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A total of 14 additional Indel markers developed in the narrower region flanked by markers M23 and RM135 were subjected to the third round of linkage analysis (Additional file 2: Table S2).
The second round was carried out with additional SSR markers in the target region flanked by the closest markers derived from the first round of linkage analysis, which were developed on the basis of reference sequence of cv.
Additional nine polymorphic SSR markers developed in the region defined by the flanking markers RM16 and RM55 were subjected to the second round of linkage analysis (Additional file 2: Table S2).
The first round of linkage analysis with 750 viable F2 plants revealed that there were 64 and 37 recombinants, respectively, at RM487 and RM16 loci on the centromere side, 35 and 22 distinct recombinants, respectively, at RM168 and RM55 loci on the telomere side, indicating that the four candidate markers were indeed linkage markers with the target Pi gene (Fig. 2a).
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Genomic map of target gene were established through three rounds of linkage analysis using genomic position-ready molecular markers (He et al. 2012).
However, it is worth mentioning for larger genes, successive rounds of linkage can be carried out [ 10].
By three rounds of linkage mapping using the NILs and the NIL-derived population, the Qcrs.cpi-3B locus was mapped to an interval of 0.7 cM covering a physical distance of about 1.5 Mb.
The follow-up ended at the date of the next (second round) linkage of addresses, the date of diagnosis of colorectal cancer, or 31 December 2007 (the latest date with follow-up for cancer incidence), whichever came first.
The first round of mapping produced 28 linkage groups comprising a total map length of 2956 cM.
This was followed by a second round of hierarchical clustering, using single linkage, euclidean distance and a distance threshold of 1% of the data range.
The second round of mapping produced a 2556.6-cM linkage map composed of 25 linkage groups, each consisting of 25 to 171 markers, ranging in length from 31.18 to 142.78 cM, with mean intermarker distances ranging from 0.47 to 3.66 cM.
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