA(0,100 and 200 mg·L-1)being daubed on the surface at the mature green fruit stage. The soluble sugars,organic acids and volatile compounds were determined during maturation of tomato fruits. The results indicated that 200 mg·L -1 exogenous ALA promoted the maturity of tomato fruits,advanced the tomato fruits maturation period by 4 days,and significantly increased the content of fructose,glucose and total soluble sugar in tomato fruits. Meanwhile,the tartaric acid,citric acid,oxalic acid,quinic acid,shikimic acid and total organic acid content were significantly reduced through the application of 200 mg·L-1 exogenous ALA,thereby,the sugar acid ratio of the tomato fruits increased markedly. 96 volatile compounds were detected in tomato fruits,including 35 alcohols,25 aldehydes,12 hydrocarbons,nine ketones,six esters,five phenols and four other compounds. The content of volatile compounds in 0(control),100 and 200 mg·L -1ALA treatment were:1 065.72,1 478.60 and 2 374.50 µg·kg -1. The total contents of alcohols,aldehydes,hydrocarbons,ketones,esters,phenols and other compounds in tomato fruits were significantly increased by 200 mg·L -1 ALA,while the species of alcohols,ketones,hydrocarbons and other substances were increased by 100 mg·L -1 ALA. Ten characteristic volatile compounds were detected in tomato fruits,among which 200 mg·L -1 ALA treatment had the highest content(1 074.78 µg·kg -1). In conclusion,applying an appropriate concentration of exogenous ALA(200 mg·L -1)during maturation of tomato not only enhance the flavor quality of the fruits,but also advance the tomato fruits maturation period.
Based on the genome data of pepper,the chromosomal localization,gene structure,phylogenetic relationships and tissue-specific expression of B-box genes in pepper were analyzed by bioinformatics method. Moreover,the transcription level of the family genes under hormone and abiotic stress was detected by qRT-PCR. A total of 26 BBX members,which were distributed on 12 chromosomes,have been identified in pepper genome. Phylogenetic analysis showed that the CaBBX proteins could further be divided into five groups:groupⅠ-Ⅴ. Among them,members in groupⅠcontains two B-box domains. All the proteins in groupⅡandⅢcontained B-box1,B-box2 and CCT domains,except for CaBBX25. The proteins in group Ⅳ only had a B-box domain while members in group Ⅴ contained a B-box and a CCT domain. A same or similar number of exons has been found in members of the same clade usually. At the same time,a higher similar of the composition of motifs was also observed. Tissue specific expression analysis showed that the other 17 genes were highly expressed in different organs or at different devolpment stages of fruits,except for the class D genes. The result of qRT-PCR showed that 10 CaBBX genes could be promoted or repressed by the hormones and abiotic stresses.
The floral characteristics and flowering process ofPaphiopedilum dianthum under greenhouse cultivation condition were investigated. Meanwhile,the pollen viability and stigma acceptability determination,hybridization index estimation,and artificial controlled pollination test were conducted to analyze floral characteristics and breeding system of the P. dianthum. The results showed that P. dianthum blossomed from June to September,with a single flowering period of 45 to 55 d,a single plant flowering period of 75 to 90 d. Each plant generally produced one peduncle bearing with two to five flowers. During the entire blooming process,the pollinia formed by anthers were persistently located on both sides of the staminode and the relative positions remained unchanged. The pollen viability and stigma receptivity reached the highest in the full-flowering period(flowering 6 to 34 d),and the pollen and sitgma have a longer life span. The hybridization index(OCI)ofP. dianthum was four. Regardless of emasculation or not,the seed setting rates of artificial self-pollination,artificial cross-pollination of the same plant,and artificial cross-pollination of different plants were 83.30%,90.00%,and 96.70%,respectively. No significant difference was observed among these rates,which were all significantly higher than that of natural pollination. These results demonstrate that there is no apomixis but highly passive self-crossing and out-crossing abilities in the P. dianthum,and the breeding system is a mixed mating system of selfing and out-crossing that requires pollinators.
In this study,we searched the SSR loci in the whole loquat genome and analyzed its distribution characteristics. The resequencing data of 10 cultivars were used to analyze the genotypes of these loci. The results showed that 103 509 SSR sites were obtained,of which 73 604 SSRs were dinucleotide repeat,accounting for 71.1% of total discovered SSRs. The main dinucleotide repeat motif was AT/TA. The trinucleotide repeat type of SSRs accounts for 15.0%,with AAG/CTT as the main type. Ten cultivars of loquat were resequenced,and 32-206 Mb reads were obtained with a depth of 12.9×-81.3×. Two new scripts(Flank and SSRtype)were developed to analyze the length of SSR sites embedded in the resequencing data reads. The sequencing depth of Zaozhong 6 loquat was 12.9×,covering 80% of SSR sites,indicating that a sequencing depth of more than 12 times was enough to the analysis of the SSR loci. A large number of SSR loci in loquat are multiple copies(accounting for 78.4%),producing more than three bands in a locus in a cultivar. There are 12 962 SSR loci with two copies in all cultivars(accounting for 12.5%). The polymorphic bands generated by these loci range from two to 10. The median of the polymorphism information content(PIC)is 0.269. The PIC value of 526 SSR sites was more than 0.5. Twenty pairs of primers were screened to perform fluorescent PCR. The results of polymorphism of these primers were consistent with the resequencing analysis.
The minor coat protein(CPm)of citrus tristeza virus(CTV)is necessary in virus transmission by the brown citrus aphid[Toxoptera citricida(Kirkaldy)]. The expression levels of CPm in three isolates with high,middle and low aphid transmissibility were conducted by Western blot technique. Then,the CPm sequences of CTLJ and CT16-2 isolates were acquired by RT-PCR,cloning and sequencing. Finally,these amino acid sequences were used to analyze the multiple alignments by the DNAStar program. The result showed that the identity of amino acid sequence of CPm for CTLJ with low aphid transmissibility was only 93.3%-94.2%,when compared with those of other CTV isolates. Comparison of the CPm of these isolates showed that nine of fourteen amino acid mutation site were unique in the CTLJ isolate. Particularly,some mutations led to substitution of new amino acids with a different charge and polarity,which resulted in changes of the secondary structure of the CPm protein in CTLJ. However,the expression of CPm for CTLJ,CT16-2 and CT11A did not show a significant difference. The results suggest that the aphid transmission efficiency of CTV might be not related to the expression level of CPm,but might be associated with amino acid site variation.
In recent years,various types of cytoplasmic male sterile(CMS)lines and their restorer lines have been reported in vegetables,and a number of restorer genes have been located and cloned,and the markers closely linked with restorer genes have been developed and used in vegetable breeding. Here,recent progress on CMS restorer lines were reviewed,as well as the mapping and cloning of restorer genes,the marker development of restorer genes,and the application of restorer lines in breeding of vegetables. The existing problems and the development trend in the future were discussed.
‘Zhongnong Wanzhenzhu’is a new late ripening nectarine cultivar bred from the seedlings of ‘Qiuhongzhu’. The tree posture is half open. The flower is showy,pink. The fruit is round with average weight about 131.2 g. Its flesh and pericap are light green. The soluble soilds content is 16.0%;the titratable acid content is 0.34%;the vitamin C content is 52.1 μg · g-1. It has a good tolerance for storage and transportation. The fruit ripens in mid-late September in Juxian,Rizhao City,Shandong Province. The yield in the first and second growth cycle were 10 500 and 22 500 kg · hm -2,and the yield in the fruit-bearing stage was controlled at 37 500 kg · hm -2.
‘Mobao’is a hybrid of wax gourd[Benincasa hispida(Thumb)Cogn. How]with small fruit. It has strong growth potential and good continuous fruit setting. The fruit is in short cylinder shape and well proportioned. The average fruit weight is 1.8 kg. The fruit length is about 25.0 cm and the diameter is about 13.0 cm. The pericarp color is dark green,with no wax powder or furrow. The flesh is firm and it is resistant to storage and transportation. The yield is 48 906 kg · hm-2. This cultivar is resistant to Fusarum wilt and moderately resistant toPhytophthora blight wilt. It is suitable for growing in spring and autumn cultivation in South China.
The new ornamental lotus cultivar‘Lingyun’was bred from the seedings of‘Youyi Mudan lian’. The plant is tall and straight,with erect leaf height of 130-195 cm,long leaf diameter of 37-62 cm and short leaf diameter of 36-56 cm. Flower color is yellowish green. The outer petal number is 19-22,and the inner petal number is 53-56. The diameter of corolla is 23-26 cm. The density of flowers is 5-9 pers quare meter.