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| Development of InDels Markers and Their Usage in Detection of Residual Heterozygous Lines in Chinese Cabbage (Brassica rapa ssp. pekinensis) |
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Abstract The completion of Chinese cabbage whole genome and the reduced cost of re-sequencing provide possibility for high-throughput development of InDel markers. In the present study, two Chinese cabbage inbred lines He102 and 06-247 with significant differences in phenotype such as bolting tolerance, virus resistance and tip-burn tolerance were re-sequenced for genome-wide InDels identification. The sequencing depth of these two lines was 10×and 8×respectively. A total of 330 218 polymorphic InDels loci were predicted based on the sequence data. The frequency of InDels occurrence was 1.2/kb, among which 11 238 loci were located in deduced coding region and represented for 5184 genes. We randomly selected 933 predicted polymorphic InDels loci that distributed on the 10 chromosomes of He102 and 06-247 respectively for PCR validation. Results showed that the positive rate varied greatly with sequencing depth. When the choosed loci from a higher sequencing depth material He102, the false positive rate was higher either, and vise versa. In the present study, the average false positive rate was 51.9% and 22.5% for the selected loci from He102 and 06-247, respectively. In other words, higher false positive rate based on higher-depth material reflected that the false positive loci were missed in lower-depth material; contrarily, lower false positive rate based on lower-depth material reflected that the positive loci were detected in higher-depth material. A total of 593 verified loci including 375 loci located in coding sequence (CDS), were identified. More than 90% of the polymorphism loci were co-dominant while about 5% of them were dominant. The rest couldn't produce clear band or multiple bands were generated. The detected polymorphic loci were not evenly distributed on ten chromosomes. Chromosome A01 contained the most number (86) of loci, while A08 contains only 29 loci. The co-dominant polymorphic loci were used to detect the residual heterozygous line of F7 which was generated by single seed descend from F2 of He102×06-247. The results showed that in F7 population, more than 90% of the alleles were homogenous while less than 10% of the them were remain heterogenous. The hemogenous/heterogenous state and the physical position of each locus in all 217 lines of F7 were confirmed. The residual heterozygous lines and the identified polymorphic markers provided useful information for elaborate decompose and fine mapping of QTLs/genes related to bolting, virus resistance and tip-burn tolerance in Chinese cabbage.
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Received: 29 September 2015
Published: 24 February 2016
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| Fund:the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences |
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