| Cultivated Capsicum fruits are used as a source of vegetables, spice, colorant and for some medical applications. The genus is native to Central and South America and includes the species C.chinense, C.baccatum, C.frutescens, C.pubescens and C.annuum. Of these five species, C.annuum is the most important one because it is cultivated in both tropical and temperate area in the world and it is the most versatile of the five species0.In contrast, the other four species are cultivated in limited regions in the world or only in tropical areas and they are mainly used as spices. Pepper blight(Phytophthora capsici.L) is a world-class disease and impact on the yield and quality of pepper seriously. The test used the resistant materials PI201234 and susceptible materials G29 and its construction of F2 population as the research object, using the EST-SSR primers previously screened and SSR molecular marker technology to construct a genetic linkage map and then combined with the identification of resistance to phytophthora capsici in F2 population to detect QTLs related to disease resistance. We also conduct the transcriptome sequencing analysis by using the F2 population. Main conclusions are as follows:1. Identification of phytophthora capsici resistance in pepperThe experiment adopted the zoospore filling root method to identify the resistance to phytophthora blight. The pathogen isolated from pepper of huai’an, jiangsu province which named Ph1 belongs to one small kind of phytophthora capsici. After activating on PDA medium and then transfer them to Vg medium,28℃ in dark conditions for 5-7 days, and then given long daylight for 3 days to induce the sporangium. The next step was adding some sterile water to the medium, lay aside for 40 min under 4℃ and then 30 minutes under normal temperature to release zoospores. An F2 population was developed by the intraspecific cross between resistant (PI201234) and susceptible (G29) lines of pepper (Capsicum annuum L.). The F2 progeny were bioassayed with zoospores of phytophthora capsici in root at 6-8 leaf stages. After inoculating 24 h and then giving 28℃,90% relative humidity,4000 lx,12 h/d culture condition to induce the disease. We observed the vaccination results after 15 days and found that disease incidence trends appeared skewed Gaussian distribution, among them 10 plants belong to 0 class,16 plants belong to 1 class, 30 plants belong to 2 class,33 plants to 3 class,31 plants belong to 4 class,28 plants to 5 class.2. Construction of genetic linkage map for pepper disease resistanceThe experiment used the resistant materials PI201234 and susceptible materials G29 and its construction of F2 population as the research object, using the EST-SSR molecular marker to construct the linkage map. First used the parents to identify 664 pairs of SSR primers, a total of 114 pairs polymorphic primers was obtained, then used part of F2 population to validation, finally choose 65 pairs primers which behaved stability, clear for genotyping of F2 population. Application of JoinMap4.0 software to analyze the results, setting the LOD (Logarithm of Odds) score of 2 or more as a threshold, used "Kosambi" mapping function to build a linkage group which contains 10 linkage groups,49 EST-SSR pepper genetic markers. The total length of the linkage map is 418.6 cM, the average genetic distance between the two markers is 8.55 cM.3. QTL mapping of pepper disease resistanceThe test used MQM (multiple QTL model) method of MapQTL4.0 software to detect quantitative trait loci. One QTL on N3 linkage map between EST451 and EST191 markers which can explain 14.9% variation of the phenotypic were identified based on the genetic linkage map combined with the results of the phytophthora blight resistance identification of F2 population.4. Transcriptome sequencing analysis of pepperFirst extracted the total mRNA of pepper, purified and then interrupted the mRNA reverse transcribed into cDNA fragments of different sizes which performed by using Illumina HiSeqTM:2000. Reused the original reads (double end sequence) to assemble Unigene Library of this species and then undertook the work of genetic structure, gene expression analysis, gene function annotation and so on. A total length of 44.6 Gb data sequencing which contains 220,810,807 reads was obtained, CycleQ20 reached 100%. After de novo assembly, we received 77,333 Unigenes, including 16,181 Unigenes which length was more than 1 Kb. Among all the Unigenes,64.90%(50,188) was annotated into the databases of NR, NT, Swiss-Prot, KEGG, COG and GO database. By gene annotation, 30,936 Unigenes were annotated into NR database,45,322 into NT database,18,212 into Swiss-Prot database,5,2484 into KEGG pathway database,8,257 into GOG database and 22,960 into GO database. We also analyzed 22043-2303bp and 22043 sequences about nucleotide and protein by the BLAXT software, the results showed that the two sequences have a large degree of homology with potato resistance genes. All the above annotations enriched the transcript-tome information in pepper. |
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