| The species of Casuarinaceae is characterized by fast growing and high resistance against wind,drought and saline,and thus is extensively used in coast protection forest in Guangdong.Casuarinaceae bacterial wilt was detected since 2012 probably due to the application of only a few clones in afforestation,and has largely damaged the coast forest.Bacterial wilt is caused by Ralstonia solanacearum,which spreads out via soil,and is one of the most harmful bacteria diseases worldwide.Breeding for disease resistance is considered as the most effective way to control bacterial wilt.Based on the isolation and identification of R.solanacearum,this work studied the resistance identification of Casuarinaceae germplasm resources in China by reliable and effective resistance identification method,tested the physiological changes of infected Casuarinaceae,developed 235 EST-SSR markers to analyze genetic diversity and group structure of Casuarinaceae core sources,and analyzed the association of molecular markers and resistant traits.The study found that:(1)Two methods,dilution isolation and root overflow,were used in strains isolation,and totally 31 strains were isolated.The method of root overflow is simple to operate and the isolation rate was about 60%,thus was supplementary to regular dilution method.Only 22 strains were amplified from the 31 strains via 16 S rRNA sequencing,and the amplified strains were identified as R.solanacearum.The results of pathogenicity test showed that the pathogenicity between clones,strains,and interaction between strains and clones were significantly different(P<0.01).The correlation analysis showed that pathogenicity of these strains under different inoculation approaches was significant(P<0.05)or very significant(P<0.01)with the correlation coefficients between 0.497 and 0.731.After comparing the pathogenicity of these strains,strains GL-2,H,M,TC-1,F and Q were selected for resistance identification.(2)Using clone A8 as the tested material,eight inoculation approaches were designed to explore the effect of different inoculation approaches on the bacterial wilt resistance identification.The inoculation approaches included hydroponic rooting seedlings,twig,lignified green branch,lignified brown branch,bacterial wilt crude toxin,potted seedlings root damage and without damage.The results showed that the injured root inoculation of potted seedling and hydroponic inoculation of lignified brown branch were the best two approaches for identifying resistance.Resistant and infected clones were effectively distinguished through the two approaches.There was a significant positive correlation between the two methods(r = 0.857).The other inoculation approaches,i.e.,no-injured root inoculation of potted seedling,hydroponic inoculation of rooting seedlings,tender branch,lignified green branch and crude toxin were not suitable as the resistance identification approaches.Using hydroponic inoculation of lignified brown branch,the resistance of 53 Casuarinaceae clones were tested,and X1?45?Ping5?30?W6?Zajiao?501?G1?503?41?A1 and A1-3 of high resistance clones were screened out.(3)The content of soluble proteins and activities of peroxidas(POD),catalase(CAT),superoxide dismutase(SOD),polyphenol oxidase(PPO)and phenylalanine ammonia lyase(PAL)were measured after R.solanacearum inoculated on the injured root of Casuarinaceae by the potted seedling method.The result showed that the activity of protective enzymes in A8 clone had been changed significantly after inoculation.The activity of SOD,PPO and PAL showed a waved trend(up-down-up-down trend),and end up with low enzyme activity before senescence.In contrast,the activity of CAT and POD was prohibited and was much lower than that in the control.In addition,the contents of soluble proteins increased,which trend was consistent with the activity changes of SOD,PPO and PAL.(4)Using the 37902 Casuarina equisetifolia stem transcriptome sequence in NCBI,3861 sequences containing 4187 SSR loci were obtained and the detection rate was 10.2%.On average,there was one SSR loci in 8.3 kb.Dinucleotide and trinucleotide motifs were dominant in the detected SSR loci,accounting for 39.5% and 24.2% of the total SSR loci.Dinucleotide motifs were dominated by AG/CT,and trinucleotide motifs were dominated by AAG/CTT.Dinucleotide motifs were dominated by 6,7 and 8 replicates,and pentanucleotide and hexanucleotide motifs were dominated by 3 and 4 replicates.235 out of 404 pairs of designed primers can be effectively amplified,and EST sequences of 151 out of the 235 pairs was homologous to the known genes,which functions can be classified as biological progress,molecular function and cellular competent,and lots of the sequences involve in multiple functions.The developed 235 markers had a high universality in 4 species of Casuarina,and the deleted degree(DD)was lower than 5%.223 out of the 235 markers had a high polymorphism: the average observed heterozygosity(Ho)was 0.8558.The average expected heterozygosity(HE)was 0.7243.The average number of alleles(Na)was 5.4 and the average polymorphi information content(PIC)was 0.63.The average genetic distance of the studied core sources of Casuarinaceae was 0.5523,and the 63 clones were classified as 3 groups at the genetic distance of 0.30.(5)Genome scanning was conducted on the Casuarinaceae germplasm resources of China by using 235 pairs of EST-SSR markers.49 differential expression marker was obtained in resistance/susceptible gene pool.The population was constituted by 2 sub-populations,who displayed no associations with their geographical origins.Population structure was relatively simple and the heritable variations were high,and thus suitable for genetic correlation analysis.Totally 10 markers(CeSSR253,CeSSR022,CeSSR359,CeSSR241,CeSSR242,CeSSR056,CeSSR263,CeSSR052,CeSSR176 and CeSSR258)were detected via MLM(Q + K)model,and they significantly associated with the disease index of the studied clones(P<0.05),with the phenotypic variation explained per marker being 12.6% ~ 60.7%,suggesting that these markers were linked with some unknown important resistant/susceptible loci.Alleles of CeSSR052-237 bp and CeSSR052-255 bp displayed the highest positive AAE(37.1)and AN(70.1%)in the analysis of bacterial wilt resistance.The positive AAE of alleles of CeSSR359-370 bp,CeSSR359-386 bp and CeSSR241-265 bp was higher than 20.0.Alleles of CeSSR022-190 bp and CeSSR022-196 bp had a negaitive AAE> 40.0 and AN> 40%.The negative AAE of alleles of CeSSR253-186 bp,CeSSR253-202 bp,CeSSR242-227 bp and CeSSR176-204 bp was higher than 20.0. |
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