Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library from the Japanese Malting Barley variety 'Haruna Nijo'

Abstract

Cultivated barley (Hordeum vulgare L.) is well known as one of the most widely cultivated crops in the world and as an extensively studied plant species in the field of genetics. In recent years, despite its very large genome size (ca. 5,000 Mb), the research resources needed for barley genomic studies have become available, including a large number of expressed sequence tags (ESTs). These have been widely used for barley genome analyses, such as DNA marker-generation and the construction of microarrays. However, the availability of a large-insert genomic library, which is also essential for genomic studies, has been relatively limited in the barley research community. We described here the construction and characterization of a barley bacterial artificial chromosome (BAC) library, using the Japanese malting barley variety 'Haruna Nijo'. The BAC library consisted of 294,912 clones arrayed in 768 384-well microtiter plates. The average size of each cloned insert was estimated to be 115.2 kb, with approximately 0.5% of the clones lacking inserts. Chroloplast DNAs were present in about 1.7% of the library. Thus, the genomic coverage of the 'Haruna Nijo' BAC library was estimated to be about 6.6 genome-equivalents. In order to rapidly identify specific BAC clones, we developed a screening strategy that combined PCR analysis of pooled BAC DNAs with colony hybridization. Using this screening scheme, we investigated the genomic coverage of this BAC library, using 13 locus-specific ESTs and a sequence-tagged site marker. By screening the whole library with individual markers, we identified an average of 5.1 clones per marker. This screening scheme also enabled us to rapidly construct a physical contig spanning a region of approx. 190 kb around the HvBRI1 locus, where the mutation responsible for the semi-dwarf plant type 'uzu' is located. These results indicate that the 'Haruna Nijo' BAC library will be useful for barley genomic studies.

Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway

“…AK061163 and AK121264) within the collinear region were successfully used as vehicles to develop closer barley markers (ABRS3 and ABRS9). Starting with markers sKT9 and ABRS3, we screened the bacterial artificial chromosome (BAC) library of the covered barley cultivar Haruna Nijo (19). Seven rounds of chromosome walks selected 20 BAC clones [supporting information (SI) Table 1], and a 500 kb-contig spanning the nud locus was constructed (see SI Table 2 for markers used for BAC contig assembly).…”

“…BAC clones near the nud locus were selected from the Haruna Nijo BAC library (19). Chromosome walking was repeated until a recombinant on the proximal side was identified (see SI Methods and SI Table 1).…”

Phosphate Utilization Efficiency Correlates with Expression of Low-Affinity Phosphate Transporters and Noncoding RNA, IPS1, in Barley

“…We isolated two additional HvPHT1;1 paralogs from a Haruna Nijo bacterial artificial chromosome (BAC) library (Saisho et al, 2007) using a 388-bp probe derived from the coding sequence of HvPHT1;1. The first gene encoding a protein was identical to HvPHT1;1 (Fig.…”

“…AY188394; 945-1,332 bp) was used for screening a Haruna Nijo BAC library (Saisho et al, 2007). Two unique BAC clones were isolated, one containing HvPHT1;10 and the other containing HvPHT1;1, HvPHT1;9, and HvPHT1;2.…”

A high-density transcript linkage map of barley derived from a single population

“…Hori et al (2005) developed and used recombinant chromosome substitution lines (RCSLs) derived from these two accessions. Haruna Nijo is the recurrent parent in each of the 10 new RCSL populations developed at Okayama University (Sato, unpublished Single population derived barley transcript map K Sato et al constructed from both Haruna Nijo (Saisho et al, 2007) and H602 (Sato, unpublished). Haruna Nijo is the basis of a new TILLING system comprised of 10 000 individuals (Sato, unpublished (Close et al, 2004).…”