Toward the positional cloning of qBlsr5a, a QTL underlying resistance to bacterial leaf streak, using overlapping sub-CSSLs in rice.

Bacterial leaf steak (BLS) is one of the most destructive diseases in rice. Studies have shown that BLS resistance in rice is quantitatively inherited, controlled by multiple quantitative trait loci (QTLs). A QTL with relatively large effect, qBlsr5a, was previously mapped in a region of ∼ 380 kb on chromosome 5. To fine map qBlsr5a further, a set of overlapping sub-chromosome segment substitution lines (sub-CSSLs) were developed from a large secondary F2 population (containing more than 7000 plants), in which only the chromosomal region harboring qBlsr5a was segregated. By genotyping the sub-CSSLs with molecular markers covering the target region and phenotyping the sub-CSSLs with artificial inoculation, qBlsr5a was delimited to a 30.0-kb interval, in which only three genes were predicted. qRT-PCR analysis indicated that the three putative genes did not show significant response to the infection of BLS pathogen in both resistant and susceptible parental lines. However, two nucleotide substitutions were found in the coding sequence of gene LOC_Os05g01710, which encodes the gamma chain of transcription initiation factor IIA (TFIIAγ). The nucleotide substitutions resulted in a change of the 39th amino acid from valine (in the susceptible parent) to glutamic acid (in the resistant parent). Interestingly, the resistant parent allele of LOC_Os05g01710 is identical to xa5, a major gene resistant to bacterial leaf blight (another bacterial disease of rice). These results suggest that LOC_Os05g01710 is very possibly the candidate gene of qBlsr5a

Physical map of the target region and graphical genotypes and resistance phenotypes of the sub-CSSLs.

<p>The open bars, solid bars and gray bars represent H359, H359-BLSR5A and recombined segments, respectively. The vertical solid/dotted lines indicate molecular marker positions. The lowercase and uppercase letters after the lesion length indicate statistical difference at 0.05 and 0.01 significance levels, respectively. R/S: resistant/susceptible to BLS.</p

Comparison of the coding sequences of gene LOC_Os05g01710 from the two parents.

<p>Asterisks indicate the substituted nucleotides.</p

InDel markers developed in this study.

<p>InDel markers developed in this study.</p

Primers used for coding sequence amplification and qRT-PCR analysis.

<p>Note: For each putative gene, primers for coding sequence amplification are shown in the upper row; primers for qRT-PCR are shown in the lower row. The primers for Actin gene were used for qRT-PCR.</p

Expression response of LOC_Os05g01700 (A), LOC_Os05g01710 (B) and LOC_Os05g01730 (C) to BLS pathogen in the two parents.

<p>The expression is thought to be significantly up−/down-regulated when the relative expression intensity, defined as log₂[(expression intensity in treatment)/(expression intensity in control)], is greater/smaller than 1/−1.</p