Wheat - Physical mapping of chromosome 5A


Mission statement
Genome sequencing is widely recognized as an efficient way to achieve a more rapid crop genetic improvement. Regarding wheat, due to the huge genome size (17Gb, about five times the human genome), an international, collaborative effort has been started via the foundation of the IWGSC (International Wheat Genome Sequencing Consortium). The Consortium has been developed on a "chromosome by chromosome" based strategy that should ultimately result in the construction of a complete map of hexaploid wheat that is the starting point for the subsequent sequencing step. The Italian project coordinated by CRA - Genomics Research Centre is involved in the development of the physical map of chromosome 5A in the reference cultivar Chinese Spring.

Updates of the results

Physical map

The DNA from purified single 5AS and 5AL chromosome arms has been used to produce two BAC libraries in the lab of Jaroslav Doležel (Institute of Experimental Botany, Czech republic):

  1. 5AS BAC library: 46,080 clones (120 “384 well plates”); mean insert size = 120kb; coverage = 16.5x
  2. 5AL BAC library: 90,240 clones (235 “384 well plates”); mean insert size = 123kb; coverage = 18.3x

44,744 BAC clones for 5AS, and 51,072 BAC clones for 5AL, were fingerprinted using SnaPshot method and the useful fingerprints were assembled into contigs using two different softwares: FPC and LTC, in collaboration with IGA-Institute of Applied Genomics, Udine. With FPC a first minimal tiling path (MTP) consisting of 4,201 for 5AS and 6,560 for 5AL overlapping BAC clones was defined, and organized in three dimensional pools to increase the efficiency of further anchoring. The anchoring of contigs is in progress, the 14.4% of the short arm and the 2.5% long arm has been completed so far. With LTC an improved MTP consisting of 5,412 for 5AS and 8,709 for 5AL overlapping BAC clones was defined, and organized in three dimensional pools.

Genetic map

A consensus genetic map has been developed for 5A employing four mapping populations belonging to different Triticum species:

  1. 383 F2-F3 lines from Chinese Spring (CS) x Renan (T. aestivum x T. aestivum)
  2. 124 RILs from Latino (Triticum turgidum ssp. durum) x MG5323 (Triticum turgidum ssp. dicoccum)
  3. 188 RILs from Chinese Spring x CS-T. dicoccoides Disomic Substitution 5A TDIC (5A CS) (T. aestivum x T. turgidum dicoccoides)
  4. 132 RILs from DV92 x G3116 (T. monococcum x T.monococcum)

Different categories of molecular markers have been used. About 200 unique and 5A specific markers have been mapped including ESTs (expressed sequence tags), COS (conserved ortholog set), TE junction (transposable elements) based markers and SSRs (simple sequence repeat). These latter were obtained either from literature or by screening a 2x coverage of 454 sequences run on flow sorted DNA. The physical position of these markers has been also assigned to the 5A deletion bins.

Sequencing

A massively parallel 454 pyrosequencing has been used to obtain a 2x coverage of wheat chromosome 5A short and long arms. The resulting sequence assembly was used to identify TEs, genes and miRNAs, as well as to infer a virtual gene order based on the synteny with other grass genomes. Repetitive elements accounted for more than 75% of the genome. Gene content was estimated considering non-redundant reads showing at least one match to ESTs or proteins. The results indicated that the coding fraction represents 1.08% and 1.3% of the short and long arm respectively, projecting the genic fration of the whole chromosome to approximately 5,000 genes. miRNA precursors belonging to 16 miRNA families were identified, accounting for a total of 195 candidates. The 5A genes were used to search for syntenic relationships between grass genomes. The short arm is closely related to Brachypodium chromosome 4, sorghum chromosome 8 and rice chromosome 12; the long arm to regions of Brachypodium chromosomes 4 and 1, sorghum chromosomes 1 and 2 and rice chromosomes 9 and 3. From these similarities it was possible to infer the virtual gene order (Genome Zipper) of 392 (5AS) and 1,480 (5AL) genes of chromosome 5A, which was compared to, and found to be largely congruent with the available physical map of this chromosome.

Next steps

  • The screening of the new MTP, realized with LTC, will be performed using Agilent Microarray Technology. A 15K specific array has been produced in collaboration with University of Verona using the 5A Genome Zipper (Vitulo et al., PloS One 2011), all the 5A available ESTs and all the markers already genetically mapped on 5A.
  • A chromosome 5A radiation-hybrid panel is in the pipeline and will be subjected to genotyping using TE-based markers and SSRs derived from the sequencing of 5,000 BAC-ends obtained from BACs of the FPC-MTP.

Publications

International Wheat Genome Sequencing Consortium, 2014. A chromosome based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science, 345 (6194): 1251788.

Gadaleta A, Giancaspro A, Nigro D, Giove SL, Simeone R, Piarulli L, Colasuonno P, Valè G, Cattivelli L, Blanco A, 2014. A high resolution genetic and deletion map of wheat chromosome 5A to detect candidate genes for quantitative traits. Molecular Breeding, 34: 1599–1611.

Vitulo N, Albiero A, Forcato C, Campagna C, Dal Pero F, Bagnaresi P, Colaiacovo M, Faccioli P, Lamontanara A, Simkova H, Kubálákova M, Perrotta G, Facella P, Lopez L, Pietrella M, Gianese G, Dolezel J, Giuliano G, Cattivelli L. Valle G, Stanca AM (2011). First survey of the wheat chromosome 5A composition through a next generation sequencing approach. PLoS ONE, 6(10), e26421.

Barabaschi D, Guerra D, Lacrima K, LainoP, Michelotti V, Urso S, Valè G, Cattivelli L, 2012. Emerging knowledge from genome sequencing of crop species. Molecular Biotechnology, 50: 250–266.

Gadaleta A, Giancaspro A, Giove SL, Zacheo S, Incerti O, Colasuonno P, Nigro D, Valè G, Cattivelli L, Stanca AM, Blanco A, 2012. Development of a deletion and genetic linkage map for the 5A and 5B chromosome of wheat (Triticum aestivum L.). Genome, 55: 417-427.

Project deliverables

  • BAC-based physical map for subsequent 5A genome sequencing;
  • genetically anchored physical map that will expedite map-based cloning of genes and QTL (Quantitative Trait Loci) of interest
  • marker development (in particular SNPs, Single Nucleotide Polymorphism) suitable for fine mapping, positional cloning, QTL identification, and Marker Assisted Selection (MAS);
  • a more comprehensive understanding of the wheat plant biology that can lead to access genomic regions involved in the control of important agronomic traits.

Members

  1. CRA - Genomic Research Centre (Fiorenzuola d'Arda)
  2. CRA - Cereal Research Centre (Foggia)
  3. CRA - Genomic Research Unit (Metaponto)
  4. CRA - Unità di Ricerca per la Valorizzazione Qualitativa dei Cereali (Roma)
  5. Istituto di Genetica Applicata (Udine)
  6. ENEA - Centro di Ricerca della Casaccia
  7. University of Padova
  8. University of Bari
  9. University of Viterbo
  10. University of Reggio Emilia
  11. Metapontum AgroBios
  12. University of Bologna
  13. University of Lecce

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The Genomics Research Centre holds a collection of more than one hundred barley mutants.

Our contacts

Genomics Research Centre
Via S. Protaso, 302
29017 Fiorenzuola d'Arda (PC) - ITALY
Phone:
+39 0523 983758
Fax:
+39 0523 983750
E-mail:
gpg@entecra.it
How to find us

We thank Renzo Alberici, Donata Pagani and Gianni Tacconi for setting up the picture collection.