Progress 09/01/08 to 08/31/11
Outputs
OUTPUTS: This proposal is part of joint effort by a team of scientists affiliated with International Wheat Genome Sequencing Consortium (IWGSC) focusing on the development of a physical map of chromosome 3A of wheat. The outputs of the project are 1. Development of BAC libraries from telosomic chromosome arms 3AS (25x) and 3AL (15x) isolated by flow cytometry and then assembled FPC map by high information content fingerprinting (HICF) SNaPshot technology. 2. Six-dimensional BAC pools developed for anchoring of expressed sequence tags (ESTs) and simple sequence repeats (SSRs) of individual BACs and then integration of markers data with fingerprint data to develop an anchored FPC map. Further, a three-dimensional pools for the minimum tiling path (MTP) were developed. 3. Sequence data from 10,000 random BAC-ends were used to develop more than 1,453 new simple satellite repeat (SSR), insertion site based polymorphism (ISBP) and genomic markers. Nearly 8,000 SNPs have been identified between parents of the mapping populations involving ~1,700 genes from the large contigs of 3A assembly. Of these 8,000 SNPs, 1536 SNPs will be used on a high-throughput genotyping platform to generate the integrated genetic/physical map of the 3A chromosome. The project started in September of 2008, and the results were disseminated through conferences, workshop and media. This included Plant and Animal Genome XVII (Jan, 2009, 2010, 2011), and International Triticeae mapping initiative (ITMI, August, 2009). PARTICIPANTS: Participants at Kansas State University: Bikram S. Gill (PI), Wanlong Li (co-PI), Eduard Akhunov (co-PI), Sunish K. Sehgal (project scientist), Gaganpreet Kaur (postdoc), Vasile Catana (Programmer); Jayachandran Pillamari (Graduate student) ; USDA-ARS, Fargo ND: J Faris (co-PI), L Reddy (Grad Student); University of California, Davis: MC Luo (co-PI), YQ Ma (postdoc); Institute of Experimental Botany, Olomuc, Czech Republic: J Dolezel (co-PI). TARGET AUDIENCES: Academic personnel, high school students, producers and consumers. PROJECT MODIFICATIONS: To overcome the bottleneck of low polymorphism and further time and labor involved in gel based genetic mapping in wheat, we have decided to develop high density genetic map by identification of SNPs in parents of the mapping population for anchored ESTs and use Illumina's GoldenGate technology to map them.
Impacts
BAC library construction: Two BAC libraries each for chromosome arms 3AS and 3AL with total arm coverage of 25x (110,592 clones) and 15x (79,872) were developed and will be an important resource for gene discovery and to analyze the structural and functional organization of 3A chromosome. BAC fingerprinting and contigs assembly: A total of 110,592 BAC clones representing chromosome arm 3AS and 79,872 BAC clones representing chromosome arm 3AL were fingerprinted using the SNaPshot-based high-throughput, high-info-content BAC fingerprinting technique and assembled using FPCv9.3. For 3AS, a total of 45,739 clones were assembled into 404 contigs with an average contig length of 842Kb and 11,938 singletons. The chromosome am 3AL was assembled into 700 contigs having 51,405 clones with an average contig length of 570Kb and 12,064 singletons. The 3AS contig assembly represents 90 percent of the chromosome arm with 15x coverage and the 3AL contig assembly represents 87 percent of the chromosome arm with 12x coverage. A minimum tilling path (MTP) of ~5,100 BAC clones from 3AS and 5,300 from 3AL has been identified. The 3A-MTP will be used for sequencing of the 3A chromosome for comparative mapping and gene discovery. Marker development and anchoring: Synteny between wheat group-3 and rice chromosome 1 was exploited and wheat ESTs corresponding to rice chromosome 1 were utilized to design primer pairs from nearly 1400 unique ESTs. Further, BESs were used to develop 598 SSRs, 500 ISBPs and 200 genic markers. To anchor these markers to BAC contigs, 26,112 clones from the 3AS BAC library was pooled into 6 dimensions, 190 BAC pools distributed to the mapping labs. More than 500 EST-STS markers have been mapped to individual BACs and further to BAC contigs of 3AS FPC assembly. To further improve the marker density three-dimensional pools were developed from 2,743 BAC clones spaced along the MTP. 2,743 BACs in three dimensions were sequenced using 454 Titanium technology with 6-7x coverage. The sequence was assembled into ~194,000 contigs and masked to identify gene coding regions. Nearly, 55 % of the 194,000 contigs and over 70 % gene containing contigs were traced back to individual BAC clones. Nearly 3,000 primers were designed from MTP sequence and ~8,000 SNPs have been identified among parents of mapping population T. monococcum subsp. aegilopoides and subsp. monococcum. A high-throughput genotyping by Golden GateTM assay with 1,536 SNPs and 480 RILs will be performed to generate an anchored physical map of the 3A chromosome. The new website has a Chromosome 3A Genome Browser and WebFPC browser to serve as a browsing tool for information on BAC contigs, and markers and web BLAST for chromosome 3A. Further, a web searchable marker database has also been developed which will house all primers sequences and BAC pools scoring, BAC contig information, mapping populations and comparative mapping with rice and Brachypodium. The information regarding workshops, protocols and the BAC libraries have been uploaded on the web site for free public access to the scientific community.
Publications
- SehgalSK, AknunovE, Li W, Kaur G, Catana V, Pillamari J, Faris J, Reddy L, Devos KM, Rabinowicz PD, Chan A , Maiti R, Dolezel J, Simkova H, Safar J, Luo MC, Ma Y, You F, Gill BS(2011). Towards a physical and genetic framework map of chromosome 3A of bread wheat (Triticum aestivum L.). PAG XIX Abstracts P019 Akhunov E, Sehgal SK, Akhunova A, Liang H, Kaur G, Luo M-C, Simkova H, Dolezel J, Gill BS (2011) Sequencing and analysis of the wheat chromosome 3A gene space. PAG XIX Abstracts W352
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: This proposal is part of joint effort by a team of scientists affiliated with International Wheat Genome Sequencing Consortium (IWGSC) focusing on the development of a physical map of chromosome 3A of wheat. The outputs of the projects are: 1. Development of BAC libraries from telosomic chromosome arms 3AS (25x) and 3AL (15x) isolated by flow cytometry and then assembled FPC map by high information content fingerprinting (HICF) SNaPshot technology. 2. Six-dimensional BAC pools developed for anchoring of expressed sequence tags (ESTs) and simple sequence repeats (SSRs) of individual BACs and then integration of markers data with fingerprint data to develop an anchored FPC map. Further, a three dimensional pools for the minimum tiling path (MTP) would be developed. 3. Sequence data from 10,000 random BAC-ends were used to develop more than 1,000 new simple satellite repeat (SSR), insertion site based polymorphism (ISBP) and genomic markers. Further, 1536, SNPs markers would be developed based of sequence of the MTP of large contigs and a high-throughput genotyping platform would be used to generate the integrated genetic/physical map of the 3A chromosome. The project started last year in Sept. of 2008, the results were disseminated through conferences, workshop and media. This included Plant and Animal Genome XVII (Jan, 2009, 2010, 2011), and International Triticeae mapping initiative (ITMI, August, 2009). PARTICIPANTS: Participants at Kansas State University : Bikram S. Gill (PI), Wanlong Li (co-PI), Eduard Akhunov (co-PI), Sunish K. Sehgal (project scientist), Gaganpreet Kaur (postdoc), Vasile Catana (Programer); USDA-ARS, Fargo ND: J Faris (co-PI), L Reddy (Grad Student); University of California, Davis: MC Luo (co-PI), YQ Ma (postdoc); Institute of Experimental Botany, Olomuc, Czech Republic: J Dolezel (co-PI). TARGET AUDIENCES: Academic personnel, high school students, producers and consumers. PROJECT MODIFICATIONS: To overcome the bottleneck of low polymorphism and further time and labor involved in gel based genetic mapping in wheat, we have decided to develop high density genetic map by identification of SNPs in parents of the mapping population for anchored ESTs and use Illumina's GoldenGate technology to map them.
Impacts
BAC library construction: Two BAC libraries each for chromosome arms 3AS and 3AL with total arm coverage of 25x (110,592 clones) and 15x (79,872) were developed. BAC fingerprinting and contigs assembly: A total of 110,592 BAC clones representing chromosome arm 3AS and 79,872 BAC clones representing chromosome arm 3AL were fingerprinted using the SNaPshot-based high-throughput, high-info-content BAC fingerprinting technique and assembled using FPCv9.3. For 3AS, a total of 45,739 clones were assembled into 404 contigs with an average contig length of 842Kb and 11,938 singletons. The chromosome am 3AL was assembled into 700 contigs having 51,405 clones with an average contig length of 570Kb and 12,064 singletons. The 3AS contig assembly represents 90 percent of the chromosome arm with 15x coverage and the 3AL contig assembly represents 87 percent of the chromosome arm with 12x coverage. A minimum tilling path (MTP) of ~5,100 BAC clones from 3AS and 5,300 from 3AL has been identified for sequencing. Marker development and anchoring: Synteny between wheat group-3 and rice chromosome 1 was exploited and wheat ESTs corresponding to rice chromosome 1 were utilized to design primer pairs from nearly 1400 unique ESTs. Further, BESs were used to develop 598 SSRs, 500 ISBPs and 200 genic markers. To anchor these markers to BAC contigs, 26,112 clones from the 3AS BAC library was pooled into 6 dimensions, 190 BAC pools distributed to the mapping labs. More than 500 EST-STS markers have been mapped to individual BACs and further to BAC contigs of 3AS FPC assembly. To further improve the marker density three dimensional pools were developed from 2,743 BAC clones spaced along the MTP. 2,443 BACs in three dimensions were sequenced using 454 Titanium technology with ~6x coverage. The sequence was assembled into ~194,000 contigs and masked to identify gene coding regions. Nearly, 55 % of the 194,000 contigs and over 70 % gene containing contigs were traced back to individual BAC clones. Nearly 3,000 primers were designed from MTP sequence and are being used for SNP development among parents of mapping population T. monococcum subsp. aegilopoides and subsp. monococcum. A high-throughput genotyping by Golden GateTM assay with 1,536 SNPs and 480 F2 will be performed to generate an anchored physical map of the 3A chromosome. The new website has a Chromosome 3A Genome Browser and WebFPC browser to serve as a browsing tool for information on BAC contigs, and markers and web BLAST for chromosome 3A. Further, a web searchable marker database has also been developed which will house all primers sequences and BAC pools scoring, BAC contig information, mapping populations and comparative mapping with rice and Brachypodium. The information regarding workshops, protocols and the BAC libraries have been uploaded on the web site for free public access to the scientific community.
Publications
- SehgalSK, AknunovE, Li W, Kaur G, Catana V, Pillamari J, Faris J, Reddy L, Devos KM, Rabinowicz PD, Chan A , Maiti R, Dolezel J, Simkova H, Safar J, Luo MC, Ma Y, You F, Gill BS(2011). Towards A Physical And Genetic Framework Map Of Chromosome 3A Of Bread Wheat (Triticum aestivum L.). PAG XIX Abstracts P019.
- Ming-Cheng Luo, Yaqin Ma, Frank M You, Olin D Anderson, David Kopecky, Hana Simkova, Jan Safar, Jaroslav Dolezel, Bikram S Gill, Patrick E McGuire and Jan Dvorak (2010) Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species. BMC Genomics 11:122. doi:10.1186/1471-2164-11-122.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: This proposal is part of joint effort by a team of scientists affiliated with International Wheat Genome Sequencing Consortium (IWGSC) focusing on the development of a physical map of chromosome 3A of wheat. The outputs of the projects are: 1. Development of BAC libraries from telosomic chromosome arms 3AS (25x) and 3AL (15x) isolated by flow cytometry and then assembled FPC map by high information content fingerprinting (HICF) SNaPshot technology. 2. Six-dimensional BAC pools developed for anchoring of expressed sequence tags (ESTs) and simple sequence repeats (SSRs) of individual BACs and then integration of markers data with fingerprint data to develop an anchored FPC map. Further, a three dimensional pools for the minimum tiling path (MTP) would be developed. 3. Sequence data from 10,000 random BAC-ends were used to develop more than 1,000 new simple satellite repeat (SSR), insertion site based polymorphism (ISBP) and genomic markers. Further, 1536, SNPs markers would be developed based of sequence of the MTP of large contigs and a high-throughput genotyping platform would be used to generate the integrated genetic/physical map of the 3A chromosome. The project started last year in Sept. of 2008, the results were disseminated through conferences, workshop and media. This included Plant and Animal Genome XVII (Jan, 2009), and International Triticeae mapping initiative (ITMI, August, 2009). PARTICIPANTS: Participants at Kansas State University : Bikram S. Gill (PI), Wanlong Li (co-PI), Eduard Akhunov (co-PI), Sunish K. Sehgal (project scientist), Gaganpreet Kaur (postdoc), Vasile Catana (Programer); USDA-ARS, Fargo ND: J Faris (co-PI), L Reddy (Grad Student); University of California, Davis: MC Luo (co-PI), YQ Ma (postdoc); Institute of Experimental Botany, Olomuc, Czech Republic: J Dolezel (co-PI). TARGET AUDIENCES: Academic personnel, high school students, producers and consumers. PROJECT MODIFICATIONS: With availability of longer, better and cheaper sequence reads on 454 Titanium sequencing platform we plan to use it for MTP sequencing instead of proposed ABI solid technology.
Impacts
BAC library construction: Two BAC libraries each for chromosome arms 3AS and 3AL with total arm coverage of 25x (110,592 clones) and 15x (79,872) respectively were developed from the flow sorted telosomic chromosome arms. BAC fingerprinting and contigs assembly: We have accomplished the first major task of library fingerprinting. A total of 110,592 BAC clones representing chromosome arm 3AS and 79,872 BAC clones representing chromosome arm 3AL were fingerprinted using the SNaPshot-based high-throughput, high-info-content BAC fingerprinting technique. The first draft assembly from the first 3AS library is ready with 36,000 clones assembled into 1,677 contigs covering the 77 percent of the chromosome arm. The second draft with 94,534 (86%) successfully fingerprinted clones is under construction. The first draft assembly from the 3AL libraries with 67,092 (84%) successfully fingerprinted clones is also under construction. Marker development and anchoring: Synteny between wheat group-3 and rice chromosome 1 was exploited and wheat ESTs corresponding to rice chromosome 1 were utilized to design primer pairs from nearly 1400 unique ESTs. Further, BESs were used to develop 598 SSRs, 500 ISBPs and 200 genic markers. To anchor these markers to BAC contigs, 26,112 clones from the 3AS BAC library was pooled in 6 dimensions, 190 BAC pools distributed to the mapping labs. More than 500 EST-STS markers have been mapped to individual BACs and further to BAC contigs of 3AS FPC assembly. In order to orient the anchored BAC contigs on genetic maps polymorphisms are being screened (for the markers anchored on BAC contigs) between the parental lines T. monococcum subsp. aegilopoides and subsp. monococcum, from which a large F2 population was derived. Polymorphic markers are being mapped. MTP for all the contigs of 3AS and 3AL FPC assembly are being sequenced for identification of genic/unique sequences which will be used to identify SNPs among parents of the mapping population. A high-throughput genotyping by Golden GateTM assay would be used to generate the anchored physical map of the 3A chromosome. Bioinformatics: The project homepage was developed and is constantly being updated http://www.k-state.edu/wgrc/Projects/3%20A%20Project/3%20A_index.html . The website will soon have the Chromosome 3A Genome Browser (under testing) and will serve as a browsing tool for project outputs including BAC contigs, and markers. Furthermore, a web searchable database is under construction which will house all primers sequences and scoring of BAC pools, BAC contigs information and mapping populations and comparative mapping with rice and Brachypodium. The libraries, BAC pools and contigs, BESs, markers, and mapping population are very unique genomics resources for map-based cloning and marker - assisted selection for agriculturally important genes from chromosome 3A. Research with collaborators is undergoing on cloning of many genes or resistance QTLs for scab, leaf blotch, tan spot resistance, and pre-harvest sprouting.
Publications
- Safar J, Simkova H, Kubalakova M, Suchankova P, Cihalikova J, Bartos J, Fiocchetti F, Roselli M, Gill BS, Dolezel J, Lucretti S (2007): Generating resources for genomics of wheat homoeologous chromosome group 3: 3AS- and 3DS-specific BAC libraries. J Genet Breed 61:151-160.
- Sehgal SK, Li W, Rabinowicz P, Luo MC, Choulet F, Paux E, Feuillet C, Akhunov E, Gill B (2009) Sequence based comparison of Mega-base homoeologous regions of A and B genomes of Bread wheat. 19th International Triticeae Mapping Initiative (ITMI) Abstracts P63
- Sehgal SK, Li W, Rao HS, Rabinowicz P, Dolezel J, Luo, M-C, and Gill BS. 2008. Chinese Spring wheat chromosome arm 3AS-specific six-dimensional BAC pools facilitate efficient anchoring of BAC contigs. PAG XVI Abstracts P8, p. 121.
- Akhunova A, Catana V, Sehgal SK, Dolezel J, Simkova H, Kubalakova M, Gill B, Akhunov E (2009) Using next-generation sequencing technology to characterize the gene space of the wheat chromosome 3A.19th International Triticeae Mapping Initiative (ITMI) Abstracts P24
- Sehgal SK, Li WL, Rao HS, Faris JD, Reddy L, Devos KM, Xu X, Wu L, Rabinowicz PD, O'Brien K, Maiti R, Chan AP, Dolezel J, Safar J, Simkova H, Ma YQ, Luo MC, and Gill BS. 2009. Anchoring EST-STS markers to BAC-contigs and deletion bins: the physical map of the 3AS chromosome arm of hexaploid wheat. PAG XVII Abstracts P019
- Gill BS, Li W, Sehgal SK, Faris J, Reddy L, Devos KM, Buell R, Gornicki P, Rabinowicz PD, Dolezel J, Simkova H, Safar J, Ma Y, Chen F, Lucretti S, You FM, and Luo MC. 2008. Progress towards the construction of a sequence-ready physical map of the 3AS chromosome arm of hexaploid wheat. PAG XVI Abstracts P9, p. 121
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