Association Analysis On Adaptability And Leptocybe Invasa Resistance In Eucalyptus Grandis And E. Tereticornis

Forests serve as the main part of terrestrial ecosystems and play an important role in human ecological civilization, economic development and environmental protection. In china, the low forest coverage results in a severe challenge for national timber resource strategy and tree breeding work. Though traditional hybrid breeding has made considerable achievements, breeding programs have been hindered in trees largely by high heterozygosity, long generation times and quantitative nature of economic traits. Moreover, early efforts of tree improvement have focused on growth and productivity traits rather than adaptability and biotic or abiotic resistances. Therefore, studies on tree adaptability and resistances are valuable for effective management and utilization of forest resources and for acceleration of breeding advancement. Here, Eucalyptus grandis and E. tereticornis populations were analyzed for natural adaptability and Leptocybe invasa resistance using genome-wide low-density simple sequence repeats(SSR) markers. Candidate genes differentially expressed in transcriptomes between resistant and susceptible genotypes were identified through RNA-seq. Association analysis on a candidate gene and L. invasa resistance indicated that there were alleles significantly associated with the resistance. Also, the possibility of marker assisted selection was explored in such traits as adaptability and L. invasa resistance.The main contents and results are as follows:(1) Genetic diversity and selective loci in E. grandis nature populations. A total of 159 individuals sampled from 16 E. grandis populations were analyzed using 110 low-density SSR markers spanning the E. grandis genome, including 45 genomic SSRs and 65 expressed sequence tag(EST) derived SSRs. High levels of population diversity was detected with 31 neutral SSR loci, e.g. mean expected heterozygosity(HE) being 0.744 and mean allelic richness(AR) being 4.929. Population differentiation was extremely low, with mean FST=0.037 as well as among-population variation percentage of 3.7% in analysis of molecular variance(AMOVA), indicating within-population accounting for the majority of genetic variations. A total of 58 FST outliers were identified as selective loci from all the 110 SSR loci. Three significant associations were tested through univariate linear regression analysis between the allelic frequency and the climatic factor. Embra180-120 bp associated with mean annual temperature climatic factors EUCeSSR0755-276 bp associated with mean annual temperature and isothermality(P <0.05). The results plays an important role in understanding the genetic basis of perennial trees adaption, and in conservation and utilization of E. grandis germplasm.(2) Genetic diversity and selective loci in E. tereticornis nature populations. A total of 77 individuals from nine E. tereticornis populations were analyzed using 108 simple sequence repeats(SSR) markers spanning the E. grandis genome, including 44 genomic SSRs and 64 expressed sequence tag(EST) derived SSRs. Population diversity was high based on 25 neutral SSR loci, e.g. HE being 0.800 and AR 3.246. Population differentiation was extremely low, with mean FST = 0.012 as well as among-population variation percentage of 1.2% in analysis of molecular variance(AMOVA), indicating within-population accounting for the majority of genetic variations. A total of 78 FST outliers were identified as selective loci form all the 108 SSR loci, and the numbers of outliers related with mean annual temperature, mean annual precipitation, maximum temperature of the warmest month and precipitation seasonality. EUCeSSR485-140 bp associated with precipitation seasonality climatic factors was validated with univariate linear regression(P < 0.05).(3) SSR markers associated with L. invasa resistance in E. grandis and their verification in E. tereticornis. A total of 816 published SSR markers covering the whole genome of E. grandis were screened for PCR success and allelic frequency difference between high-susceptible and high-resistant genotype pools(8 genotypes in each pool) of E. grandis. Eighty-seven SSRs screened out were used in association analysis against 470 samples of 16 populations of E. grandis, and seven SSR markers were revealed to be significantly associated with L. invasa resistance, with the phenotypic variation explained per marker being 3.3-37.8%. Of the seven significant markers, four were verified in 303 samples of 9 populations of E. tereticornis, with the phenotypic variation explained per marker being 24.3-48.5%. All these significant markers will have great potentials for molecular breeding in L. invasa resistance of Eucalyptus.(4) Transcriptome analysis on the response of E. grandis to L. invasa. Transcriptome sequencing in susceptible(S-2 with galls naturally infected with L. invasa and S-1 without galls) and resistant(R) genotypes from each of four populations of E. grandis showed that there were 19 genes differentially expressed between S-2 and S-1 genotypes, and they were significantly enriched in ten KEGG Pathways. There were zero and seven genes expressed differentially between S-1 and R and between S-2 and R genotypes, respectively. Most of these genes were closely related with respiration, ET pathway, GA pathway and a plurality of plant stress response metabolic pathways. Specifically, Eucgr.G02880 may be an important candidate gene in defence response to L. invasa in E. grandis.(5) Association analysis on candidate gene Eucgr.G02880 with L. invasa resistance in E. grandis. The candidate gene Eucgr.G02880 was amplicon sequenced across the 470 samples of 16 E. grandis populations, and 38 single nucleotide polymorphisms(SNPs) and two insert/deletions(Indels) were found within the 767 bp sequence. Of the nine SNPs with allele frequency ≥ 5%, three were significantly associated with resistance to L. invasa, with the resistance variation explained per significant marker being 0.876-1.959%. The results indicate the efficiency of association studies combined with transcriptome analysis in dissecting genomic loci related with economic traits.(6) Molecular marker-assisted selection of adaptability and L. invasa resistance in E. grandis and E. tereticornis. Superior genotypes with the most plus-allele-block were predicted for adaptability and/or L. invasa resistance in both species. There was no genotype in E. grandis combining all the plus alleles, and it was suggested to make one or more controlled pollinations and carry out marker-assisted selection of superior genotypes in the progeny. The genotype 111 of E. tereticornis combines all the plus alleles and could have potentials for further deployment.