Unigene derived microsatellite markers for the cereal genomes

Unigene derived microsatellite (UGMS) markers have the advantage of assaying variation in the expressed component of the genome with unique identity and positions. We characterized the microsatellite motifs present in the unigenes of five cereal species namely, rice, wheat, maize, Sorghum and barley and compared with those in Arabidopsis. The overall UGMS frequency in the five cereal species was 1/7.6 kb. The maximum UGMS frequency was in rice (1/3.6 kb) and the lowest in wheat (1/10.6 kb). GC-rich trinucleotide repeat motifs coding for alanine followed by arginine and the dinucleotide repeat motif GA were found to be abundant UGMS classes across all the five cereal species. Primers could be designed for 95% (wheat and barley) to 97% (rice) of the identified microsatellites. The proportion and frequency of occurrence of long hypervariable class I (≥20 nucleotides) and potentially variable class II (12–20 nucleotides) UGMS across five cereal species were characterized. The class I UGMS markers were physically mapped in silico on to the finished rice genome and bin-mapped in wheat. Comparative mapping based on class I UGMS markers in rice and wheat revealed syntenic relationships between the two genomes. High degree of conservation and cross-transferability of the class I UGMS markers were evident among the five cereal species, which was validated experimentally. The class I UGMS-conserved orthologous set (COS) markers identified in this study would be useful for understanding the evolution of genes and genomes in cereals. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Identification and characterization of annexin gene family in rice

Plant annexins are Ca²⁺-dependent phospholipid-binding proteins and are encoded by multigene families. They are implicated in the regulation of plant development as well as protection from drought and other stresses. They are well characterized in Arabidopsis, however no such characterization of rice annexin gene family has been reported thus far. With the availability of the rice genome sequence information, we have identified ten members of the rice annexin gene family. At the protein level, they share 16–64% identity with predicted molecular masses ranging from 32 to 40 kDa. Phylogenetic analysis of rice annexins together with annexins from other monocots led to their classification into five different orthologous groups and share similar motif patterns in their protein sequences. Expression analysis by real-time RT-PCR revealed differential temporal and spatial regulation of these genes. The rice annexin genes are also found to be regulated in seedling stage by various abiotic stressors including salinity, drought, heat and cold. Additionally, in silico analysis of the putative upstream sequences was analyzed for the presence of stress-responsive cis-elements. These results provide a basis for further functional characterization of specific rice annexin genes at the tissue/developmental level and in response to abiotic stresses.     

Mapping in the realm of polyploidy: The wheat model

Wheat is an allopolyploid containing three distinct but genetically related (homoeologous) genomes, A, B and D. Because of polyploid inheritance and large genome size (16×10¹² bp), the wheat genome is thought to be intractable to map-based cloning of agronomic and other genes of interest. We propose a targeted geneti mapping strategy that combines linkage and physical mapping and may facilitate map-based cloning. High-density linkage maps are either generated in wheat or in diploid Triticum tauschii, the donor of the D genome to wheat. Molecular marker-based chromosome maps are constructed, using an array of deletion lines in wheat. The conventional genetic linkage maps are aligned with chromosome maps to construct cytogenetic ladder maps (CLMs). The CLMs allow region-specific mapping and convert genetic distances into physical distances. The information from CLMs suggests that many genes in wheat are present in clusters that are highly recombiogenic, small, and may be amenable to cloning by chromosome walking. Therefore, the effective genome size of wheat is relatively small in comparison to the whole genome. The utility of using the smaller genome of rice for mapping and homologous gene cloning is discussed.     

Comparative genetic analysis of a wheat seed dormancy QTL with rice and <Emphasis Type="Italic">Brachypodium</Emphasis> identifies candidate genes for ABA perception and calcium signaling

Wheat preharvest sprouting (PHS) occurs when seed germinates on the plant before harvest resulting in reduced grain quality. In wheat, PHS susceptibility is correlated with low levels of seed dormancy. A previous mapping of quantitative trait loci (QTL) revealed a major PHS/seed dormancy QTL, QPhs.cnl-2B.1, located on wheat chromosome 2B. A comparative genetic study with the related grass species rice (Oryza sativa L.) and Brachypodium distachyon at the homologous region to the QPhs.cnl-2B.1 interval was used to identify the candidate genes for marker development and subsequent fine mapping. Expressed sequence tags and a comparative mapping were used to design 278 primer pairs, of which 22 produced polymorphic amplicons that mapped to the group 2 chromosomes. Fourteen mapped to chromosome 2B, and ten were located in the QTL interval. A comparative analysis revealed good macrocollinearity between the PHS interval and 3 million base pair (mb) region on rice chromosomes 7 and 3, and a 2.7-mb region on Brachypodium Bd1. The comparative intervals in rice were found to contain three previously identified rice seed dormancy QTL. Further analyses of the interval in rice identified genes that are known to play a role in seed dormancy, including a homologue for the putative Arabidopsis ABA receptor ABAR/GUN5. Additional candidate genes involved in calcium signaling were identified and were placed in a functional protein association network that includes additional proteins critical for ABA signaling and germination. This study provides promising candidate genes for seed dormancy in both wheat and rice as well as excellent molecular markers for further comparative and fine mapping.     

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice–wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Collinearity-based marker mining for the fine mapping of <Emphasis Type="Italic">Pm6</Emphasis>, a powdery mildew resistance gene in wheat

The genome sequences of rice (Oryza sativa L.) and Brachypodium distachyon and the comprehensive Triticeae EST (Expressed Sequence Tag) resources provide invaluable information for comparative genomics analysis. The powdery mildew resistance gene, Pm6, which was introgressed into common wheat from Triticum timopheevii, was previously mapped to the wheat chromosome bin of 2BL [fraction length (FL) 0.50–1.00] with limited DNA markers. In this study, we saturated the Pm6 locus in wheat using the collinearity-based markers by extensively exploiting these genomic resources. All wheat ESTs located in the bin 2BL FL 0.50–1.00 and their corresponding orthologous genes on rice chromosome 4 were firstly used to develop STS (Sequence Tagged Site) markers. Those identified markers that flanked the Pm6 locus were then used to identify the collinear regions in the genomes of rice and Brachypodium. Triticeae ESTs with orthologous genes in these collinear regions were further used to develop new conserved markers for the fine mapping of Pm6. Using two F₂ populations derived from crosses of IGVI-465 × Prins and IGVI-466 × Prins, we mapped a total of 29 markers to the Pm6 locus. Among them, 14 markers were co-segregated with Pm6 in the IGVI-466/Prins population. Comparative genome analysis showed that the collinear region of the 29 linked markers covers a ~5.6-Mb region in chromosome 5L of Brachypodium and a ~6.0-Mb region in chromosome 4L of rice. The marker order is conserved between rice and Brachypodium, but re-arrangements are present in wheat. Comparative mapping in the two populations showed that two conserved markers (CINAU123 and CINAU127) flanked the Pm6 locus, and an LRR-receptor-like protein kinase cluster was identified in the collinear regions of Brachypodium and rice. This putative resistance gene cluster provides a potential target site for further fine mapping and cloning of Pm6. Moreover, the newly developed conserved markers closely linked to Pm6 can be used for the marker-assisted selection (MAS) of Pm6 in wheat breeding programs.     

In silico comparative analysis reveals a mosaic conservation of genes within a novel colinear region in wheat chromosome 1AS and rice chromosome 5S

Comparative RFLP mapping has revealed extensive conservation of marker order in different grass genomes. However, microcolinearity studies at the sequence level have shown rapid genome evolution and many exceptions to colinearity. Most of these studies have focused on a limited size of genomic fragment and the extent of microcolinearity over large distances or across entire genomes remains poorly characterized in grasses. Here, we have investigated the microcolinearity between the rice genome and a total of 1,500 kb from physical BAC contigs on wheat chromosome 1AS. Using ESTs mapped in wheat chromosome bins as an additional source of physical data, we have identified 27 conserved orthologous sequences between wheat chromosome 1AS and a region of 1,210 kb located on rice chromosome 5S. Our results extend the orthology described earlier between wheat chromosome group 1S and rice chromosome 5S. Microcolinearity was found to be frequently disrupted by rearrangements which must have occurred after the divergence of wheat and rice. At the Lr10 orthologous loci, microrearrangements were due to the insertion of mobile elements, but also originated from gene movement, amplification, deletion and inversion. These mechanisms of genome evolution are at the origin of the mosaic conservation observed between the orthologous regions. Finally, in silico mapping of wheat genes identified an intragenomic colinearity between fragments from rice chromosome 1L and 5S, suggesting an ancestral segmental duplication in rice.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Sequence Analysis of a 34.7-kb DNA Segment from the Genome of Buchnera aphidicola (Endosymbiont of Aphids) Containing groEL, dnaA, the atp operon, gidA, and rho

Buchnera aphidicola is a prokaryotic endosymbiont of the aphid Schizaphis graminum. From past and present nucleotide sequence analyses of the B. aphidicola genome, we have assembled a 34.7-kilobase (kb) DNA segment. This segment contains genes coding for 32 open reading frames (ORFs), which corresponded to 89.9% of the DNA. All of these ORFs could be identified with homologous regions of the Escherichia coli genome. The order of the genes with established functions was groELS–trmE–rnpA–rpmH–dnaA–dnaN–gyrB–atpCDGAHFEB–gidA–fdx–hscA– hscB–nifS–ilvDC–rep–trxA–rho. The order of genes in small DNA fragments was conserved in both B. aphidicola and E. coli. Most of these fragments were in approximately the same region of the E. coli genome. The latter organism, however, contained many additional inserted genes within and between the fragments. The results of the B. aphidicola genome analyses indicate that the endosymbiont has many properties of free-living bacteria. Received: 15 August 1997 / Accepted: 29 August 1997     

Functional mapping of quantitative trait loci associated with rice tillering

Several biologically significant parameters that are related to rice tillering are closely associated with rice grain yield. Although identification of the genes that control rice tillering and therefore influence crop yield would be valuable for rice production management and genetic improvement, these genes remain largely unidentified. In this study, we carried out functional mapping of quantitative trait loci (QTLs) for rice tillering in 129 doubled haploid lines, which were derived from a cross between IR64 and Azucena. We measured the average number of tillers in each plot at seven developmental stages and fit the growth trajectory of rice tillering with the Wang–Lan–Ding mathematical model. Four biologically meaningful parameters in this model––the potential maximum for tiller number (K), the optimum tiller time (t ₀), and the increased rate (r), or the reduced rate (c) at the time of deviation from t ₀––were our defined variables for multi-marker joint analysis under the framework of penalized maximum likelihood, as well as composite interval mapping. We detected a total of 27 QTLs that accounted for 2.49–8.54% of the total phenotypic variance. Nine common QTLs across multi-marker joint analysis and composite interval mapping showed high stability, while one QTL was environment-specific and three were epistatic. We also identified several genomic segments that are associated with multiple traits. Our results describe the genetic basis of rice tiller development, enable further marker-assisted selection in rice cultivar development, and provide useful information for rice production management.     

Comparative mapping of HKT genes in wheat, barley, and rice, key determinants of Na+ transport, and salt tolerance

Salt tolerance of plants depends on HKT transporters (High-affinityK⁺ Transporter), which mediate Na⁺-specific transport or Na⁺-K⁺co-transport. Gene sequences closely related to rice HKT geneswere isolated from hexaploid bread wheat (Triticum aestivum)or barley (Hordeum vulgare) for genomic DNA southern hybridizationanalysis. HKT gene sequences were mapped on chromosomal armsof wheat and barley using wheat chromosome substitution linesand barley–wheat chromosome addition lines. In addition,HKT gene members in the wild diploid wheat ancestors, T. monococcum(A^m genome), T. urartu (A^u genome), and Ae. tauschii (D^t genome)were investigated. Variation in copy number for individual HKTgene members was observed between the barley, wheat, and ricegenomes, and between the different wheat genomes. HKT2;1/2-like,HKT2;3/4-like, HKT1;1/2-like, HKT1;3-like, HKT1;4-like, andHKT1;5-like genes were mapped to the wheat–barley chromosomegroups 7, 7, 2, 6, 2, and 4, respectively. Chromosomal regionscontaining HKT genes were syntenic between wheat and rice exceptfor the chromosome regions containing the HKT1;5-like gene.Potential roles of HKT genes in Na⁺ transport in rice, wheat,and barley are discussed. Determination of the chromosome locationsof HKT genes provides a framework for future physiological andgenetic studies investigating the relationships between HKTgenes and salt tolerance in wheat and barley. Key words: Barley, comparative mapping, HKT, rice, salt tolerance, sodium transport, wheat     

Fine scale genetic and physical mapping using interstitial deletion mutants of <Emphasis Type="Italic">Lr34</Emphasis><Emphasis Type="Italic">/Yr18</Emphasis>: a disease resistance locus effective against multiple pathogens in wheat

The Lr34/Yr18 locus has contributed to durable, non-race specific resistance against leaf rust (Puccinia triticina) and stripe rust (P. striiformis f. sp. tritici) in wheat (Triticum aestivum). Lr34/Yr18 also cosegregates with resistance to powdery mildew (Pm38) and a leaf tip necrosis phenotype (Ltn1). Using a high resolution mapping family from a cross between near-isogenic lines in the “Thatcher” background we demonstrated that Lr34/Yr18 also cosegregated with stem rust resistance in the field. Lr34/Yr18 probably interacts with unlinked genes to provide enhanced stem rust resistance in “Thatcher”. In view of the relatively low levels of DNA polymorphism reported in the Lr34/Yr18 region, gamma irradiation of the single chromosome substitution line, Lalbahadur(Parula7D) that carries Lr34/Yr18 was used to generate several mutant lines. Characterisation of the mutants revealed a range of highly informative genotypes, which included variable size deletions and an overlapping set of interstitial deletions. The mutants enabled a large number of wheat EST derived markers to be mapped and define a relatively small physical region on chromosome 7DS that carried Lr34/Yr18. Fine scale genetic mapping confirmed the physical mapping and identified a genetic interval of less than 0.5 cM, which contained Lr34/Yr18. Both rice and Brachypodium genome sequences provided useful information for fine mapping of ESTs in wheat. Gene order was more conserved between wheat and Brachypodium than with rice but these smaller grass genomes did not reveal sequence information that could be used to identify a candidate gene for rust resistance in wheat. We predict that Lr34/Yr18 is located within a large insertion in wheat not found at syntenic positions in Brachypodium and rice. W. Spielmeyer and R. P. Singh contributed equally to the study through the “Thatcher” and “Lalbahadur” genetic stocks, respectively.     

Fine genetic mapping fails to dissociate durable stem rust resistance gene Sr2 from pseudo-black chaff in common wheat (Triticum aestivum L.)

The broad-spectrum stem rust resistance gene Sr2 has provided protection in wheat against Puccinia graminis Pers. f. sp. tritici for over 80 years. The Sr2 gene and an associated dark pigmentation trait, pseudo-black chaff (PBC), have previously been localized to the short arm of chromosome 3B. In a first step towards the positional-based cloning of Sr2, we constructed a high-resolution map of this region. The wheat EST (wEST) deletion bin mapping project provided tightly linked cDNA markers. The rice genome sequence was used to infer the putative gene order for orthologous wheat genes and provide additional markers once the syntenic interval in rice was identified. We used this approach to map six wESTs that were collinear with the physical order of the corresponding genes on rice chromosome 1 suggesting there are no major re-arrangements between wheat and rice in this region. We were unable to separate by recombination the tightly linked morphological trait, PBC from the stem rust resistance gene suggesting that either a single gene or two tightly linked genes control both traits.     

Cereal asparagine synthetase genes

Asparagine synthetase catalyses the transfer of an amino group from glutamine to aspartate to form glutamate and asparagine. The accumulation of free (nonprotein) asparagine in crops has implications for food safety because free asparagine is the precursor for acrylamide, a carcinogenic contaminant that forms during high‐temperature cooking and processing. Here we review publicly available genome data for asparagine synthetase genes from species of the Pooideae subfamily, including bread wheat and related wheat species (Triticum and Aegilops spp.), barley (Hordeum vulgare) and rye (Secale cereale) of the Triticeae tribe. Also from the Pooideae subfamily: brachypodium (Brachypodium dIstachyon) of the Brachypodiae tribe. More diverse species are also included, comprising sorghum (Sorghum bicolor) and maize (Zea mays) of the Panicoideae subfamily and rice (Oryza sativa) of the Ehrhartoideae subfamily. The asparagine synthetase gene families of the Triticeae species each comprise five genes per genome, with the genes assigned to four groups: 1, 2, 3 (subdivided into 3.1 and 3.2) and 4. Each species has a single gene per genome in each group, except that some bread wheat varieties (genomes AABBDD) and emmer wheat (Triticum dicoccoides; genomes AABB) lack a group 2 gene in the B genome. This raises questions about the ancestry of cultivated pasta wheat and the B genome donor of bread wheat, suggesting that the hybridisation event that gave rise to hexaploid bread wheat occurred more than once. In phylogenetic analyses, genes from the other species cluster with the Triticeae genes, but brachypodium, sorghum and maize lack a group 2 gene, while rice has only two genes, one group 3 and one group 4. This means that TaASN2, the most highly expressed asparagine synthetase gene in wheat grain, has no equivalent in maize, rice, sorghum or brachypodium. An evolutionary pathway is proposed in which a series of gene duplications gave rise to the five genes found in modern Triticeae species.     

Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice (Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome

The International Rice Genome Sequencing Project has recently announced the high-quality finished sequence that covers nearly 95% of the japonica rice genome representing 370 Mbp. Nevertheless, the current physical map of japonica rice contains 62 physical gaps corresponding to approximately 5% of the genome, that have not been identified/represented in the comprehensive array of publicly available BAC, PAC and other genomic library resources. Without finishing these gaps, it is impossible to identify the complete complement of genes encoded by rice genome and will also leave us ignorant of some 5% of the genome and its unknown functions. In this article, we report the construction and characterization of a tenfold redundant, 40 kbp insert fosmid library generated by random mechanical shearing. We demonstrated its utility in refining the physical map of rice by identifying and in silico mapping 22 gap-specific fosmid clones with particular emphasis on chromosomes 1, 2, 6, 7, 8, 9 and 10. Further sequencing of 12 of the gap-specific fosmid clones uncovered unique rice genome sequence that was not previously reported in the finished IRGSP sequence and emphasizes the need to complete finishing of the rice genome. Fosmid library reported here is publicly available from our web site     

Genomic content and new insights on the origin of the B chromosome of the cichlid fish <Emphasis Type="Italic">Astatotilapia latifasciata</Emphasis>

B chromosomes are additional chromosomes widely studied in a diversity of eukaryotic groups, including fungi, plants and animals, but their origin, evolution and possible functions are not clearly understood. To further understand the genomic content and the evolutionary history of B chromosomes, classical and molecular cytogenetic analyses were conducted in the cichlid fish Astatotilapia latifasciata, which harbor 1–2 B chromosomes. Through cytogenetic mapping of several probes, including transposable elements, rRNA genes, a repeated DNA genomic fraction (C ₀ t − 1 DNA), whole genome probes (comparative genomic hybridization), and BAC clones from Oreochromis niloticus, we found similarities between the B chromosome and the 1st chromosome pair and chromosomes harboring rRNA genes. Based on the cytogenetic mapping data, we suggest the B chromosome may have evolved from a small chromosomal fragment followed by the invasion of the proto-B chromosome by several repeated DNA families.     

Genomic location and expression analysis of expansin gene family reveals the evolutionary and functional significance in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

The plant-specific expansin proteins constitute an ancient and major gene family known to have roles in regulating diverse biological processes in plants. Although the functions of many expansin genes have been identified in wheat and other species, little is known about the evolution and genomic locations of the expansin genes in wheat (Triticum aestivum). In this study, a total of 87 expansin genes were identified in the wheat genome, including 52 EXPAs, 42 EXPBs and 4 EXLAs. The EXLB gene was not found in the wheat genome. Phylogenetic tree and comparative analysis revealed amplification of the EXPBs in rice, maize and wheat. The predicted wheat expansins were distributed across 14 of 21 chromosomes with different densities, 3 tightly co-located clusters and 15 paralogous pairs, indicating that tandem duplication and segmental duplication events also played roles in the evolution of expansins in wheat. In addition, the gene structures and conserved protein domains of wheat expansins suggest high levels of conservation within the phylogenetic subgroups. Analysis of a published microarray database showed that most wheat expansin genes exhibit different expression levels in different tissues and developmental stages. To our knowledge, this is the first report of a genome-wide analysis of the wheat expansin gene family, which should provide valuable information for further elucidating the classification and putative functions of the entire gene family.