Identification of a xanthomonas euvesicatoria resistance gene from pepper (capsicum annuum) and method for generating plants with resistance
a technology of xanthomonas euvesicatoria and resistance gene, which is applied in the field of identification of a xanthomonas euvesicatoria resistance gene from pepper (capsicum annuum) and generating plants with resistance, can solve the problems of reducing yield or yield loss, affecting the growth of plants, etc., to achieve the effect of increasing the resistance of xanthomonas
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example 1
Genetic Crosses and Analysis of the F2 Progeny of the Xcv Plant
[0146]For the generation of F1 individuals, commercially available C. annuum var. Feherozon sensitive to Xanthomonas euvesicatoria (Xe) was used as the father parent (marked as F0), and Capsicum annuum var. T1 / 1 carrying Xe resistance—an individual of Gene Bank Accession No. PI163192—was used as the mother parent (T1 / 1). After the crossing, 45 F1 seeds from the mother plant were sown and F2 plants were grown from them. When plants reached the 8-leaved age, a Xanthomonas euvesicatoria infection test was used to determine the sensitivity of the plants to Xe. Finally, 20 F2 individuals—8 resistant and 12 sensitive individuals (see Table 1)—were selected for the general mapping experiments; on the other hand, more than 3000 F2 individuals were used for the fine-mapping of the Xe resistance gene (xcv-1).
TABLE 1Phenotypes of 20 F2 individuals of the segregating populationafter infection by Xanthomonaseuvesicatoria (xcv phenoty...
example 2
Identification of Markers Linked to the Xcv-1 Gene of the T1 / 1 Mutant Plant
[0147]Identification by genetic mapping of molecular markers mapping close to the mutated xcv gene, i.e., those linked to Xcv resistance, was carried out using the 20 F2 progeny mentioned in Example 1. Total DNA from fresh leaves was subjected to PCR amplification using specific primers designed on the basis of pepper sequences available in the databases, and the resulting fragments were subjected to electrophoresis on agarose gels or on so-called SSCP acrylamide gels. In order to visualise the DNA fragments, agarose gels and acrylamide gels were stained using ethidium bromide and silver, respectively. The linkage of markers showing polymorphism on the agarose or SSCP gels was determined by colour mapping (Kiss et al., Acta Biologica Hungarica 49:47-64, 1998) with respect to the xcv / Xcv phenotype after ascertaining the homozygote or heterozygote status. As a result of the systematic mapping, a single marker d...
example 3
Isolation of BAC Clones Overlapping with the Xcv-1 Mutation, Contig Building
[0150]A primary BAC clone (No. 279) was isolated using the molecular marker showing the strongest linkage to the xcv-1 mutation, i.e., CaCY. The primary and other BAC clones are isolated using multiplex PCR from a BAC library comprising 380,000 BACs, which was prepared from an Xcv resistant pepper (Capsicum annuum) plant and ensures a 22-fold coverage of the pepper genome (Bukovinszki et al., VII. Hungarian Congress on Genetics, Abstract Book, p. 91, 2007).
[0151]Both ends of BAC Clone No. 279 were sequenced and additional two BAC clones were identified using primer pairs specific to these sequences: BAC Clone No. 1248 using primers Pr_279 op F1 (SEQ ID NO:3) and Pr_279 op R1 (SEQ ID NO:4), and BAC Clone No. 632 using primer pair Pr_279-40 F1 (SEQ ID NO:5) plus Pr_279-40 R1 (SEQ ID NO:6).
[0152]Primer pairs specific to the terminal sequences of BAC No. 1248 were used to identify BAC Clone No. 1191: the pair co...
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