High synteny and colinearity among Eucalyptus genomes revealed by high-density comparative genetic mapping

Understanding genome differentiation is important to compare and transfer genomic information between taxa, such as from model to non-model organisms. Comparative genetic mapping can be used to assess genome differentiation by identifying similarities and differences in chromosome organization. Following release of the assembled Eucalyptus grandis genome sequence (January 2011; ), a better understanding of genome differentiation between E. grandis and other commercially important species belonging to the subgenus Symphyomyrtus is required. In this study, comparative genetic mapping analyses were conducted between E. grandis, Eucalyptus urophylla, and Eucalyptus globulus using high-density linkage maps constructed from Diversity Array Technology and microsatellite molecular markers. There were 236–393 common markers between maps, providing the highest resolution yet achieved for comparative mapping in Eucalyptus. In two intra-section comparisons (section Maidenaria–E. globulus and section Latoangulatae–E. grandis vs. E. urophylla), ∼1% of common markers were non-syntenic and within chromosomes 4.7–6.8% of markers were non-colinear. Consistent with increasing taxonomic distance, lower synteny (6.6% non-syntenic markers) was observed in an inter-section comparison between E. globulus and E. grandis × E. urophylla consensus linkage maps. Two small chromosomal translocations or duplications were identified in this comparison representing possible genomic differences between E. globulus and section Latoangulatae species. Despite these differences, the overall high level of synteny and colinearity observed between section Maidenaria–Latoangulatae suggests that the genomes of these species are highly conserved indicating that sequence information from the E. grandis genome will be highly transferable to related Symphyomyrtus species.     

AFLP genetic maps of Eucalyptus globulus and E. tereticornis

 Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient technique for detecting large numbers of DNA markers in eucalypts. We have used AFLP markers in a two-way pseudo-testcross strategy to generate genetic maps of two clones of different Eucalyptus species (E. tereticornis and E. globulus). Of 606 polymorphic fragments scored, 487 segregated in a 1 : 1 ratio, corresponding to DNA polymorphisms heterozygous in one parent and null in the other. In the maternal E. tereticornis map, 268 markers were ordered in 14 linkage groups (919 cM); the paternal E. globulus map had 200 markers in 16 linkage groups (967 cM). Results from PGRI software were compared with MAPMAKER. The average density of markers was approximately 1 per 3.9 cM. Framework markers were ordered with an average confidence level of 90%, covering 80–100% of the estimated Eucalyptus genome size. In order to investigate the homologies between the E. tereticornis and the E. globulus genetic linkage maps, we included 19 markers segregating 3 : 1 in the analysis. Some homeologous linkage groups were recognized. The linkage data developed in these maps will be used to detect loci controlling commercially important traits. Received: 17 July 1997 / Accepted: 13 October 1997     

Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla

 We report on the development, genetic characterization and linkage mapping of a battery of SSR (simple sequence repeat) loci in Eucalyptus grandis and E. urophylla. This study reveals the abundance of SSRs in Eucalyptus, the very high information content of these markers for mapping and individual identification, and demonstrates the feasibility of constructing a comprehensive microsatellite-based linkage map for Eucalyptus. Primer sequence for a set of 20 highly informative EMBRA (Eucalyptus microsatellites from Brazil) loci are made available together with their map position and estimates of the expected heterozygosity and allele size range in these two species. Using genomic library enrichment and anchored-PCR screening prior to sequencing, the efficiency of SSR marker locus development was 63% from sequencing data to operationally useful SSR loci. Absolute transportability between the two species and very high levels of allelic variability and expected heterozygosity (H) were seen at all SSR loci surveyed. The number of alleles per locus ranged from 9 to 26 with an average of 16.3±4.8. The average H of 15 loci was 0.86±0.04, 0.83±0.08 and 0.89±0.04, respectively, for E. urophylla, E. grandis and the combined two-species estimate. In the mapping analysis 16 out of 20 marker loci segregated in a fully informative configuration, allowing the determination of synteny of six homologous linkage groups between the two species. The availability of transportable, multiallelic, PCR-based co-dominant SSR loci represents a dramatic improvement in our ability to carry out detailed population genetic analysis and to search, understand, and manipulate allelic variation at QTLs (quantitative trait loci) in species of Eucalyptus. Received: 16 March 1998 / Accepted: 22 March 1998     

Development,characterization, validation,and mapping of SSRs derived from <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.<Emphasis Type="Italic">–Moniliophthora perniciosa</Emphasis> interaction ESTs

In this study, we report results of the detection and analysis of SSR markers derived of cacao–Moniliophthora perniciosa expressed sequence tags (ESTs) in relation to cacao resistance to witches’ broom disease (WBD), and we compare the polymorphism of those ESTs (EST-simple sequence repeat (SSR)) with classical neutral SSR markers. A total of 3,487 ESTs was used in this investigation. SSRs were identified in 430 sequences: 277 from the resistant genotype TSH 1188 and 153 from the susceptible one Catongo, totalizing 505 EST-SSRs with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%), and tetranucleotides (0.6%). EST-SSRs were classified into 16 main categories; most of the EST-SSRs belonged to “Unknown function” and “No homology” categories (45.82%). A high frequency of SSRs was found in the 5’UTR and in the ORF (about 27%) and a low frequency was observed in the 3’UTR (about 8%). Forty-nine EST-SSR primers were designed and evaluated in 21 cacao accessions, 12 revealed polymorphism, having 47 alleles in total, with an average of 3.92 alleles per locus. On the other hand, the 11 genomic SSR markers revealed a total of 47 alleles, with an average of 5.22 alleles per locus. The association of EST-SSR with the genomic SSR enhanced the analysis of genetic distance among the genotypes. Among the 12 polymorphic EST-SSR markers, two were mapped on the F₂ Sca 6 × ICS 1 population reference for WBD resistance.     

High-density genetic linkage maps with over 2,400 sequence-anchored DArT markers for genetic dissection in an F2 pseudo-backcross of Eucalyptus grandis × E. urophylla

Traits that differentiate cross-fertile plant species can be dissected by genetic linkage analysis in interspecific hybrids. Such studies have been greatly facilitated in Eucalyptus tree species by the recent development of Diversity Arrays Technology (DArT) markers. DArT is an affordable, high-throughput marker technology for the construction of high-density genetic linkage maps. Eucalyptus grandis and Eucalyptus urophylla are commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions. We analysed 7,680 DArT markers in an F2 pseudo-backcross mapping pedigree based on an F1 hybrid clone of E. grandis and E. urophylla. A total of 2,440 markers (31.7%) were polymorphic and could be placed in linkage maps of the F1 hybrid and two pure-species backcross parents. An integrated genetic linkage map was constructed for the pedigree resulting in 11 linkage groups (n = 11) with 2,290 high-confidence (LOD ≥ 3.0) markers and a total map length of 1,107.6 cM. DNA sequence analysis of the mapped DArT marker fragments revealed that 43% were located in protein coding regions and 90% could be placed in the recently completed draft genome assembly of E. grandis. Together with the anchored genomic sequence information, this linkage map will allow detailed genetic dissection of quantitative traits and hybrid fitness characters segregating in the F2 progeny and will facilitate the development of markers for molecular breeding in Eucalyptus.     

Recurrent nuclear DNA introgression accompanies chloroplast DNA exchange between two eucalypt species

Numerous studies within plant genera have found geographically structured sharing of chloroplast (cp) DNA among sympatric species, consistent with introgressive hybridization. Current research is aimed at understanding the extent, direction and significance of nuclear (nr) DNA exchange that accompanies putative cpDNA exchange. Eucalyptus is a complex tree genus for which cpDNA sharing has been established between multiple species. Prior phylogeographic analysis has indicated cpDNA introgression into the widespread forest species Eucalyptus globulus from its rare congener E. cordata. In this study, we use AFLP markers to characterize corresponding nrDNA introgression, on both a broad and fine spatial scale. Using 388 samples we examine (i) the fine‐scale spatial structure of cp and nrDNA introgression from E. cordata into E. globulus at a site in natural forest and (ii) broad‐scale patterns of AFLP marker introgression at six additional mixed populations. We show that while E. globulus and E. cordata retain strongly differentiated nuclear gene pools overall, leakage of nrDNA occurs at mixed populations, with some AFLP markers being transferred to E. globulus recurrently at different sites. On the fine scale, different AFLP fragments show varying distances of introgression into E. globulus, while introgression of cpDNA is extensive. The frequency of E. cordata markers in E. globulus is correlated with spatial proximity to E. cordata, but departs from expectations based on AFLP marker frequency in E. cordata, indicating that selection may be governing the persistence of introgressed fragments in E. globulus.     

Gene flow of the canker pathogen Botryosphaeria australis between Eucalyptus globulus plantations and native eucalypt forests in Western Australia

Abstract The eucalypt plantation industry in Western Australia provides a unique opportunity to study the movement of pathogens between closely related host taxa. Eucalyptus globulus, a native to Tasmania and south‐eastern Australia, is the predominant species in Western Australian plantations, often being planted adjacent to native forest containing Eucalyptus marginata and Eucalyptus diversicolor. Since the commencement of the plantation industry 20 years ago, several fungal species, previously known only to eastern Australia or overseas, have been reported on E. globulus in Western Australia. Botryosphaeria australis is a newly described species, recently found causing cankers on Acacia spp. in eastern Australia. However, during a routine survey, B. australis was found to be the predominant species associated with E. globulus plantations and native Eucalyptus spp. in Western Australia. In this study, six short simple repeat markers were used to evaluate genetic diversity and gene flow between collections of B. australis from native eucalypt forest and E. globulus plantations at two locations in south‐western Australia. In both cases, there was no restriction to gene flow between the plantations and the adjacent native forest. Botryosphaeria australis has now been isolated from a wide range of hosts across south‐western Australia and was not isolated from E. globulus in Tasmania or South Australia. This extensive distribution and host range suggests B. australis is native to Western Australia. This study demonstrates the ability of a pathogen to move between plantation and forests.     

Puccinia psidii infecting cultivated Eucalyptus and native myrtaceae in Uruguay

Eucalyptus or guava rust caused by Puccinia psidii is a serious disease of Eucalyptus and other Myrtaceae. In Uruguay, it has been previously found on Eucalyptus globulus and Psidium brasiliensis. Almost nothing is known regarding the occurrence of this pathogen on other Eucalyptus species or native Myrtaceae in that country. In this study, we determined the presence of P. psidii on Eucalyptus species and native Myrtaceae trees in Uruguay and evaluated the pathogenicity of specimens from native myrtaceous hosts on E. globulus and E. grandis. Phylogenetic analyses based on the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA operon were used to confirm pathogen identity. Comparisons of ITS sequences confirmed the identity of P. psidii on Eucalyptus globulus, E. grandis, Myrcianthes pungens, and Myrrhinium atropurpureum var. octandrum. This is the first report of P. psidii on M. atropurpureum var. octandrum. Pathogenicity tests showed that isolates from native Myrtaceae could infect both Eucalyptus species tested, indicating a strong biological relationship between both introduced and native Myrtaceae. This study supplies relevant field data, morphological information, molecular phylogenetic analyses and infection studies that contribute to a better understanding of an important and little studied pathogen.     

Frequency, type, and distribution of EST-SSRs from three genotypes of Lolium perenne, and their conservation across orthologous sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa

Background  

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies in several plant species. They are used for cultivar identification, variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Currently, a limited number of SSR markers are publicly available for perennial ryegrass (Lolium perenne). We report on the exploitation of a comprehensive EST collection in L. perenne for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of L. perenne, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of L. perenne, Festuca arundinacea, Brachypodium distachyon, and O. sativa, 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding.     

Genotyping systems for Eucalyptus based on tetra-, penta-, and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests

Eucalypts are keystone species in their natural ranges and are extensively planted worldwide for high-quality woody biomass. A novel set of 21 polymorphic and interspecifically transferable microsatellite markers based on tetra-, penta- and hexanucleotide repeats were developed and tested for high-precision genotyping of species of Eucalyptus. These microsatellites were characterized in population samples of four species, Eucalyptus grandis, Eucalyptus globulus, Eucalyptus urophylla, and Eucalyptus camaldulensis, representing three phylogenetic sections of subgenus Symphyomyrtus. These markers provide a clear advantage for accurate allele calling due to their larger allele size difference. Two multiplexed microsatellite combinations, a 14-locus/four-dye and an 18-locus/five-dye set, analyzable in single lanes were designed, providing resolution and throughput analogous to those routinely used in human DNA profiling. This set of microsatellites was shown to have high resolution for clone fingerprinting, inter-individual genetic distance estimation, species distinction, and assignment of hybrid individuals to their most likely ancestral species. These systems will be particularly useful for comparative population genetics and molecular breeding applications that require consistent allele calling across different points in time or laboratories.     

Genetic diversity of Eucalyptus hybrids estimated by genomic and EST microsatellite markers

The knowledge of breeding impacts on the genetic diversity of hybrids of Eucalyptus is crucial to the exploration of genetic resources. We estimated genetic polymorphic parameters of 112 hybrids of Eucalyptus spp. using 10 genomic simple sequence repeats (SSR) markers and 10 expressed sequence tags (EST) microsatellite markers. According to Student’s t-test, there were no significant differences between genomic SSR and EST-SSR markers. Our results also revealed high polymorphism in the hybrids analyzed, indicating that both markers are appropriate for use in genetic breeding programs.     

Construction of a genetic linkage map using simple sequence repeat markers from expressed sequence tags for cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta) is an economically important crop that is grown in tropical and sub-tropical regions. Use of molecular technology for genetic improvement of cassava has been limited by the lack of a large set of DNA markers and a genetic map. Therefore, the aims here were to develop additional simple sequence repeat (SSR) markers from the public expressed sequence tags (ESTs), and to construct a genetic linkage map. In this study, we designed 425 EST-SSR markers from sequences obtained from the cassava EST database in GenBank, and integrated them with 667 SSR markers from a microsatellite-enriched genomic sequence received from the International Center for Tropical Agriculture (CIAT). Of these, 107 EST-SSR and 500 genomic SSR primer pairs showed polymorphic patterns when screened in two cassava varieties, Hauy Bong 60 and Hanatee, which were used as female and male parental lines, respectively. Within the 107 and 500 primer pairs, 81 and 226 EST-SSR and SSR primer pairs were successfully genotyped with 100 samples of F₁ progeny, respectively. The results showed 20 linkage groups consisting of 211 markers—56 EST-SSR and 155 SSR markers—spanning 1,178 cM, with an average distance between markers of 5.6 cM and about 11 markers per linkage group. These novel EST-SSR markers provided genic PCR-based co-dominant markers that were useful, reliable and economical. The EST-SSRs were used together with SSR markers to construct the cassava genetic linkage map which will be useful for the identification of quantitative trait loci controlling the traits of interest in cassava breeding programs.     

SSR mining in coffee tree EST databases: potential use of EST-SSRs as markers for the Coffea genus

Expressed sequence tags (ESTs) from Coffea canephora leaves and fruits were used to search for types and frequencies of simple sequence repeats (EST–SSRs) with a motif length of 1–6 bp. From a non-redundant (NR) EST set of 5,534 potential unigenes, 6.8% SSR-containing sequences were identified, with an average density of one SSR every 7.73 kb of EST sequences. Trinucleotide repeats were found to be the most abundant (34.34%), followed by di- (25.75%) and hexa-nucleotide (22.04%) motifs. The development of unique genic SSR markers was optimized by a computational approach which allowed us to eliminate redundancy in the original EST set and also to test the specificity of each pair of designed primers. Twenty-five EST–SSRs were developed and used to evaluate cross-species transferability in the Coffea genus. The orthology was supported by the amplicon sequence similarity and the amplification patterns. The >94% identity of flanking sequences revealed high sequence conservation across the Coffea genus. A high level of polymorphic loci was obtained regardless of the species considered (from 75% for C. liberica to 86% for C. canephora). Moreover, the polymorphism revealed by EST–SSR was similar to that exposed by genomic SSR. It is concluded that Coffea ESTs are a valuable resource for microsatellite mining. EST-SSR markers developed from C. canephora sequences can be easily transferred to other Coffea species for which very little molecular information is available. They constitute a set of conserved orthologous markers, which would be ideal for assessing genetic diversity in coffee trees as well as for cross-referencing transcribed sequences in comparative genomics studies.     

Root-promoting rhizobacteria in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> cuttings

Cloned Eucalyptus spp. plantations are based in greenhouse production of plants generated by vegetative propagation. Diverse studies have demonstrated that rhizospheric bacteria can stimulate plant growth, and more recently that they can increase rooting in vegetative material. Considering this potential, the objective of this study was to verify the effect of bacterial strains on rooting Eucalyptus globulus. A total of 132 bacterial strains isolated from the rhizosphere of E. globulus and Eucalyptus nitens were studied. The bacterial inoculums in a concentration of 4 × 10⁸ cfu/ml were applied to the rooting substrate at the cutting installation and 45 days after by irrigation. Rooting was evaluated on days 60 and 75 after cutting installation, considering the number of roots as well as their fibrosity and roots biomass. Of the 132 strains evaluated, 26 significantly increased cutting rooting in a range of 191.4–69.4% with respect to the control. Additionally, some strains stimulated the development of fine roots and incremented the roots biomass. The strains identificated that produced a rooting effect were: Bacillus firmus, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, B. subtilis/amyloliquefaciens, Bacillus circulans, Brevibacillus brevis, Paenibacillus lautus and Stenotrophomona maltophilia. These first trials suggest the potential of these bacteria to be used in clonal production programs for E. globulus.     

A genetic map of pineapple (Ananas comosus (L.) Merr.) including SCAR, CAPS, SSR and EST-SSR markers

Despite the paramount importance of pineapple (Ananas comosus L.) in world production and trade of tropical fruits, the genomics of this crop is still lagging behind that of other tropical fruit crops such as banana or papaya. A genetic map of pineapple was constructed using an F2 segregating population obtained from a single selfed F1 plant of a cross A. comosus var. comosus (cv. Rondon, clone BR 50) × A. comosus var. bracteatus (Branco do mato, clone BR 20). Multiple randomly amplified markers (RAPD, ISSR and AFLP) were brought together with SSR and EST-SSR markers identified among sequences uploaded to public databases and with sequence-specific markers (SCAR, SSR and CAPS) derived from random amplified markers. Sixty-three randomly amplified markers (RAPD, ISSR and AFLP) were selected and cloned, resulting in 71 sequences which were used to generate sequence-specific SCAR and CAPS markers. The present map includes 492 DNA markers: 57 RAPD, 22 ISSR, 348 AFLP, 20 SSR, 12 EST-SSR, 25 SCARs, 8 CAPS, and the morphological trait locus “piping”, gathered into 33 linkage groups that integrate markers inherited from both botanical varieties, four linkage groups with markers only from var. comosus and three linkage groups with markers exclusively from var. bracteatus. The relatively higher mapping efficiency of sequence-specific markers derived from randomly amplified markers (50.7%) versus SSR (31.4%) and EST-SSR (28.9%) markers is discussed. Spanning over 80% of the 2,470 cM estimated average length of the genome, the present map constitutes a useful research tool for molecular breeding and genomics projects in pineapple and other Bromeliaceae species.     

桉属植物非挥发性化学成分和药理活性研究进展

桉属(Eucalyptus L. Herit)是桃金娘科(Myrtaceae)的大属,该属约600余种,主要分布于世界各地热带亚热带地区。我国引入品种较多,主要分布于华南地区,其中广东和广西为桉树的主要种植基地。桉属植物具有较多的工业价值,其木材、叶、果实等是化学工业、香料、医药领域的重要原料,可用作开发高性能桉木重组材、竹桉复合材料、造浆与造纸等。桉属植物作为民间药材被使用,具有抑菌消炎、疏风解热、防腐止痒等功效,其药理研究表明,桉属植物具有良好的抗氧化、抗炎、抗菌、抗病毒、抗肿瘤、抗心血管疾病等药理活性。该研究通过查阅近三十年桉属植物相关的国内外文献报道,对桉属植物不同部位的421个非挥发性化学成分及其药理活性等进行了较详细的分类阐述,其中黄酮类化合物共73个、有机酸化合物共61个、萜类化合物共45个、多酚类化合物共229个、脂肪醇类化合物共13个,药理活性多集中在抗氧化、抗菌、抗病毒、抗肿瘤等,但相关机制仍需进一步阐明。该文重点关注桉属植物的药用部位,充分发掘其药用价值,开展临床转化和新药研究工作,为今后桉属植物的进一步研究、开发和利用提供科学依据。