RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and -susceptible alleles

Bacterial leaf pustule (BLP) disease is caused by Xanthomonas axonopodis pv. glycines (Xag). To investigate the plant basal defence mechanisms induced in response to Xag, differential gene expression in near-isogenic lines (NILs) of BLP-susceptible and BLP-resistant soybean was analysed by RNA-Seq. Of a total of 46 367 genes that were mapped to soybean genome reference sequences, 1978 and 783 genes were found to be up- and down-regulated, respectively, in the BLP-resistant NIL relative to the BLP-susceptible NIL at 0, 6, and 12h after inoculation (hai). Clustering analysis revealed that these genes could be grouped into 10 clusters with different expression patterns. Functional annotation based on gene ontology (GO) categories was carried out. Among the putative soybean defence response genes identified (GO:0006952), 134 exhibited significant differences in expression between the BLP-resistant and -susceptible NILs. In particular, pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the BLP-resistant NIL. Additionally, pathogenesis-related (PR)-1 and -14 were highly expressed at 0 hai, and PR-3, -6, and -12 were highly expressed at 12 hai. There were also significant differences in the expression of the core JA-signalling components MYC2 and JASMONATE ZIM-motif. These results indicate that powerful basal defence mechanisms involved in the recognition of PAMPs or DAMPs and a high level of accumulation of defence-related gene products may contribute to BLP resistance in soybean.     

Fine mapping of a resistance gene to bacterial leaf pustule in soybean

Soybean bacterial leaf pustule (BLP) is a prevalent disease caused by Xanthomonas axonopodis pv. glycines. Fine mapping of the BLP resistant gene, rxp, is needed to select BLP resistant soybean cultivars by marker-assisted selection (MAS). We used a total of 227 recombinant inbred lines (RILs) derived from a cross between ‘Taekwangkong’ (BLP susceptible) and ‘Danbaekkong’ (BLP resistant) for rxp fine mapping and two different sets of near isogenic lines (NILs) from Hwangkeumkong × SS2-2 and Taekwangkong × SS2-2 were used for confirmation. Using sequences between Satt372 and Satt486 flanking rxp from soybean genome sequences, eight simple sequence repeats (SSR) and two single nucleotide polymorphism (SNP) markers were newly developed in a 6.2-cM interval. Linkage mapping with the RILs and NILs allowed us to map the rxp region with high resolution. The genetic order of all markers was completely consistent with their physical order. QTL analysis by comparison of the BLP phenotyping data with all markers showed rxp was located between SNUSSR17_9 and SNUSNP17_12. Gene annotation analysis of the 33 kb region between SNUSSR17_9 and SNUSNP17_12 suggested three predicted genes, two of which could be candidate genes of BLP resistance: membrane protein and zinc finger protein. Candidate genes showed high similarity with their paralogous genes, which were located on the duplicated regions obtaining BLP resistance QTLs. High-resolution map in rxp region with eight SSR and two SNP markers will be useful for not only MAS of BLP resistance but also characterization of rxp.     

Mode of amplification and reorganization of resistance genes during recent Arabidopsis thaliana evolution.

The NBS-LRR (nucleotide-binding site plus leucine-rich repeat) genes represent the major class of disease resistance genes in flowering plants and comprise 166 genes in the ecotype Col-0 of Arabidopsis thaliana. NBS-LRR genes are organized in single-gene loci, clusters, and superclusters. Phylogenetic analysis reveals nine monophyletic clades and a few phylogenetic orphans. Most clusters contain only genes from the same phylogenetic lineage, reflecting their origin from the exchange of sequence blocks as a result of intralocus recombination. Multiple duplications increased the number of NBS-LRR genes in the progenitors of Arabidopsis, suggesting that the present complexity in Col-0 may derive from as few as 17 progenitors. The combination of physical and phylogenetic analyses of the NBS-LRR genes makes it possible to detect relatively recent gene rearrangements, which increased the number of NBS-LRR genes by about 50, but which are almost never associated with large segmental duplications. The identification of 10 heterogeneous clusters containing members from different clades demonstrates that sequence sampling between different resistance gene loci and clades has occurred. Such events may have taken place early during flowering plant evolution, but they generated modules that have been duplicated and remobilized also more recently.     

Evidence of Multiple Disease Resistance (MDR) and Implication of Meta-Analysis in Marker Assisted Selection

Meta-analysis was performed for three major foliar diseases with the aim to find out the total number of QTL responsible for these diseases and depict some real QTL for molecular breeding and marker assisted selection (MAS) in maize. Furthermore, we confirmed our results with some major known disease resistance genes and most well-known gene family of nucleotide binding site (NBS) encoding genes. Our analysis revealed that disease resistance QTL were randomly distributed in maize genome, but were clustered at different regions of the chromosomes. Totally 389 QTL were observed for these three major diseases in diverse maize germplasm, out of which 63 QTL were controlling more than one disease revealing the presence of multiple disease resistance (MDR). 44 real-QTLs were observed based on 4 QTL as standard in a specific region of genome. We also confirmed the Ht1 and Ht2 genes within the region of real QTL and 14 NBS-encoding genes. On chromosome 8 two NBS genes in one QTL were observed and on chromosome 3, several cluster and maximum MDR QTL were observed indicating that the apparent clustering could be due to genes exhibiting pleiotropic effect. Significant relationship was observed between the number of disease QTL and total genes per chromosome based on the reference genome B73. Therefore, we concluded that disease resistance genes are abundant in maize genome and these results can unleash the phenomenon of MDR. Furthermore, these results could be very handy to focus on hot spot on different chromosome for fine mapping of disease resistance genes and MAS.     

Genetic mapping revealed two loci for soybean aphid resistance in PI 567301B

The soybean aphid (Aphis glycines Matsumura) is the most damaging insect pest of soybean [Glycine max (L.) Merr.] in North America. New soybean aphid biotypes have been evolving quickly and at least three confirmed biotypes have been reported in USA. These biotypes are capable of defeating most known aphid resistant soybean genes indicating the need for identification of new genes. Plant Introduction (PI) 567301B was earlier identified to have antixenosis resistance against biotype 1 and 2 of the soybean aphid. Two hundred and three F_7:9 recombinant inbred lines (RILs) developed from a cross of soybean aphid susceptible cultivar Wyandot and resistant PI 567301B were used for mapping aphid resistance genes using the quantitative trait loci (QTL) mapping approach. A subset of 94 RILs and 516 polymorphic SNP makers were used to construct a genome-wide molecular linkage map. Two candidate QTL regions for aphid resistance were identified on this linkage map. Fine mapping of the QTL regions was conducted with SSR markers using all 203 RILs. A major gene on chromosome 13 was mapped near the previously identified Rag2 gene. However, an earlier study revealed that the detached leaves of PI 567301B had no resistance against the soybean aphids while the detached leaves of PI 243540 (source of Rag2) maintained aphid resistance. These results and the earlier finding that PI 243540 showed antibiosis resistance and PI 567301B showed antixenosis type resistance, indicating that the aphid resistances in the two PIs are not controlled by the same gene. Thus, we have mapped a new gene near the Rag2 locus for soybean aphid resistance that should be useful in breeding for new aphid-resistant soybean cultivars. Molecular markers closely linked to this gene are available for marker-assisted breeding. Also, the minor locus found on chromosome 8 represents the first reported soybean aphid-resistant locus on this chromosome.     

Homoeologous set of NBS-LRR genes located at leaf and stripe rust resistance loci on short arms of chromosome 1 of wheat

Homoeologous group 1 chromosomes of wheat contain important genes that confer resistance to leaf, stem and stripe rusts, powdery mildew and Russian wheat aphid. A disease resistance gene analog encoding nucleotide binding site-leucine rich repeat (NBS-LRR), designated RgaYr10, was previously identified at the stripe rust resistant locus, Yr10, located on chromosome 1BS distal to the storage protein, Gli-B1 locus. RgaYr10 identified gene members in the homoeologous region of chromosome 1DS cosegregating with the leaf rust resistance gene, Lr21, which originally was transferred from a diploid D genome progenitor. Four RgaYr10 gene members were isolated from chromosome 1DS and compared to two gene members previously isolated from the chromosome 1BS homeologue. NBS-LRR genes tightly linked to stripe rust resistance gene Yr10 on chromosome 1BS were closely related in sequence and structure to NBS-LRR genes tightly linked to leaf rust resistance gene Lr21 located within the homoeologous region on chromosome 1DS. The level of sequence homology was similar between NBS-LRR genes that were isolated from different genomes as compared to genes from the same genome. Electronic Publication     

Detection and fine-mapping of SC7 resistance genes via linkage and association analysis in soybean

Soybean mosaic virus (SMV) disease is one of the most serious and broadly distributed soybean (Glycine max (L.) Merr.) diseases. Here, we combine the advantages of association and linkage analysis to i...     

Isolation and characterization of disease resistance gene homologues from rice cultivar IR64

We initiated a search for disease resistance (R) gene homologues in rice cultivar IR64, one of the most agronomically important rice varieties in the world, with the assumption that some of these homologues would correspond to previously identified disease resistance loci. A family of rice R gene homologues was identified using the Arabidopsis NBS-LRR disease resistance gene RPS2 as a hybridization probe. Because member genes of this rice R gene family exhibit features characteristic of the NBS-LRR class of resistance genes, the family was given the name NRH (for NBS-LRR resistance gene homologues). Three members of the NRH family, NRH1, NRH2, and NRH3, were cloned and studied in detail. In IR64, NRH1 and NRH2 appear to encode full-length polypeptides, whereas NRH3 is prematurely truncated with a stop codon generated by a frameshift. NRH1 maps on chromosome 5, and NRH2 and NRH3 are less than 48kb apart on chromosome 11. Although NRH1, NRH2, and NRH3 map to regions of the rice genome where disease resistance loci to Xanthomonas oryzae pv. oryzae (Xoo) have been identified, susceptible rice varieties transformed with either NRH1 or NRH2 failed to exhibit increased resistance to a set of well-characterized Xoo strains.     

小麦Mlo及NBS-LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因Mlo cDNA序列及一个来源于栽培一粒小麦(Triticum monococcum L.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选.结果获得两个表达基因的cDNA克隆.其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%.另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类.白粉菌诱导前后,该片段RT-PCR扩增产物存在差异,表明该片段可能与小麦抗病性相关.利用"中国春"缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上.     

Fine mapping and identification of candidate genes controlling the resistance to southern root-knot nematode in PI 96354

Meloidogyne incognita (Kofoid and White) Chitwood (Mi) is the most economically damaging species of the root-knot nematode to soybean and other crops in the southern USA. PI 96354 was identified to carry a high level of resistance to galling and Mi egg production. Two Quantitative Trait Locus (QTLs) were found to condition the resistance in PI 96354 including a major QTL and a minor QTL on chromosome 10 and chromosome 18, respectively. To fine map the major QTL on chromosome 10, F_5:6 recombinant inbred lines from the cross between PI 96354 and susceptible genotype Bossier were genotyped with Simple Sequence Repeats (SSR) markers to identify recombinational events. Analysis of lines carrying key recombination events placed the Mi-resistant allele on chromosome 10 to a 235-kb region of the ‘Williams 82’ genome sequence with 30 annotated genes. Candidate gene analysis identified four genes with cell wall modification function that have several mutations in promoter, exon, 5′, and 3′UTR regions. qPCR analysis showed significant difference in expression levels of these four genes in Bossier compared to PI 96354 in the presence of Mi. Thirty Mi-resistant soybean lines were found to have same SNPs in these 4 candidate genes as PI 96354 while 12 Mi-susceptible lines possess the ‘Bossier’ genotype. The mutant SNPs were used to develop KASP assays to detect the resistant allele on chromosome 10. The four candidate genes identified in this study can be used in further studies to investigate the role of cell wall modification genes in conferring Mi resistance in PI 96354.     

Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping

The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals.     

Genome-level evolution of resistance genes in Arabidopsis thaliana

Pathogen resistance genes represent some of the most abundant and diverse gene families found within plant genomes. However, evolutionary mechanisms generating resistance gene diversity at the genome level are not well understood. We used the complete Arabidopsis thaliana genome sequence to show that most duplication of individual NBS-LRR sequences occurs at close physical proximity to the parent sequence and generates clusters of closely related NBS-LRR sequences. Deploying the statistical strength of phylogeographic approaches and using chromosomal location as a proxy for spatial location, we show that apparent duplication of NBS-LRR genes to ectopic chromosomal locations is largely the consequence of segmental chromosome duplication and rearrangement, rather than the independent duplication of individual sequences. Although accounting for a smaller fraction of NBS-LRR gene duplications, segmental chromosome duplication and rearrangement events have a large impact on the evolution of this multigene family. Intergenic exchange is dramatically lower between NBS-LRR sequences located in different chromosome regions as compared to exchange between sequences within the same chromosome region. Consequently, once translocated to new chromosome locations, NBS-LRR gene copies have a greater likelihood of escaping intergenic exchange and adopting new functions than do gene copies located within the same chromosomal region. We propose an evolutionary model that relates processes of genome evolution to mechanisms of evolution for the large, diverse, NBS-LRR gene family.     

Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.     

A resistance gene analog useful for targeting disease resistance genes against different pathogens on group 1S chromosomes of barley,wheat and rye

Comparative sequence analysis of the resistance gene analog (RGA) marker locus aACT/CAA (originally found to be tightly linked to the multiallelic barley Mla cluster) from genomes of barley, wheat and rye revealed a high level of relatedness among one another and showed high similarity to a various number of NBS-LRR disease resistance proteins. Using the sequence-specific polymerase chain reaction (PCR), RGA marker aACT/CAA was mapped on group 1S chromosomes of the Triticeae and was associated with disease resistance loci. In barley and rye, the marker showed linkage to orthologous powdery mildew resistance genes Mla1 and Pm17, respectively, while in wheat linkage with a QTL against fusarium head blight (FHB) disease was determined. The use of RGA clones for R gene mapping and their role in the expression of qualitative and quantitative resistance is discussed.     

Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.)

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.     

Conditional QTL underlying resistance to late blight in a diploid potato population

A large number of quantitative trait loci (QTL) for resistance to late blight of potato have been reported with a "conventional" method in which each phenotypic trait reflects the cumulative genetic effects for the duration of the disease process. However, as genes controlling response to disease may have unique contributions with specific temporal features, it is important to consider the phenotype as dynamic. Here, using the net genetic effects evidenced at consecutive time points during disease development, we report the first conditional mapping of QTL underlying late blight resistance in potato under five environments in Peru. Six conditional QTL were mapped, one each on chromosome 2, 7 and 12 and three on chromosome 9. These QTL represent distinct contributions to the phenotypic variation at different stages of disease development. By comparison, when conventional mapping was conducted, only one QTL was detected on chromosome 9. This QTL was the same as one of the conditional QTL. The results imply that conditional QTL reflect genes that function at particular stages during the host-pathogen interaction. The dynamics revealed by conditional QTL mapping could contribute to the understanding of the molecular mechanism of late blight resistance and these QTL could be used to target genes for marker development or manipulation to improve resistance.     

DNA marker analysis of loci conferring resistance to peanut root-knot nematode in soybean

 Peanut root-knot nematode [Meloidogyne arenaria (Neal) Chitwood] (Ma) is a serious pathogen of soybean, Glycine max L. Merrill, in the southern USA. Breeding for root-knot nematode resistance is an important objective in many plant breeding programs. The inheritance of soybean resistance to Ma is quantitative and has a moderate-to-high variance-component heritability on a family mean basis. The objectives of the present study were to use restriction fragment length polymorphism (RFLP) markers to identify quantitative trait loci (QTLs) conferring resistance to Ma and to determine the genomic location and the relative contribution to resistance of each QTL. An F₂ population from a cross between PI200538 (Ma resistant) and ‘CNS’ (Ma susceptible) was mapped with 130 RFLPs. The 130 markers converged on 20 linkage groups spanning a total of 1766 cM. One hundred and five F_2:3 families were grown in the greenhouse and inoculated with Ma Race 2. Two QTLs conferring resistance to Ma were identified and PI200538 contributed the alleles for resistance at both QTLs. One QTL was mapped at 0-cM recombination with marker B212V-1 on linkage group-F (LG-F) of the USDA/ARS-Iowa State University RFLP map, and accounted for 32% of the variation in gall number. Another QTL was mapped in the interval from B212D-2 to A111H-2 on LG-E, and accounted for 16% of the variation in gall number. Gene action for the QTL located on LG-F was additive to partially dominant, whereas the gene action for the QTL on LG-E was dominant with respect to resistance. The two QTLs, when fixed on the framework map, accounted for 51% of the variation in gall number in a two-QTL model. The two QTLs for Ma resistance were found in duplicated regions of the soybean genome, and the major QTL for Ma resistance on LG-F is positioned within a cluster of eight diverse disease-resistance loci. Received: 10 June 1996 / Accepted: 18 April 1997