Retrotransposon-based molecular markers for linkage and genetic diversity analysis in wheat

Retrotransposon-based molecular markers have been developed to study bread wheat ( Triticum aestivum) and its wild relatives. SSAP (Sequence-Specific Amplification Polymorphism) markers based on the BARE-1/ Wis-2-1A retrotransposons were assigned to T. aestivum chromosomes by scoring nullisomic-tetrasomic chromosome substitution lines. The markers are distributed among all wheat chromosomes, with the lowest proportion being assigned the D wheat genome. SSAP markers for BARE-1/ Wis-2-1A and three other wheat retrotransposons, Thv19 , Tagermina and Tar1, are broadly distributed on a wheat linkage map. Polymorphism levels associated with these four retrotransposons vary, with BARE-1/ Wis-2-1A and Thv19 both showing approximately 13% of bands polymorphic in a mapping population, Tagermina showing approximately 17% SSAP band polymorphism and Tar1 roughly 18%. This suggests that Tagermina and Tar1 have been more transpositionally active in the recent evolutionary past, and are potentially the more useful source of molecular markers in wheat. Lastly, BARE-1 / Wis-2-1A markers have also been used to characterise the genetic diversity among a set of 35 diploid and tetraploid wheat species including 26 Aegilops and 9 Triticum accessions. The SSAP-based diversity tree for Aegilops species agrees well with current classifications, though the Triticum tree shows several significant differences, which may be associated with polyploidy in this genus.Communicated by M.-A. Grandbastien     

Resistance gene analogues of Arabidopsis thaliana: recognition by structure

Following completion of Arabidopsis thaliana sequencing projects, multiple resistance gene analogues (RGAs) have been identified. In this work a review of the current state of knowledge available in protein databases and scientific articles is presented. Putative resistance genes were identified by using BLAST searches as well as HMM fingerprints (the latter to infer existence of characteristic domains). The representation of all five classes of putative resistance genes in Col-0 ecotype was examined, along with the statistics on RGAs present on all five chromosomes of Arabidopsis thaliana.     

Puroindolines: the molecular genetic basis of wheat grain hardness

The variation in grain hardness is the single most important trait that determines end-use quality of wheat. Grain texture classification is based primarily on either the resistance of kernels to crushing or the particle size distribution of ground grain or flour. Recently, the molecular genetic basis of grain hardness has become known, and it is the focus of this review. The puroindoline proteins a and b form the molecular basis of wheat grain hardness or texture. When both puroindolines are in their `functional' wild state, grain texture is soft. When either one of the puroindolines is absent or altered by mutation, then the result is hard texture. In the case of durum wheat which lacks puroindolines, the texture is very hard. Puroindolines represent the molecular-genetic basis of the Hardness locus on chromosome 5DS and the soft (Ha) and hard (ha) alleles present in hexaploid bread wheat varieties. To date, seven discrete hardness alleles have been described for wheat. All involve puroindoline a or b and have been designated Pina-D1b and Pinb-D1b through Pinb-D1g. A direct role of a related protein, grain softness protein (as currently defined), in wheat grain texture has yet to be demonstrated.     

Resistance gene analogues of chickpea ( Cicer arietinum L.): isolation,genetic mapping and association with a Fusarium resistance gene cluster

Resistance gene analogues (RGAs) of Cicer were isolated by different PCR approaches and mapped in an inter-specific cross segregating for fusarium wilt by RFLP and CAPS analysis. Initially, two pairs of degenerate primers targeting sequences encoded at nucleotide-binding sites (NBS), which are conserved in plant disease resistance genes such as RPS2, L6 and N, were selected for amplification. Cloning and sequence analysis of amplified products from C. arietinum DNA revealed eight different RGAs. Additionally, five RGAs were identified after characterisation of the presumptive RGA alleles from C. reticulatum. Therefore, a total of 13 different RGAs were isolated from Cicer and classified through pair-wise comparison into nine distinct classes with sequence similarities below a 68% amino acid identity threshold. Sequence comparison of seven RGA alleles of C. arietinum and C. reticulatum revealed polymorphisms in four RGAs with identical numbers of synonymous and non-synonymous substitutions. An NlaIII site, unique in the RGA-A allele of C. arietinum, was exploited for CAPS analysis. Genomic organisation and map position of the NBS-LRR candidate resistance genes was probed by RFLP analysis. Both single-copy as well as multi-copy sequence families were present for the selected RGAs, which represented eight different classes. Five RGAs were mapped in an inter-specific population segregating for three race-specific Fusarium resistances. All RGAs mapped to four of the previously established eight linkage groups for chickpea. Two NBS-LRR clusters were identified that could not be resolved in our mapping population. One of these clusters, which is characterised by RFLP probe CaRGA-D, mapped to the linkage group harbouring two of three Fusarium resistance genes characterised in the inter-specific population. Our study provides a starting point for the characterisation and genetic mapping of candidate resistance genes in Cicer that is useful for marker-assisted selection and as a pool for resistance genes of Cicer.     

Acanthamoeba castellanii: gene profile of encystation by ESTs analysis and KOG assignment

The trophozoite of Acanthamoeba transforms into a cyst, the resistant form under harmful environments such as starvation, cold and certain chemicals used in medical treatment. To investigate the factors mediating encystation, ESTs of encystation-induced A. castellanii were analysed and compared to those of trophozoites. Each EST was compared by the predicted proteins from the ESTs, to the cyst and the trophozoite by reciprocal BLAST analysis, KOG assignment, and gene annotation. In addition to the genes previously reported to encystation mediate such as cyst specific protein 21, protein kinase C, proteasome and heat shock protein, several genes like cullin 4, autophage protein 8 and ubiquitin-conjugating enzymes were identified to be related to encystation. Five kinds of proteinase genes were detected in cyst ESTs. The information of the genes expressed during encystation may open the way to further study on differentiation and resistance of cyst-forming pathogenic protozoa.     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998     

Targeted molecular mapping of a major wheat QTL for Fusarium head blight resistance using wheat ESTs and synteny with rice.

A major QTL for resistance to Fusarium head blight (FHB) in wheat, Qfhs.ndsu-3BS, has been identified and verified by several research groups. The objective of this study was to increase the marker density in this QTL region using STS (sequence-tagged site) markers developed from wheat expressed sequence tags (ESTs) near Qfhs.ndsu-3BS. Because wheat chromosome 3BS and rice chromosome 1S are syntenous, the sequences of P1-derived artificial chromosome (PAC) and (or) bacterial artificial chromosome (BAC) clones covering the sub-distal portion of rice chromosome 1S were used as queries for a BLASTn search to identify wheat ESTs most likely near Qfhs.ndsu-3BS. Sixty-eight out of 79 STS primer pairs designed from wheat ESTs amplified PCR products from the genomic DNA of Triticum aestivum 'Chinese Spring'. Twenty-eight STS markers were localized on chromosome 3BS by aneuploid analysis. Six out of the nine STS markers that could be mapped in the T. aestivum 'Sumai 3'/T. aestivum 'Stoa' population had higher R2 and LOD values for this QTL than the most significant marker reported previously. Therefore, leveraging genome sequence information available in rice for wheat genetics is an effective strategy to develop DNA markers for Qfhs.ndsu-3BS, and this strategy may have broad applications for targeted mapping of other traits in cereal crops.     

Comparative DNA sequence analysis of mapped wheat ESTs reveals the complexity of genome relationships between rice and wheat

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to related large-genome species has revolutionized molecular genetics and breeding strategies for improving those crops. Comparative sequence analysis methods can be used to cross-reference genes between species maps, enhance the resolution of comparative maps, study patterns of gene evolution, identify conserved regions of the genomes, and facilitate interspecies gene cloning. In this study, 5,780 Triticeae ESTs that have been physically mapped using wheat (Triticum aestivum L.) deletion lines and segregating populations were compared using NCBI BLASTN to the first draft of the public rice (Oryza sativa L.) genome sequence data from 3,280 ordered BAC/PAC clones. A rice genome view of the homoeologous wheat genome locations based on sequence analysis shows general similarity to the previously published comparative maps based on Southern analysis of RFLP. For most rice chromosomes there is a preponderance of wheat genes from one or two wheat chromosomes. The physical locations of non-conserved regions were not consistent across rice chromosomes. Some wheat ESTs with multiple wheat genome locations are associated with the non-conserved regions of similarity between rice and wheat. The inverse view, showing the relationship between the wheat deletion map and rice genomic sequence, revealed the breakdown of gene content and order at the resolution conferred by the physical chromosome deletions in the wheat genome. An average of 35% of the putative single copy genes that were mapped to the most conserved bins matched rice chromosomes other than the one that was most similar. This suggests that there has been an abundance of rearrangements, insertions, deletions, and duplications eroding the wheat-rice genome relationship that may complicate the use of rice as a model for cross-species transfer of information in non-conserved regions.     

An intraspecific gene duplication polymorphism of the urate oxidase gene of Drosophila virilis: a genetic and molecular analysis

Nineteen strains of Drosophila virilis from diverse geographic locations were examined by genetic and molecular analyses, revealing (a) 12 strains with a single copy of the urate oxidase (UO) gene per haploid genome and (b) 7 strains with a tandem duplication of the UO locus. The D. virilis strains with the UO duplication appear to have identical restriction maps of this region, implying either a single origin for the duplication or several similar events occurring at a hot spot. On the basis of the location of the duplication breakpoints and the restriction sites flanking these breakpoints, this duplication probably arose through nonhomologous recombination involving either a breakage and rejoining event or replication slippage. because documented cases of intraspecific gene duplication polymorphism are rare, the D. virilis UO duplication will be useful in identifying the molecular event giving rise to a gene duplication.      

An abiotic stress response gene TaSnRK2.7-B in wheat accessions: genetic diversity analysis and gene mapping based on SNPs

TaSnRK2.7, a SnRK2 (sucrose non-fermenting1-related protein kinase 2) member of wheat, confers enhanced multi-stress tolerance in carbohydrate metabolism. Here, the nucleotide diversity of TaSnRK2.7 was investigated in 60 wheat accessions with different stress-tolerant features. One copy, named TaSnRK2.7-B, was detected in 32 accessions, which mainly exhibited stress tolerance. The TaSnRK2.7-B evolutionary dynamics were examined in these cultivars, which have been subjected to population bottlenecks and intensive selection in breeding. Nucleotide diversity (π) in the entire TaSnRK2.7-B region was 0.00148. Sliding-window analysis demonstrated that 3932-4535bp was a variation-enriched region, covering the eighth intron, the ninth exon and the 3'-flanking region. As predicted, neutrality tests revealed that the TaSnRK2.7-B protein was relatively conservative, and population bottlenecks or purifying selection may have acted on TaSnRK2.7-B. Furthermore, linkage disequilibrium between SNP loci extends across the entire TaSnRK2.7-B region. A SNP marker of TaSnRK2.7-B was developed and mapped on chromosome 2AL flanked by WMC179.4 and WMC401, which were co-located in the same or adjacent chromosome intervals with QTLs for phosphorus utilization efficiency and accumulation efficiency of stem water-soluble carbohydrates.     

FunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression

Background  

While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes.     

Isolation of resistance gene analogues to powdery mildew resistance sequences in hexaploid wheat

This paper reports the characterization of the powdery mildew resistance homologous genes family of Triticum aestivum. Using degenerate primer pair for wheat resistance genes, we have cloned seven 3′ truncated powdery mildew resistance gene homologous fragments Tpc5a, Tp25a, Tp25b, Tp3a5a, Tp3a5b, Tp4b5a and Tp4b5b. These fragments were sequenced. The deduced amino acid sequences showed that six of them have premature stop codons. All these sequences had a very high level of similarity to known Pm resistance genes such as Pm3a, Pm3b, Pm3d and pm3f in hexaploid wheat. By ignoring the stop codons in the sequences, their deduced protein sequences were of coiled-coil (CC)-nucleotide binding site (NBS)-leucine repeat rich (LRR) structure. These results suggest that there are many powdery mildew resistance gene analogues in both resistant and susceptible wheat. Among them, small insertion/deletion events and point mutations can result in the diversity of wheat Pm resistance homologous genes.     

Targeted mapping of ESTs linked to the adult plant resistance gene Lr46 in wheat using synteny with rice

The gene Lr46 has provided slow-rusting resistance to leaf rust caused by Puccinia triticina in wheat (Triticum aestivum), which has remained durable for almost 30 years. Using linked markers and wheat deletion stocks, we located Lr46 in the deletion bin 1BL (0.84–0.89) comprising 5% of the 1BL arm. The distal part of chromosome 1BL of wheat is syntenic to chromosome 5L of rice. Wheat expressed sequence tags (ESTs) mapping in the terminal 15% of chromosome 1BL with significant homology to sequences from the terminal region of chromosome 5L of rice were chosen for sequence-tagged site (STS) primer design and were mapped physically and genetically. In addition, sequences from two rice bacterial artificial chromosome clones covering the targeted syntenic region were used to identify additional linked wheat ESTs. Fourteen new markers potentially linked to Lr46 were developed; eight were mapped in a segregating population. Markers flanking (2.2 cM proximal and 2.2 cM distal) and cosegregating with Lr46 were identified. The physical location of Lr46 was narrowed to a submicroscopic region between the breakpoints of deletion lines 1BL-13 [fraction length (FL)=0.89–1] and 1BL-10 (FL=0.89–3). We are now developing a high-resolution mapping population for the positional cloning of Lr46.     

水稻全基因组编码抗病基因同源序列分析

利用模糊搜索的方法,在TIGR水稻日本晴基因组数据库(TIGR Rice Genome Annotation-Release5)中识别出565个编码抗病蛋白质的同源序列;利用识别出565个编码抗病蛋白质序列分别与籼稻基因组数据库进行BLASTP联配,共确定320个对应的等位基因。通过在线生物信息学软件,识别了这565个抗病基因的保守结构域、保守模体和DNA序列内转座子元件,其中有14个抗病基因同源序列注释错误。同时绘出了这些基因的基因组分布,并基于这些基因的同源树分析和基因组物理分布,认为基因的原位和远程复制事件产生了抗病基因的现存分布和多样性,其中转座子在复制过程中扮演了重要角色。这些对抗病机制研究和抗病基因进化研究以及抗病基因的转育具有重要意义。