| Potato(Solanum tuberosum L.)is the third most important food crop in the world after rice and wheat in terms of human consumption.It is also a crucial agricultural economic crop for domestic food security and healthy diet in China as the advancing of “Potato Staple Food Development” by the Ministry of Agriculture.Phytophthora infestans((Mont.)de Bary),the causal agent of late blight disease,is the most devastating pathogen of potato,the excessive use of fungicides increases the costs and poses detrimental risks to potato production and to the environment,more importantly,it would not prevent and stop the disease.Thus,potato varieties with durable resistance to late blight are needed urgently for the potato breeding program.However,comprehensive understanding of molecular mechanisms of the durability of resistance is a challenge,so that little progress has been made on potato breeding for late blight resistance in over a hundred years.Following the often short-lived protection that major nucleotide binding,leucine-rich-repeat resistance genes offer against the potato pathogen P.infestans,field resistance was thought to provide a more durable alternative to prevent late blight disease.We previously identified the QTL d PI09 c on potato Chromosome 9 as a more durable field resistance source against late blight in potato dihaploid population B3C1HP100 by collaborating with the International Potato Center.In this study,we reported the molecular characterization of the d PI09 c locus through map-based cloning,d Ren Seq,allele mining.Comparative genomics revealed the putative evolution mechanism of this locus.The main results are listed as follows:1.P.infestans isolates screening.Detached leaf assays were conducted on ramdonly selected individuals of B3C1HP100 with six different virulence P.infestans isolates which originated from different places.The resistant individuals showed complete resistance to the 3 moderate isolates 88069,HB16-1 and Ljx18,while the susceptible ones developed severe disease symptons;when inoculated with the 3 aggressive isolates UK3928 a,HB14-2 and EC-1,the resistant individual showed varied resistance degree,the susceptible ones were also susceptible to these isolates.These results further confirmed the broad spectrum of population B3C1HP100 against late blight disease,isolate 88069 was selected for further study.2.Developing and screening polymorphic maker in d PI09 c.Based on the initial mapping results,a total number of 651 markers(304 gene markers developed by former colleague according to the end of Chromosome 9,and 347 markers newly developed based on the sequence from marker At3g24160f2(58.72M)to the end of Chromosome 9(61.54M))were used to screen the polymorphism between the resistance female plant and bulk consisted by progenies,susceptible male plant and bulk,and resulting in 44 polymorphic primers to integrate on the d PI09 c interval.3.Fine mapping of QTL d PI09 c.A total number of 206 individuals from initial population B3C1HP100 and B3C1HP106/4000 newly developed by this study were genotyped,and combined with the phenotypes,resulting in 3 recombinants and narrowed d PI09 c in a 389 kb genomic region in the potato reference genome of DM1-3.4.BAC library constructing and screeeing.One of the resistant progeny of B3C1HP100,304413.40,was used to construct a BAC library which consisted 10 times of the genome coverage.Three flanking markers 3233-1,8384-1 and 8586-1,and four co-segregating markers jr38,5455-1,jr69 and jr78-2 were used for BAC library screening.Seven positive BAC clones were isolated,and two clones covering the whole d PI09 c region were sequenced,resulted in a length of 186 kb of physical distance,compared with the 389 kb in the reference genome,a distance of 200 kb was reduced.There were 10 puptative tomato tospovirus resistance gene Sw-5 in this region based on sequence prediction.Thus,this result laid foundation of cloning the candidate gene responsible for the resistance of d PI09 c.5.The gene composition analysis of d PI09 c interval.According to the BAC sequence predicetion result,a NB-LRR gene cluster was located in the d PI09 c region.In this study,two resistance and two susceptible pool were constructed,two resistant pools included the resistant female 301071.3 and 27 resistant progenies of B3C1HP100,respectively;the susceptible pools contained susceptible male 703308 and 27 susceptible progenies of B3C1HP100,respectively.The newly developed d Ren Seq analysis was conducted on these pools,the results showed full sequence representation of R8,while the susceptible ones did not.These results suggested that R8 could possibly confer the resistance of d PI09 c.6.R8 allele mining and functional analysis.R8 was validated in QTL d PI09 c using long-range PCR allele mining in both resistant female plant 301071.3 and 2 resistant progenies 304413.40 and 304413.74.By using the same approach,R8 gene was found in B3C1 population and diverse Chinese late blight resistant materials,this suggested that R8 played an important role in diverse potato genotypes from different area in conferring resistance to late blight disease.In addition,a functional R8 homologue R8-like was identified in potato wild species S.demissum,functional anlysis indicated that it is resistant to P.infestans,and could trigger HR in the recognition of Avr8.The intial mapping population and many modern cultivars have the resistance background from S.demissum,thus,R8 is speculated to come from wild species S.demissum.7.Genomic comparison analysis of d PI09 c.A sequence alignment of five Solanaceous [DM1-3 516 R44(S.phureja),Ma R8(1/2 S.demissum),304413.40(B3C1HP100),Heinz1706(S.lycopersicum)and S.pennellii] in the d PI09 c interval was conducted.The results demonstrated that severe rearrangements between all the haplotypes from different species in this interval,but,R8 protein and its homologues were conserved according to sequence similarity,suggesting that R8 analogues probably originated from the same ancestor and underwent distinct evolution in different species in d PI09 c interval.8.Preliminary study on Avr8.Avr8 was cloned from 15 P.infestans isolates,the results showed that it was widely existed in this pathogen and very conserved in sequence.A deletion experiment showed that C terminal of Avr8 was essential for the recognition with R8.Subcellular localization showed the potential functional site of Avr8 was plasma membrane and nuclear.These results provided a helpful clue in the research on the durability and broad-specturm of R8. |
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