Studies On Resistance Gene Analogs Conferring Resistance To Anthracnose In Walnut (Juglans Regia L.)

Anthracnose caused by C. gloeosporioides is a common disease in walnut (Juglansregia L.) cultivation of China, and it seriously hampered the development of walnut industry.There is a need to study the walnut anthracnose and to select resistant germplasm duringwalnut breeding. In this study, three different types of resistance gene analogs (containingNBS, LRR and STK) were isolated from anthracnose resistant cultivar ‘QingLin’ and their F1hybrids of ‘Yuan Lin’בQing Lin’, using a PCR strategy. The degenerate primers weredesigned to the conserved domains of plant R genes. It provide important theoretic databasefor the cloning of resistance genes conferring resistance to anthracnose from walnut, and itwould facilitate walnut molecular-assisted breeding. The main results are as follows:1. Eight degenerate primer pairs, designed based on the NBS, LRR and STK domains ofknown R genes, were used to isolate RGAs from walnut, the results showed that onlythree pairs of could give specific bands, namely P-loop/GLPL, NLRR inv1/inv2and Ptokin1/kin2. The PCR products were purified and ligated into pMD18-T and then weretransformed into E. coli DH5α competent cells. The recombinant clones were screenedovernight on LB plates supplemented with ampicillin, x-gal andisopropyl-β-D-1-thiogalactopyranoside. Total of127sequences were obtained in thisstudy, consisting of35NBS-sequences,47LRR-sequences and45STK-sequences.2. NBS type RGA markers: The PCR reaction with primer pair P-loop/GLPL amplified aspecific band in size about500bp from walnut genomic DNA, they were designated asjrRGAPGs and deposited in GenBank under accession numbers JX646704to JX646738.BLASTn search revealed the nucleotide sequences of these jrRGAPGs were highlyhomologeous to known NBS-type RGAs or plant disease resistance (R) genes in GenBank,with similarity of67%-88%; while BLASTx indicated46%to73%similarity betweendeduced amino acid sequences of these jrRGAPGs and those reported NBS-type Rproteins from other species. Out of35jrRGAPGs,32sequences could be translated intopolypeptides without stop codons. Multiple alignments revealed32jrRGAPGs sequencescontained the characteristic motifs (P-loop, kinase-2a, kinase-3a and GLPL) which werefound in known R genes. They were subdivided into10clusters based on the nucleotidesimilarities. Phylogenetic analysis of their deduced amino acids based on neighbor-joining(NJ) method classified these32jrRGAPGs into two distinct sub-classes (TIR and non-TIR), which further confirmed the conclusion that it contained two categories Rgenes in dicots. These jrRGAPGs were clustered together into ten sorts when coefficientgot0.1by UPGMA tree. Similarity percentages of deduced amino acid among these10subgroups ranged from23.1%to75%with identifies to known R genes ranging21.6%-51.9%. Nucleotide polymorphism and diversity (Pi) of jrRGAPGs were highlyconserved at each motif than at other partial sequences, indicating their conservativestructures of NBS genes. The ratio of non-synonymous to synonymous nucleotidesubstitution (dN/dS) in the NBS domain from most of walnut RGAs varied from0to0.933(lower than one) for different classes which suggested a purifying selection, exceptthe dN/dS from RGAⅧ got1.552(bigger than one) which suggested a positive selection.It had complex protein secondary and3D structure, in which the proportion of Heliceswas53.55%, that of Coils was43.17%but that of Strands was just3.28%.3. LRR type RGA markers: The PCR reaction with primer pair NLRR inv1/inv2amplifiedspecific bands at sizes of500bp and2000bp. We just cloned the band at500bp, and47LRR sequences were obtained finally, they were named jrRGANLs, GenBank accessionnumbers KF151270-KF151316. Unfortunately, BLASTn together with BLASTx revealedthere were no homology sequences in GenBank. Multiple alignments revealed these LRRsequences were highly polymorphic. Phylogenetic analysis divided these LRRs intomultiple branchs; while amino acid grouped them into two sub-classes with three mainbranches, each branch contained different numbers of LRR sequences.4. STK type RGA markers: The PCR reaction with primer pair Pto kin1/kin2amplified aspecific band at size of750bp. They were all purified and cloned.45STK sequences wereobtained, they were designated as jrRGAPTs, GenBank accession numbersKF151225-KF151225. BLASTn revealed these STKs shared high similarities with proteinkinase receptor or regulator, with similarity of89%-93%. But BLASTx revealed there noconserved domain in GenBank. Multiple alignments analysis revealed there were eightconserved regions in these STKs, while open reading frames analysis indicated there weremore than one stop codons inner the sequences. They were likely to be “pseudogenes”.5. Position: The resistance gene analogs obtained in the study were linked closely with Rloci of walnut. The genetic distances among NBS, LRR and STK markers with R lociwere16.22cM,24.49cM and3.06cM, respectively.