Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 180

Table 6-1 Overview on the six BAC libraries in sunflower.

Gentzbittel Özdemir Knapp Feng et al. Feng et Bouzidi et

et al. 2002 et al. 2004 (CUGI) 2006 al. 2006 al. 2006

Line HA821 RHA325 HA383 HA89 HA89 YDQ

(= HA335)

Average 80 60 125 140 137 118

insert size
(kb)
Vector

pBelo pBelo pIndigo- PECBAC1 pCLD045 pBelo

BAC11 BAC11 BAC536 41(binary) BAC11

Restriction Hind III Hind III Hind III Bam HI Hind III Hind III

enzyme
Genome 4-5-fold 1.9-2-fold 8.3-fold 5-fold 3.9-fold 5-fold

coverage
Number of

150,000 104,678 202,752 107,136 84,864 147,456

clones

these BAC libraries, Feng et al. (2006) also constructed a BIBAC library.
Using a binary vector allows the immediate application of an identified
BIBAC clone in transformation experiments using Agrobacterium in order to
verify candidate genes by complementation of mutants.
With regard to the relationship of genetic maps with physical maps, 1
cM map distance represents about 2 Mb in sunflower genome assuming an
estimated haploid genome size (1C) of about 3,000 Mb to 3,500 Mb
(Arumuganathan and Earle 1991; Baack et al. 2005) and a map size for the
whole genome of about 1,500 cM (961 cM - 1,920 cM; Micic et al. 2004 and
Hu et. al. 2007, respectively).
In the ideal case, markers very closely linked to the gene of interest
would in a hybridization experiment identify a genomic clone that already
contains the target gene (“chromosome landing”; Tanksley et al. 1995).
However, a more realistic “chromosome walking” approach will lead to
cloning of the gene ( Fig. 6-1 ). Markers from both sides of the target gene
would be used to identify BAC clones through colony hybridization or PCR
screening of BAC pools. Identified BAC clones need to be arranged into
contigs by BAC fingerprinting. BAC end sequences would then be used to
develop probes for continuing hybridizations until a closed contig around
the gene of interest is formed, which is characterized by overlapping clones
coming from both the sides. A relatively large genome size and presence of
retroelements pose a major problem for chromosome walking in sunflower.
So far, map-based cloning has not led to any successful isolation of a gene in
sunflower. However, as mentioned earlier, combination of map-based cloning
and candidate gene approaches could hopefully allow isolation of genes in
the near future.