| With a F1 segregation population obtained from the cross between Xu 78-1 highly resistant to sweetpotato stem nematode (SSN) and Xushu 18 highly susceptible to SSN, molecular markers linked to SSN resistance genes were developed by using BSA (bulk segregant analysis) and SRAP (sequence-related amplification polymorphism). The results suggested that the inheritance of the resistance to SSN was controlled by a single gene.The screening of 200 SRAP primer pairs against resistant and susceptible bulks resulted in the identification of 77 pairs of primers which amplified polymorphic bands between resistant and susceptible pools. Of 77 pairs of primers, 62 were tested with F1 population and 4 markers linked to SSN resistance genes were identified. Three markers, named as Nsp1, Nsp2 and Nsp3, respectively, were obtained with the primer pair A9B4, whose genetic distance to SSN resistance genes were 4.71cM, 4.71cM and 6.32cM, respectively. One marker, named as Nsp4, was obtained with the primer pair A3B6, whose genetic distance to SSN resistance gene was 8.73 cM.A mutant, Nongda 603, resistant to SSN was obtained from the gamma-irradiated progenies of sweetpotato cv. Xushu 18 susceptible to SSN. Degenerate primers were designed from the conserved NBS (nucleotide-binding site) motifs of plant nematode resistance genes isolated from other crops. RGAP (resistance gene analog polymorphisms) and MSSAP (modified sequence-specific amplification polymorphisms) were used in the PCR of amplifying the genomic DNA of Nongda 603 and Xushu 18. Four fragments (Fragments 58, 54, 72 and 77) with NBS conserved domain of plant resistance genes were amplified from Nongda 603, not from Xushu 18. DNA and peptide-sequence similarity and homology between the sequence of fragments cloned and the resistance genes reported in other crops were determined by blast analyses through GenBank. The results indicated that these four sequences had low homology to plant resistance genes reported. Primers designed from the sequences of the four fragments cloned were used in PCR amplification with the templates of the genomic DNA of resistant and susceptible sweetpotato cultivars. Polymorphic bands were obtained by the PCR with the primers for Fragment 54, while no polymorphic band was amplified by the primers for Fragment 58, Fragment 72 and Fragment 77. Thus, it is thought that Fragment 54 might be a RGA relative to SSN resistance.The mRNAs of storage roots of Nongda 603 and Xushu 18 were used as materials. By using a degenerate primer P2 designed from the NBS conserved amino-acid sequence of plant nematode resistance genes and a specific primer P54 designed from the sequence of Fragment 54, RT-PCR analysis indicated that a cDNA fragment (483bp) was amplified and the mRNA amount of the gene was higher in Nongda 603 than in Xushu 18. The BLAST analysis in GenBank showed that the sequence of the fragment cloned had high homology with MIPS genes of other plants. The 3' cDNA of the aimed gene was amplified using 3' RACE and the 5' cDNA of the aimed gene was amplified by PCR using the specific primers of 3' cDNA and the degenerate primer designed based on the conserved amino-acidsequence of the 5' end of plant MIPS genes. The full-length cDNA of the cloned gene was 1856bp.The DNA and amino-acid sequence alignment showed that the gene cloned had high homology with MIPS genes of other plants. The identities of DNA sequences between the gene cloned and MIPS genes of soybean/tomato were 83.63 % and 83.89 %, respectively; the identities of amino-acid between the gene cloned and MIPS genes of soybean/tomato were 92.16 % and 90.59 %, respectively. So it was suggested that the cloned gene was MIPS gene of sweetpotato and the gene was related to stem nematode resistance. The cloning of sweetpotato MIPS gene is useful for the further research of the relationship between sweetpotato MIPS gene and sweetpotato stem nematode resistance.
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