Most cited article - PubMed ID 12582592
Flow karyotyping and chromosome sorting in bread wheat ( Triticum aestivum L.)
To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool1. Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43, which was crossed into bread wheat from the wild grass Thinopyrum elongatum2,3. Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.
Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.
- Keywords
- Aegilops biuncialis, Aegilops geniculata, chromosome flow sorting, flow karyotyping, genome dissecting,
- Publication type
- Journal Article MeSH
Rye (Secale cereale) is a climate-resilient cereal grown extensively as grain or forage crop in Northern and Eastern Europe. In addition to being an important crop, it has been used to improve wheat through introgression of genomic regions for improved yield and disease resistance. Understanding the genomic diversity of rye will assist both the improvement of this crop and facilitate the introgression of more valuable traits into wheat. Here, we isolated and sequenced the short arm of rye chromosome 7 (7RS) from Triticale 380SD using flow cytometry and compared it to the public Lo7 rye whole genome reference assembly. We identify 2747 Lo7 genes present on the isolated chromosome arm and two clusters containing seven and sixty-five genes that are present on Triticale 380SD 7RS, but absent from Lo7 7RS. We identified 29 genes that are not assigned to chromosomal locations in the Lo7 assembly but are present on Triticale 380SD 7RS, suggesting a chromosome arm location for these genes. Our study supports the Lo7 reference assembly and provides a repertoire of genes on Triticale 7RS.
BACKGROUND: The tall wheatgrass species Thinopyrum elongatum carries a strong fusarium head blight (FHB) resistance locus located on the long arm of chromosome 7 (7EL) as well as resistance to leaf and stem rusts, all diseases with a significant impact on wheat production. Towards understanding the contribution of Th. elongatum 7EL to improvement of disease resistance in wheat, the genomic sequence of the 7EL fragment present in the wheat Chinese Spring (CS) telosomic addition line CS-7EL was determined and the contribution and impact of 7EL on the rachis transcriptome during FHB infection was compared between CS and CS-7EL. RESULTS: We assembled the Th. elongatum 7EL chromosome arm using a reference-guided approach. Combining this assembly with the available reference sequence for CS hexaploid wheat provided a reliable reference for interrogating the transcriptomic differences in response to infection conferred by the 7EL fragment. Comparison of the transcriptomes of rachis tissues from CS and CS-7EL showed expression of Th. elongatum transcripts as well as modulation of wheat transcript expression profiles in the CS-7EL line. Expression profiles at 4 days after infection with Fusarium graminearum, the causal agent of FHB, showed an increased in expression of genes associated with an effective defense response, in particular glucan endo-1,3-beta-glucosidases and chitinases, in the FHB-resistant line CS-7EL while there was a larger increase in differential expression for genes associated with the level of fungal infection in the FHB-susceptible line CS. One hundred and seven 7EL transcripts were expressed in the smallest 7EL region defined to carry FHB resistance. CONCLUSION: 7EL contributed to CS-7EL transcriptome by direct expression and through alteration of wheat transcript profiles. FHB resistance in CS-7EL was associated with transcriptome changes suggesting a more effective defense response. A list of candidate genes for the FHB resistance locus on 7EL has been established.
- Keywords
- Alien introgression, Disease resistance, Fusarium graminearum, Non-coding RNA, RNA-seq, Thinopyrum elongatum, Transcriptome, Triticum aestivum,
- MeSH
- Chromosomes, Plant * genetics MeSH
- Fusarium * MeSH
- Genomics MeSH
- Poaceae * genetics microbiology MeSH
- Plant Diseases * genetics microbiology MeSH
- Disease Resistance * genetics MeSH
- Transcriptome MeSH
- Publication type
- Journal Article MeSH
Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
- Keywords
- DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
- MeSH
- Cell Cycle MeSH
- Chromosomes, Plant * genetics MeSH
- Metaphase MeSH
- Flow Cytometry MeSH
- Plants * genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Plasma membrane-associated and intracellular proteins and protein complexes play a pivotal role in pathogen recognition and disease resistance signaling in plants and animals. The two predominant protein families perceiving plant pathogens are receptor-like kinases and nucleotide binding-leucine-rich repeat receptors (NLR), which often confer race-specific resistance. Leaf rust is one of the most prevalent and most devastating wheat diseases. Here, we clone the race-specific leaf rust resistance gene Lr14a from hexaploid wheat. The cloning of Lr14a is aided by the recently published genome assembly of ArinaLrFor, an Lr14a-containing wheat line. Lr14a encodes a membrane-localized protein containing twelve ankyrin (ANK) repeats and structural similarities to Ca2+-permeable non-selective cation channels. Transcriptome analyses reveal an induction of genes associated with calcium ion binding in the presence of Lr14a. Haplotype analyses indicate that Lr14a-containing chromosome segments were introgressed multiple times into the bread wheat gene pool, but we find no variation in the Lr14a coding sequence itself. Our work demonstrates the involvement of an ANK-transmembrane (TM)-like type of gene family in race-specific disease resistance in wheat. This forms the basis to explore ANK-TM-like genes in disease resistance breeding.
- MeSH
- Ankyrin Repeat genetics MeSH
- Basidiomycota pathogenicity MeSH
- Gene Pool MeSH
- Haplotypes MeSH
- Membrane Proteins genetics MeSH
- Mutagenesis MeSH
- Plant Diseases genetics MeSH
- Disease Resistance genetics MeSH
- Triticum genetics MeSH
- Gene Expression Regulation, Plant MeSH
- Genes, Plant genetics MeSH
- Plant Proteins genetics MeSH
- Plant Breeding MeSH
- Nicotiana genetics MeSH
- Gene Silencing MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Membrane Proteins MeSH
- Plant Proteins MeSH
Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.
- MeSH
- Cell Cycle physiology MeSH
- Time Factors MeSH
- Chromosomes, Plant * metabolism MeSH
- Genome, Plant genetics MeSH
- Genomics methods MeSH
- Hydroxyurea MeSH
- Metaphase MeSH
- Mitotic Index MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds MeSH
- Flow Cytometry methods MeSH
- Buffers MeSH
- Saccharum cytology genetics MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- amiprophos methyl MeSH Browser
- Hydroxyurea MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds MeSH
- Buffers MeSH
Durum wheat (Triticum turgidum) derives from a hybridization event approximately 400,000 years ago which led to the creation of an allotetraploid genome. The evolutionary recent origin of durum wheat means that its genome has not yet been fully diploidised. As a result, many of the genes present in the durum genome act in a redundant fashion, where loss-of-function mutations must be present in both gene copies to observe a phenotypic effect. Here, we use a novel set of induced variation within the cv. Kronos TILLING population to identify a locus controlling a dominant, environmentally dependent chlorosis phenotype. We carried out a forward screen of the sequenced cv. Kronos TILLING lines for senescence phenotypes and identified a line with a dominant early senescence and chlorosis phenotype. Mutant plants contained less chlorophyll throughout their development and displayed premature flag leaf senescence. A segregating population was classified into discrete phenotypic groups and subjected to bulked-segregant analysis using exome capture followed by next-generation sequencing. This allowed the identification of a single region on chromosome 3A, Yellow Early Senescence 1 (YES-1), which was associated with the mutant phenotype. While this phenotype was consistent across 4 years of field trials in the United Kingdom, the mutant phenotype was not observed when grown in Davis, CA (United States). To obtain further SNPs for fine-mapping, we isolated chromosome 3A using flow sorting and sequenced the entire chromosome. By mapping these reads against both the cv. Chinese Spring reference sequence and the cv. Kronos assembly, we could identify high-quality, novel EMS-induced SNPs in non-coding regions within YES-1 that were previously missed in the exome capture data. This allowed us to fine-map YES-1 to 4.3 Mb, containing 59 genes. Our study shows that populations containing induced variation can be sources of novel dominant variation in polyploid crop species, highlighting their importance in future genetic screens. We also demonstrate the value of using cultivar-specific genome assemblies alongside the gold-standard reference genomes particularly when working with non-coding regions of the genome. Further fine-mapping of the YES-1 locus will be pursued to identify the causal SNP underpinning this dominant, environmentally dependent phenotype.
- Keywords
- TILLING, bulked-segregant analysis, chlorosis, durum wheat, genomics, mapping-by-sequencing, senescence,
- Publication type
- Journal Article MeSH
Leaf rust, caused by Puccinia triticina, threatens global wheat production due to the constant evolution of virulent pathotypes that defeat commercially deployed all stage-resistance (ASR) genes in modern cultivars. Hence, the deployment of combinations of adult plant resistance (APR) and ASR genes in new wheat cultivars is desirable. Adult plant resistance gene Lr49 was previously mapped on the long arm of chromosome 4B of cultivar VL404 and flanked by microsatellite markers barc163 (8.1 cM) and wmc349 (10.1 cM), neither of which was sufficiently closely linked for efficient marker assisted selection. This study used high-density SNP genotyping and flow sorted chromosome sequencing to fine-map the Lr49 locus as a starting point to develop a diagnostic marker for use in breeding and to clone this gene. Marker sunKASP_21 was mapped 0.4 cM proximal to Lr49, whereas a group of markers including sunKASP_24 were placed 0.6 cM distal to this gene. Testing of the linked markers on 75 Australian and 90 European cultivars with diverse genetic backgrounds showed that sunKASP_21 was most strongly associated with Lr49. Our results also show that the Lr49 genomic region contains structural variation relative to the reference stock Chinese Spring, possibly an inverted genomic duplication, which introduces a new set of challenges for the Lr49 cloning.
- Keywords
- Infinium iSelect 90K SNP array, adult plant resistance, chromosome sorting, leaf rust, marker assisted breeding,
- Publication type
- Journal Article MeSH
Despite a long history, the production of useful alien introgression lines in wheat remains difficult mainly due to linkage drag and incomplete genetic compensation. In addition, little is known about the molecular mechanisms underlying the impact of foreign chromatin on plant phenotype. Here, a comparison of the transcriptomes of barley, wheat and a wheat-barley 7HL addition line allowed the transcriptional impact both on 7HL genes of a non-native genetic background and on the wheat gene complement as a result of the presence of 7HL to be assessed. Some 42% (389/923) of the 7HL genes assayed were differentially transcribed, which was the case for only 3% (960/35 301) of the wheat gene complement. The absence of any transcript in the addition line of a suite of chromosome 7A genes implied the presence of a 36 Mbp deletion at the distal end of the 7AL arm; this deletion was found to be in common across the full set of Chinese Spring/Betzes barley addition lines. The remaining differentially transcribed wheat genes were distributed across the whole genome. The up-regulated barley genes were mostly located in the proximal part of the 7HL arm, while the down-regulated ones were concentrated in the distal part; as a result, genes encoding basal cellular functions tended to be transcribed, while those encoding specific functions were suppressed. An insight has been gained into gene transcription in an alien introgression line, thereby providing a basis for understanding the interactions between wheat and exotic genes in introgression materials.
- Keywords
- RNA-seq, alien introgression, chromosomal rearrangement, deletion, gene transcription, transcriptome modification,
- MeSH
- Genome, Plant * MeSH
- Hordeum genetics metabolism MeSH
- Triticum genetics metabolism MeSH
- Sequence Deletion MeSH
- Transcriptome * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
