Construction and characterization of a half million clones BAC library of durum wheat (Triticum turgidum ssp. durum)

 Cenci, A.^1,2*, Chantret, N.^1,3,*, Kong, Xy.⁴, Gu, Y.⁴, Anderson, O.D.⁴, Fahima, T.^1,5, Distelfeld, A. ^1,5, Dubcovsky, J.1 

¹ Dept. of Agronomy and Range Science, University of California, One Shields Avenue, Davis CA 95616-8515, USA.

² Current address: Dip. Biologia e Chimica Agroforestale e Ambientale, Università degli Studi di Bari, Via G. Amendola 165/a, 70126 Bari, Italia.

³ Current address: CIRAD-AMIS-Biotrop, av. Agropolis, TA 40/30, 34398 Montpellier Cedex 5, France.

⁴ USDA Western Reg. Research Center, 800 Buchanan St., Albany, CA 94710, USA.

⁵ Current address: The Institute of Evolution, University of Haifa, Mt. Carmel, Haifa. Israel

Theoretical and Applied Genetics, In press

Corresponding author: Jorge Dubcovsky

ABSTRACT

            Durum wheat (Triticum turgidum ssp. durum, 2n=4x=28, genomes AB) is an economically important cereal used as the raw material to make pasta and semolina. In this paper we present the construction and characterization of a BAC library of tetraploid durum wheat cultivar 'Langdon'. This variety was selected because of the availability of substitution lines that facilitate the assignment of BACs to the A and B genome. The selected Langdon line has a 30 cM segment of chromosome 6BS from T. turgidum ssp. dicoccoides carrying a gene for high grain protein content, the target of a positional cloning effort in our laboratory.  A total of 516,096 clones were organized in 1344 384-well plates and blotted into 28 high-density filters. Ninety-eight percent of these clones have wheat inserts (0.3% chloroplast DNA, 1.4% empty clones and 0.3% empty wells). The average insert size of 500 randomly selected BAC clones was 131-kb, resulting in a coverage of 5.1 X genome equivalents for each of the two genomes, and a 99.4% probability of recovering any gene from each of the two genomes of durum wheat. Six known copy number probes were used to validate this theoretical coverage and gave an estimated coverage of 5.8 X genome equivalents. Screening of the library with eleven probes related to grain storage proteins and starch biosynthesis showed that the library contains several clones for each of these genes, confirming the value of the library in characterizing the organization of these important gene families. In addition, characterization of fingerprints from colinear BACs from the A and B genomes showed a large differentiation between the A and B genomes. This library will be a useful tool for evolutionary studies in one of the best-characterized polyploid systems and a source of valuable genes for wheat. Clones and high-density filters can be requested at agronomy.ucdavis.edu/Dubcovsky/BAC-library/ BAC_Langdon.htm.