Haplotype variability and identification of new functional alleles at the Rdg2a leaf stripe resistance gene locus

The barley Rdg2a locus confers resistance to the leaf stripe pathogen Pyrenophora graminea and, in the barley genotype Thibaut, it is composed of a gene family with three highly similar paralogs. Only one member of the gene family (called as Rdg2a) encoding for a CC-NB-LRR protein is able to confer resistance to the leaf stripe isolate Dg2. To study the genome evolution and diversity at the Rdg2a locus, sequences spanning the Rdg2a gene were compared in two barley cultivars, Thibaut and Morex, respectively, resistant and susceptible to leaf stripe. An overall high level of sequence conservation interrupted by several rearrangements that included three main deletions was observed in the Morex contig. The main deletion of 13,692 bp was most likely derived from unequal crossing over between Rdg2a paralogs leading to the generation of a chimeric Morex rdg2a gene which was not associated to detectable level of resistance toward leaf stripe. PCR-based analyses of genic and intergenic regions at the Rdg2a locus in 29 H. vulgare lines and one H. vulgare ssp. spontaneum accession indicated large haplotype variability in the cultivated barley gene pool suggesting rapid and recent divergence at this locus. Barley genotypes showing the same haplotype as Thibaut at the Rdg2a locus were selected for a Rdg2a allele mining through allele re-sequencing and two lines with polymorphic nucleotides leading to amino acid changes in the CC-NB and LRR encoding domains, respectively, were identified. Analysis of nucleotide diversity of the Rdg2a alleles revealed that the polymorphic sites were subjected to positive selection. Moreover, strong positively selected sites were located in the LRR encoding domain suggesting that both positive selection and divergence at homologous loci are possibly representing the molecular mechanism for the generation of high diversity at the Rdg2a locus in the barley gene pool.     

Genomic distribution of MITEs in barley determined by MITE-AFLP mapping.

Miniature inverted-repeat transposable elements (MITEs) represent a large superfamily of transposons that are moderately to highly repetitive and frequently found near or within plant genes. To elucidate the organization of MITEs in the barley genome, MITEs were integrated into the genetic map of barley. In this report, we describe the use of MITEs in amplified fragment length polymorphism (AFLP) mapping, and demonstrate their superiority over conventional AFLP mapping. Barley MITEs include members of the Stowaway, Barfly, and Pangrangja families. By amplifying the flanking sequences of these MITEs, a total of 214 loci were mapped from a population of 93 doubled-haploid segregating individuals between Hordeum vulgare ssp. vulgare and H. vulgare ssp. spontaneum. The 214 MITE-AFLP and 40 anchor simple sequence repeat (SSR) loci were distributed on 7 linkage groups, covering a total map distance of 1 165 cM. The average marker density on each chromosome ranged between 3.4 and 9.6 cM per locus. Only 1 MITE-based locus was frequently found to be associated with MITE loci from the same family, resulting in clusters in chromosomal subregions. In barley, it will be possible to cover the entire genome with a limited set of MITE-based primers and to build highly dense maps of specific regions.     

Resistance gene analogs in barley and their relationship to rust resistance genes.

Regions of amino acid conservation in the NBS domain of NBS-LRR resistance proteins facilitated the PCR isolation of eight resistance gene analog (RGA) sequences from genomic DNA of rice, barley, and Aegilops tauschii. These clones and other RGAs previously isolated from maize, rice, and wheat were assigned to 13 classes by DNA-sequence comparison and by their patterns of hybridisation to restricted barley DNA. Using a doubled-haploid mapping population, probes from 12 RGA classes were used to map 17 loci in the barley genome. Many of these probes have been used for mapping in wheat, and the collective data indicate that the positions of orthologous RGAs are conserved between barley and wheat. RGA loci were identified in the vicinity of barley leaf rust resistance loci Rph4, Rph7, and Rph10. Recombinants were identified between RGA loci and Rph7 and Rph10, while a cluster of RGA sequences detected by probe 5.2 cosegregated with Rph4 in 55 F2 lines.     

A large rearrangement involving genes and low-copy DNA interrupts the microcollinearity between rice and barley at the Rph7 locus

Grass genomes differ greatly in chromosome number, ploidy level, and size. Despite these differences, very good conservation of the marker order (collinearity) was found at the genetic map level between the different grass genomes. Collinearity is particularly good between rice chromosome 1 and the group 3 chromosomes in the Triticeae. We have used this collinearity to saturate the leaf rust resistance locus Rph7 on chromosome 3HS in barley with ESTs originating from rice chromosome 1S. Chromosome walking allowed the establishment of a contig of 212 kb spanning the Rph7 resistance gene. Sequencing of the contig showed an average gene density of one gene/20 kb with islands of higher density. Comparison with the orthologous rice sequence revealed the complete conservation of five members of the HGA gene family whereas intergenic regions differ greatly in size and composition. In rice, the five genes are closely associated whereas in barley intergenic regions are >38-fold larger. The size difference is due mainly to the presence of six additional genes as well as noncoding low-copy sequences. Our data suggest that a major rearrangement occurred in this region since the Triticeae and rice lineage diverged.     

High-resolution mapping of the barley leaf rust resistance gene Rph5 using barley expressed sequence tags (ESTs) and synteny with rice

The rapidly growing expressed sequence tag (EST) resources of species representing the Poacea family and availability of comprehensive sequence information for the rice (Oryza sativa) genome create an excellent opportunity for comparative genome analysis. Extensive synteny between rice chromosome 1 and barley (Hordeum vulgare L.) chromosome 3 has proven extremely useful for saturation mapping of chromosomal regions containing target genes of large-genome barley with conserved orthologous genes from the syntenic regions of the rice genome. Rph5 is a gene conferring resistance to the barley leaf rust pathogen Puccinia hordei. It was mapped to chromosome 3HS, which is syntenic with rice chromosome 1S. The objective of this study was to increase marker density within the sub-centimorgan region around Rph5, using sequence-tagged site (STS) markers that were developed based on barley ESTs syntenic to the phage (P1)-derived artificial chromosome (PAC) clones comprising the distal region of rice chromosome 1S. Five rice PAC clones were used as queries in a blastn search to screen 375,187 barley ESTs. Ninety-four non-redundant EST sequences were identified from the EST database and used as templates to design 174 pairs of primer combinations. As a result, 9 barley EST-based STS markers were incorporated into the ‘Bowman’ × ‘Magnif 102’ high-resolution map of the Rph5 region. More importantly, six markers, including five EST-derived STS sequences, were found to co-segregate with Rph5. The results of this study demonstrate the usefulness of rice genomic resources for efficient deployment of barley ESTs for marker saturation of targeted barley genomic regions.     

Comparative high resolution map of the six-rowed spike locus 1 (vrs1) in several populations of barley, Hordeum vulgare L

Multiple alleles at the vrs1 locus control the development and fertility of the lateral spikelets of barley (Hordeum vulgare L.), which is a key character in the study of yield, utilization and domestication. In this study, six linkage maps of the vrs1 locus were constructed, using different mapping populations developed from nine different barley cultivars (H. vulgare subsp. vulgare) or mutant and wild barley (H. vulgare subsp. spontaneum). A total of 8387 chromosomes (gametes) were sampled for analysis based on a hypothesis that orders of marker loci were the same over the different parental lines. The results showed that four markers and the vrs1 locus in all cases were arranged in the same order, which was in a good agreement with the hypothesis. This makes the linkage maps suitable for the positional cloning of the alleles at the vrs1 locus.     

The genomic organization and localization on the chromosomes of the HvRT family of DNA repetitive sequences in barley

HvRT family of repetitive DNA sequences from barley genome appears to have complex hierarchical organization. Tandem repetition of 118-bp monomers constitutes lower level of HvRT-family organization. Amplification units of the higher level consist of several contiguous 118-bp monomers. RFLP between different species and cultivars of barley resulted from the differences in the higher-order repeat structure. Individual chromosomes of barley contain specific HvRT subfamilies. This family also possesses separate domains differing in the restriction enzyme sites density. HvRT family is presented in the genomes of H. vulgare, H. leporinum, H. murinum, H. jubatum, but is absent in the genomes of H. marinum, H. geniculatum and wheat.     

西藏和埃塞俄比亚大麦6个同工酶位点遗传变异的对比分析

我们对比分析了埃塞俄比亚(简称埃)和西藏共777份栽培大麦材料在6个同工酶位点(Est1、Est2、Kst3、Est4、Acp1和Got1)的遗传变异。结果表明,无论是从单个位点上分析还是在多位点基因组合形式上评价,埃大麦与西藏大麦群体在遗传组成和多位点基因结构上都有着很大的差异。遗传多样性对比分析表明,从单个位点基因类型看,西藏大麦与埃大麦遗传变异程度大致相当,但从多位点基因组合形式看,西藏大麦遗传多样性程度极显著地高于埃大麦。     

Genome-Wide Association Analysis of Aluminum Tolerance in Cultivated and Tibetan Wild Barley

Tibetan wild barley (Hordeum vulgare L. ssp. spontaneum), originated and grown in harsh enviroment in Tibet, is well-known for its rich germpalsm with high tolerance to abiotic stresses. However, the genetic variation and genes involved in Al tolerance are not totally known for the wild barley. In this study, a genome-wide association analysis (GWAS) was performed by using four root parameters related with Al tolerance and 469 DArT markers on 7 chromosomes within or across 110 Tibetan wild accessions and 56 cultivated cultivars. Population structure and cluster analysis revealed that a wide genetic diversity was present in Tibetan wild barley. Linkage disequilibrium (LD) decayed more rapidly in Tibetan wild barley (9.30 cM) than cultivated barley (11.52 cM), indicating that GWAS may provide higher resolution in the Tibetan group. Two novel Tibetan group-specific loci, bpb-9458 and bpb-8524 were identified, which were associated with relative longest root growth (RLRG), located at 2H and 7H on barely genome, and could explain 12.9% and 9.7% of the phenotypic variation, respectively. Moreover, a common locus bpb-6949, localized 0.8 cM away from a candidate gene HvMATE, was detected in both wild and cultivated barleys, and showed significant association with total root growth (TRG). The present study highlights that Tibetan wild barley could provide elite germplasm novel genes for barley Al-tolerant improvement.     

Comparison of restriction fragment length polymorphisms in wild and cultivated barley.

This study was undertaken to assess the relative level of molecular diversity between cultivated barley, Hordeum vulgare ssp. vulgare (HV), and one of its wild relatives, H. vulgare ssp. spontaneum (HS), and to identify possible restriction fragment length polymorphism (RFLP) patterns that may provide information concerning the phylogenetic relationship between these two barley groups. A total of 363 barley accessions were assayed, including 95 entries of HV collected from 36 major barley growing countries of the world and 268 entries of HS from 25 natural populations in Israel and Iran. The 26 RFLP marker loci used in the survey represent single-copy, low-copy, and repetitive DNA sequences and mark all of the chromosome arms. A randomization test, on the basis of equal sample sizes, showed that HS is more polymorphic than HV, as evaluated by the number of alleles and diversity indices. The analysis also indicated extensive RFLP differentiation between these two barley groups; highly significant differences of allele frequencies were detected at the majority of the loci. The HV sample can be subdivided according to winter or spring growth habits, and two- or six-rowed spikes. Analysis of genetic polymorphisms in these subgroups showed that levels of diversity were about equal in spring and winter groups and also in the groups with two- and six-rowed spikes. However, significant differences of allelic frequencies were detected between subgroups of the two divisions.     

Association mapping of resistance to Puccinia hordei in Australian barley breeding germplasm

Key message

“To find stable resistance using association mapping tools, QTL with major and minor effects on leaf rust reactions were identified in barley breeding lines by assessing seedlings and adult plants.”

Abstract

Three hundred and sixty (360) elite barley (Hordeum vulgare L.) breeding lines from the Northern Region Barley Breeding Program in Australia were genotyped with 3,244 polymorphic diversity arrays technology markers and the results used to map quantitative trait loci (QTL) conferring a reaction to leaf rust (Puccinia hordei Otth). The F_3:5 (Stage 2) lines were derived or sourced from different geographic origins or hubs of international barley breeding ventures representing two breeding cycles (2009 and 2011 trials) and were evaluated across eight environments for infection type at both seedling and adult plant stages. Association mapping was performed using mean scores for disease reaction, accounting for family effects using the eigenvalues from a matrix of genotype correlations. In this study, 15 QTL were detected; 5 QTL co-located with catalogued leaf rust resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. The adult plant resistance gene Rph20 was identified across the majority of environments and pathotypes. The QTL detected in this study offer opportunities for breeding for more durable resistance to leaf rust through pyramiding multiple genomic regions via marker-assisted selection.     

Whole-genome association mapping in elite inbred crop varieties

We have previously shown that linkage disequilibrium (LD) in the elite cultivated barley (Hordeum vulgare) gene pool extends, on average, for <1-5 cM. Based on this information, we have developed a platform for whole genome association studies that comprises a collection of elite lines that we have characterized at 3060 genome-wide single nucleotide polymorphism (SNP) marker loci. Interrogating this data set shows that significant population substructure is present within the elite gene pool and that diversity and LD vary considerably across each of the seven barley chromosomes. However, we also show that a subpopulation comprised of only the two-rowed spring germplasm is less structured and well suited to whole genome association studies without the need for extensive statistical intervention to account for structure. At the current marker density, the two-rowed spring population is suited for fine mapping simple traits that are located outside of the genetic centromeres with a resolution that is sufficient for candidate gene identification by exploiting conservation of synteny with fully sequenced model genomes and the emerging barley physical map.     

Rapid genome evolution revealed by comparative sequence analysis of orthologous regions from four triticeae genomes

Bread wheat (Triticum aestivum) is an allohexaploid species, consisting of three subgenomes (A, B, and D). To study the molecular evolution of these closely related genomes, we compared the sequence of a 307-kb physical contig covering the high molecular weight (HMW)-glutenin locus from the A genome of durum wheat (Triticum turgidum, AABB) with the orthologous regions from the B genome of the same wheat and the D genome of the diploid wheat Aegilops tauschii (Anderson et al., 2003; Kong et al., 2004). Although gene colinearity appears to be retained, four out of six genes including the two paralogous HMW-glutenin genes are disrupted in the orthologous region of the A genome. Mechanisms involved in gene disruption in the A genome include retroelement insertions, sequence deletions, and mutations causing in-frame stop codons in the coding sequences. Comparative sequence analysis also revealed that sequences in the colinear intergenic regions of these different genomes were generally not conserved. The rapid genome evolution in these regions is attributable mainly to the large number of retrotransposon insertions that occurred after the divergence of the three wheat genomes. Our comparative studies indicate that the B genome diverged prior to the separation of the A and D genomes. Furthermore, sequence comparison of two distinct types of allelic variations at the HMW-glutenin loci in the A genomes of different hexaploid wheat cultivars with the A genome locus of durum wheat indicates that hexaploid wheat may have more than one tetraploid ancestor.     

Genomic differentiation of Hordeum chilense from H. vulgare as revealed by repetitive and EST sequences

Hordeum vulgare, cultivated barley, and its wild relative, H. chilense, have several important traits that might be useful for wheat improvement. Here, in situ hybridization and barley expressed sequence tag (EST) markers were used to characterize and compare the chromosomes of H. chilense with those of H. vulgare. FISH with four repetitive DNA sequences, AG, AAG, 5S rDNA and 45S rDNA, was applied to the mitotic chromosomes of H. vulgare, H. chilense and available wheat-H. chilense addition and substitution lines. FISH with the AAG repeat differentiated the individual chromosomes of H. chilense and H. vulgare. The patterns of FISH signals in the two species differed greatly. The 45S rDNA signals were observed on two pairs of chromosomes in both species, while the 5S rDNA signals were observed on four pairs of chromosomes in H. vulgare and on one pair in H. chilense. The AG repeat showed FISH signals at the centromeric regions of all chromosomes of H. vulgare but none of the chromosomes of H. chilense. These results indicate that the chromosomes of the two species are highly differentiated. To study the homoeology between the two species, 209 EST markers of H. vulgare were allocated to individual chromosomes of H. chilense. One hundred and forty of the EST markers were allocated to respective chromosomes of H. chilense using the wheat-H. chilense addition and substitution lines. Twenty-six EST markers on average were allocated to each chromosome except to the chromosome 2H(ch)S, to which only 10 markers were allocated. Ninety percent of the allocated EST markers in H. chilense were placed on H. vulgare chromosomes of the same homo-eologous group, indicating that the expressed sequences of the two species were highly conserved. These EST markers would be useful for detecting chromatin introgressed from these species into the wheat genome.     

Structural organization of the barley D-hordein locus in comparison with its orthologous regions of wheat genomes.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.     

Expansion and divergence of the GH locus between spider monkey and chimpanzee

Growth hormone (GH) has been previously described as showing distinct evolutionary stories between primates and other mammals. A burst of changes and successive amplification events took place in the primate lineage giving rise to a multigene family in the three Anthropoidea lineages. Polymerase chain reaction (PCR) was used to obtain the genes and the intergenic regions comprising the GH loci of the spider monkey (Ateles geoffroyi), a New-World primate, and of the chimpanzee (Pan troglodytes), an ape. The intergenic sequences of both species were screened by hybridization to detect copies of the Alu family, which have been implicated in the formation of the human GH locus. The GH locus of the spider monkey contains at least six GH-related genes, four of them were cloned. Likewise, five short intergenic sequences of approximately 3 kb were amplified and cloned. On the other hand, in the chimpanzee four new placental lactogen (PL) genes as well as four intergenic regions were amplified. Consequently, in this ape, six genes (two GHs, previously obtained, and four PLs) are clustered, separated by intergenic sequences of different lengths (two short ones of about 5 kb, and at least two long ones between 9 and 13 kb). The presence of Alu sequences within the intergenic regions of both GH loci corroborates the current hypothesis that they acted as a driving force for the locus expansion. GH sequence comparisons reveal that several gene-conversion events might have occurred during the formation of this genome region, which has undergone independent evolution in the three Anthropoidea branches. To establish the GH's evolutionary history may prove to be a difficult task due to these gene-conversion events.     

应用微卫星标记研究西藏野生大麦的遗传多样性

以西藏不同地区的106份野生大麦为材料,其中包括50份野生二棱大麦（HS）,27份野生瓶形大麦（HL）和29份野生六棱大麦（HA）,用Liu等（1996）发表的SSR连锁图的每个连锁群的两个臂的不同位置上选取3～5个共30个SSR标记,研究了西藏3类野生大麦的遗传多样性。结果表明,这3类野生大麦在遗传组成及等位变异频率分布上存在着明显的遗传分化。在总样本中,共检测到229个等位变异,平均每个SSR位点检测到7．6个等位变异,其中70个为这3类野生大麦间共同的等位变异,等位变异数在这3类野生大麦间有明显的差异,亚种问的遗传多样性明显高于亚种内的遗传多样性。其遗传多样性大小顺序为HS〉HL〉HA。聚类分析表明,野生二棱大麦、野生六棱大麦分别聚在不同的两类,而野生瓶形大麦中各有约50％的材料分别聚在这两类。根据本研究及前人研究结果,我们认为中国栽培大麦是从野生二棱大麦经野生瓶形大麦向野生六棱大麦进化的。该结果支持了栽培大麦起源的“野生二棱大麦单系起源论”的观点。