CHROMOSOME GENOMICS IN WHEAT The concept of chromosome genomics is based on dissecting nuclear genomes to individual chromosomes, whose DNA can be analyzed separately. This approach brings a dramatic reduction in DNA sample complexity and facilitates genome mapping, sequencing and positional cloning. In polyploid wheat, it helps to avoid problems due to sequence redundancy and occurrence of homoeologous sequences. CHROMOSOME GENOMICS CHROMOSOME GENOMICS IMPORTANCE OF WHEAT IMPORTANCE OF WHEAT WHAT WE ARE DOING WHAT WE ARE DOING DISSECTING THE GENOME DISSECTING THE GENOME Chromosomes: 605 - 995 Mbp (3.6 – 5.9% of the genome) Three genomes of hexaploid wheat Genome size Oryza sativa (2n = 2x = 24) 1C ~ 430 Mbp Triticum aestivum (2n = 6x = 42) 1C ~ 17,000 Mbp AA BB DD Chromosome arms: 225 - 585 Mbp (1.3 – 3.4% of the genome) D B ; A a) Flow cytometry can be used to sort chromosomes in bread wheat b) Due to similarities in DNA content, only chromosome 3B can be flow-sorted in most of bread wheat cultivars. Other chromosomes can be sorted in groups. c) All chromosome arms can be sorted from telosomic lines. In these lines, the arms are stably maintained as telocentric chromosomes. d) Flow-sorted chromosomes are intact, and high molecular weight DNA can be prepared from them, suitable for molecular biology and genomics applications WHERE WE ARE WHERE WE ARE SORTING CHROMOSOME ARMS SORTING CHROMOSOME ARMS I II III 5DS 5DL 3B Afa Tel DAPI 5DS 5DL Ditelosomic line 5D 3B I II III 1BS GAA PI 1BS Telosomic line 1BS Chromosome arms can be sorted from telosomic lines. Two different arms can be sorted simultaneously from most of ditelosomic lines 21 chromosomes of bread wheat International collaboration on International collaboration on the the wheat genome wheat genome International Wheat Genome Sequencing Consortium http://www.wheatgenome.org/ 2A 3A 4A 5A 1A 6A 7A 1D 2D 4D 5D 6D 7D 2B 3D 1B 3B 4B 5B 6B 7B BAC libraries Cytogenetic mapping Development of DArT markers Physical mapping using PCR Applications No. chrs. Sorting time 10 2 <1 min 10 3 2 min 10 4 60 min 10 5 1 day 10 6 6 weeks Isothermal DNA amplification for: Mapping on DNA microarrays Shotgun sequencing Development of DNA markers APPLICATIONS OF SORTED CHROMOSOMES APPLICATIONS OF SORTED CHROMOSOMES CHROMOSOME-SPECIFIC BAC LIBRARIES CHROMOSOME-SPECIFIC BAC LIBRARIES http://olomouc.ueb.cas.cz/dna-libraries/cereals Chromosome-specific BAC libraries have a much smaller number of clones than genomic libraries and are easy to use. They facilitate targeted development of DNA markers, positional gene cloning and assembly of ready-to-sequence physical maps. Construction of BAC libraries from all chromosomes of hexaploid wheat is in progress. To date, BAC libraries are available for 19 out of the 21 chromosomes of wheat. The chromosome-based strategy simplifies BAC contig assembly and construction of physical map of hexaploid wheat PHYSICAL BAC CONTIG MAPS PHYSICAL BAC CONTIG MAPS Ctg205 from 3DS physical map. Red BAC clones belong to 3DS Minimum Tilling Path (MTP). 1BS Localization of two pairs of BACs on mitotic and stretched telosomes1BS,respectively Advantages of using flow-sorted chromosomes for FISH: Higher throughput (mapping on large populations of chromosomes) Higher sensitivity (localization of short DNA sequences ~2 kb) Higher spatial resolution CYTOGENETIC MAPPING CYTOGENETIC MAPPING NEXT-GENERATION SEQUENCING NEXT-GENERATION SEQUENCING Comparison of Comparison of wheat chromosome wheat chromosome 4A 4A sequence with that of barley sequence with that of barley chromosomes chromosomes Next generation sequencing of flow-sorted chromosomes enables the assembly of genes and low copy regions. Syntenic relationship between wheat (barley, rye) and sequenced genomes of related species is used to produce annotated syntenic builds whereby genes are placed in an approximate order and orientation. These assemblies can be used to identify candidate genes, discover SNPs and investigate genome evolution. Bread wheat (Triticum aestivum L.) is one of the world’s most important crops providing staple food for 35% of the world’s population and 20% calories consumed (http://www.CIMMYT.org/ ). The bread wheat is grown in all areas of temperate zone and its global significance could by compared only with rice. As the World’s population is supposed to reach 9Bn by 2050, the production of wheat must increase by 60%. Wheat (Triticum aestivum, 2n = 6x = 42) is characterized by a large genome (1C ~ 17,000 Mb) and presence of three homeologous genomes. These features hamper gene isolation, mapping and genome sequencing and assembling POSITIONAL GENE CLONING POSITIONAL GENE CLONING Cloning of agronomically important genes should accelerate conventional breeding efforts and production of new efficient crop cultivars. Projects for cloning russian wheat aphid resisatance gene, powdery mildew resistance gene as well as a gene controling flowering time are underway. Russian wheat aphid resistance gene Russian wheat aphid resistance gene cloning project cloning project ALIEN INTROGRESSION ALIEN INTROGRESSION The gene pool of elite bread wheat is relatively narrow. Introgression of additional variation found in genetic resources should to increase yield stability under environmental and biotic stress and further improve wheat. To support these efforts, we have developed chromosome genomics in two wild diploid wheats, Aegilops umbellulata and Ae. comosa, and their allotetraploid hybrids. Syntenic relationships between the chromosomes of Ae. biuncialis (1U b ) Ae. umbellulata (1U, 3U, 6U), and barley FUTURE RESEARCH TARGETS FUTURE RESEARCH TARGETS • Chromosome sequencing and assembly • Functional gene analysis • 3D organization of wheat nucleus • Chromosome specific proteomics • Epigenetic status of chromosomes
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CHROMOSOME GENOMICS IN WHEAT
The concept of chromosome genomics is based on dissecting nuclear genomes to individual chromosomes, whose DNA can be analyzed separately. This approach brings a dramatic reduction in DNA sample complexity and facilitates genome mapping, sequencing and positional cloning. In polyploid wheat, it helps to avoid problems due to sequence redundancy and occurrence of homoeologous sequences.
CHROMOSOME GENOMICSCHROMOSOME GENOMICSIMPORTANCE OF WHEATIMPORTANCE OF WHEAT
WHAT WE ARE DOINGWHAT WE ARE DOING
DISSECTING THE GENOMEDISSECTING THE GENOME
Chromosomes: 605 - 995 Mbp(3.6 – 5.9% of the genome)
Three genomes of hexaploid wheatGenome size
Oryza sativa(2n = 2x = 24)1C ~ 430 Mbp
Triticum aestivum(2n = 6x = 42)
1C ~ 17,000 Mbp
AA BB
DD
Chromosome arms: 225 - 585 Mbp(1.3 – 3.4% of the genome)
DB
;
A
a) Flow cytometry can be used to sort chromosomes in bread wheat
b) Due to similarities in DNA content, only chromosome 3B can be flow-sorted in most of bread wheat cultivars. Other chromosomes can be sorted in groups.
c) All chromosome arms can be sorted from telosomic lines. In these lines, the arms are stably maintained as telocentric chromosomes.
d) Flow-sorted chromosomes are intact, and high molecular weight DNA can be prepared from them, suitable for molecular biology and genomics applications
WHERE WE AREWHERE WE ARESORTING CHROMOSOME ARMSSORTING CHROMOSOME ARMS
III
III5DS
5DL
3B
AfaTelDAPI
5DS
5DL
Ditelosomic line 5D
3B
I
II
III
1BS
GAAPI
1BS
Telosomic line 1BS
Chromosome arms can be sorted from telosomic lines. Two different arms can be sorted simultaneously from most of ditelosomic lines
21 chromosomes of bread wheat
International collaboration on International collaboration on the the wheat genomewheat genome
International
Wheat
Genome
Sequencing
Consortiumhttp://www.wheatgenome.org/
2A 3A 4A 5A1A 6A 7A
1D 2D 4D 5D 6D 7D
2B
3D
1B 3B 4B 5B 6B 7B
BAC libraries
Cytogenetic mapping
Development of DArT markers
Physical mapping using PCR
ApplicationsNo. chrs.Sorting time
102<1 min
1032 min
10460 min
1051 day
1066 weeks
Isothermal DNA amplification for: Mapping on DNA microarrays Shotgun sequencing Development of DNA markers
APPLICATIONS OF SORTED CHROMOSOMESAPPLICATIONS OF SORTED CHROMOSOMESCHROMOSOME-SPECIFIC BAC LIBRARIESCHROMOSOME-SPECIFIC BAC LIBRARIES
http://olomouc.ueb.cas.cz/dna-libraries/cereals
Chromosome-specific BAC libraries have a much smaller number of clones than genomic libraries and are easy to use. They facilitate targeted development of DNA markers, positional gene cloning and assembly of ready-to-sequence physical maps. Construction of BAC libraries from all chromosomes of hexaploid wheat is in progress. To date, BAC libraries are available for 19 out of the 21 chromosomes of wheat.
The chromosome-based strategy simplifies BAC contig assembly and construction of physical map of hexaploid wheat
PHYSICAL BAC CONTIG MAPSPHYSICAL BAC CONTIG MAPS
Ctg205 from 3DS physical map. Red BAC clones belong to 3DS Minimum Tilling Path (MTP).
1BS
Localization of two pairs of BACs on mitotic and stretched telosomes1BS,respectively
Advantages of using flow-sorted chromosomes for FISH:Higher throughput (mapping on large populations of chromosomes)Higher sensitivity (localization of short DNA sequences ~2 kb)Higher spatial resolution
CYTOGENETIC MAPPINGCYTOGENETIC MAPPING
NEXT-GENERATION SEQUENCINGNEXT-GENERATION SEQUENCING
Comparison of Comparison of wheat chromosome wheat chromosome 4A4A sequence with that of barleysequence with that of barley chromosomeschromosomes
Next generation sequencing of flow-sorted chromosomes enables the assembly of genes and low copy regions. Syntenic relationship between wheat (barley, rye) and sequenced genomes of related species is used to produce annotated syntenic builds whereby genes are placed in an approximate order and orientation. These assemblies can be used to identify candidate genes, discover SNPs and investigate genome evolution.
Bread wheat (Triticum aestivum L.) is one of the world’s most important crops providing staple food for 35% of the world’s population and 20% calories consumed (http://www.CIMMYT.org/). The bread wheat is grown in all areas of temperate zone and its global significance could by compared only with rice. As the World’s population is supposed to reach 9Bn by 2050, the production of wheat must increase by 60%. Wheat (Triticum aestivum, 2n = 6x = 42) is characterized by a large genome (1C ~ 17,000 Mb) and presence of three homeologous genomes. These features hamper gene isolation, mapping and genome sequencing and assembling
POSITIONAL GENE CLONINGPOSITIONAL GENE CLONINGCloning of agronomically important genes should accelerate conventional breeding efforts and production of new efficient crop cultivars. Projects for cloning russian wheat aphid resisatance gene, powdery mildew resistance gene as well as a gene controling flowering time are underway.
Russian wheat aphid resistance geneRussian wheat aphid resistance gene cloning project cloning project
ALIEN INTROGRESSIONALIEN INTROGRESSIONThe gene pool of elite bread wheat is relatively narrow. Introgression of additional variation found in genetic resources should to increase yield stability under environmental and biotic stress and further improve wheat. To support these efforts, we have developed chromosome genomics in two wild diploid wheats, Aegilops umbellulata and Ae. comosa, and their allotetraploid hybrids.
Syntenic relationships between the chromosomes
of Ae. biuncialis (1Ub)Ae. umbellulata (1U, 3U, 6U),
and barley
FUTURE RESEARCH TARGETSFUTURE RESEARCH TARGETS
• Chromosome sequencing and assembly• Functional gene analysis• 3D organization of wheat nucleus• Chromosome specific proteomics• Epigenetic status of chromosomes
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