Developing Markers And Fine-mapping Of Genes Conferring The Resistance To Sphacelotheca Reiliana In Maize

Head smut of maize(Zea mays L.) is a kind of worldwide disease and is also an important constraint in spring maize region of China.Head smut,caused by Sphacelotheca reiliana(K(u|¨)hn) Clint,is a soil-borne or seed-borne systemic disease.The fungus invades plants during emergence or at the seedlings stage through teliospores and grows systemically with the meristem.Infection becomes visible at a late stage of plant development,when tassels and ears are partially or totally replaced by sori filled with teliospores.Quantitive genetics studies have revealed that maize resistance to S.reiliana is under polygenic control and the mode of gene action is predominantly additive or dominant.Further,molecular biology techniques were applied to study inheritance of head smut.Many resistant quantitive trait lici(QTLs) had been detected in the germplasm of China and abroad,and the linkage markers had also been indentified.Moreover,some TUGs(tentative unique genes) had been identified by the methods of SSH and cDNA chip. All the results would be useful for finding resistance genes for S.reiliana,but could not meet for propose of both marker-assisted selection(MAS) and map-based cloning.In the dissertation,we studied head smut of maize on two subjects.One was marker developing,and the other was fine mapping the major resistant gene for S.reiliana.The main methods and results are as follows:1) Basing on inbred lines,a combination of BSA with AFLP method was applied to develop SCAR primers for the detection of resistance to Sporisorium reiliana in maize:The 10 most resistant and the 10 most susceptible inbred lines for head smut of maize were used to form the resistant and the susceptible DNA bulk,respectively.Then the two bulks were analyzed with 101 AFLP primer combinations.About 4646 bands were amplified with an average of 46 bands of each AFLP primer pairs.And 65 polymorphic fragments involving in 55 AFLP primer pairs between the resistant and the susceptible bulk were identified.Further,the 65 polymorphic bands were tested by the 20 inbred lines composed of the two bulks,andχ2 test for independence was done.There were 7 candidate fragments showing association with resistance to S.reiliana,including P54M71-148, P44M62-331,P46M37-136,P46M37-137,P44M46-147,P38M46-137 and P51M38-130. Then they were all extracted,cloned,and sequenced.P46M37-136 and P46M37-137 fragments were homologous with one nucleotide InDel and one single nucleotide transversion.Then all of 6 candidate SCAR primers were designed,including l allele-specific PCR primers(ASP).Each primer was tested by the pre-selective amplification products and genomic DNA of both the two bulks and their composed inbred lines.All the 6 candidate SCAR primers were all developed into SCAR markers.They were S126,S258,AS136/A137,S98,S131and S100.Moreover,the SCAR markers were tested by other 54 inbred lines andχ2 test for independence was done.Only 2 SCAR markers,ASP and S100,were associated with resistance to S.reilian and mapped at chromosome bin1.09 and bin3.08 in maize,respectively.2) Basing on two sets of BC3 progenies,a combination of BSA with AFLP method was applied to develop SCAR primers for the detection of resistance to Sporisorium reiliana in maize:Two sets of BC3 progenies were generated with 2 resistant inbred lines(Mo17 and Qi319) and 1 susceptible inbred line(Huangzao4) of head smut of maize.One was BC3Q, derived from the cross Qi319(donor parent,highly resistant)×Huangzao4(recurrent parent,highly susceptible),the other was BC3M,derived from the cross Mo17(donor parent,highly resistant)×Huangzao4(recurrent parent,highly susceptible).Families of BC3 progenies were evaluated for resistance to S.reiliana under artificial inoculation. Then two sets of resistant and susceptible DNA bulks were prepared respectively,with QR-bulk and QS-bulk coming from BC3Q progeny and MR-bulk and MS-bulk from BC3M progeny.The DNA bulks and corresponding parental lines were analyzed with 31 AFLP primer pairs,respectively.About 1426 bands were amplified with an average of 46 bands of each AFLP primer pairs.For BC3Q,11 polymorphic fragments involving in 8 AFLP primer pairs between QR-bulk,Qi319 and QS-bulk,Huangzao4 were identified.For BC3M,10 polymorphic bands involving in 9 AFLP primer pairs between MR-bulk,Mo17 and MS-bulk,Huangzao4 were identified.Furthermore,these 21 polymorphic bands were tested by the individuals composed of the bulks,andχ2 test for independence was done. There were 6 candidate fragments showing association with resistance to S.reiliana, including P12M48-215,P13M61-152,P39M46-137,P64M47-204,P13M49-185 and P64M47-170.Then they all were extracted,cloned,and sequenced,with 5 candidate bands except P13M49-185 were re-amplified successfully.A search for sequences homologous with the AFLP sequences was conducted in Genbank and the fragment of P64M47-204 showed significant alignments with a part of EU974082.1,a Zea mays clone 439268 mRNA sequence with unknown function.All of 7 candidate SCAR primers were designed basing on the 5 sequeces.Each primer was tested by its corresponding pre-selective amplification products and genomic DNA,both corresponding bulks and their relevant individuals.Thus,3 SCAR markers,S130,S193and S116,were developed.Moreover,the 3 SCAR markers were tested by more resistant and susceptible individuals from the corresponding BC3 progeny and 74 inbred lines,andχ2 test for independence was done respectively.Only 1 SCAR marker S130 was associated with resistance to S.reilian and mapped at chromosome bin 2.09 in maize.3) Mapping of the major QTL conferring resistance to Sporisorium reiliana using SSR and anchored SCAR markers in maize:First,a relatively fine genetic linkage map of chromosome bin 2.09 was generated by adding another 5 SSR markers and 1 SCAR marker S130,with an average distance of 4.78 cM between adjacent markers.With single marker analysis methods,all of 11 markers showed linked to the major QTL resistance to S.reiliana,including bnlg1520,umc1525, p3864185,p3946135,bnlg1893,umc1207,umc2184,umc2077,S130,p5138120 and p3762147.Also,composite interval mapping were employed,and the possible linked markers were umc1207,umc2184,umc2077,S130 and p5138120,which were lying in the linked markers determined by single marker analysis method.Then the QTL linked markers were used to analyze the most resistant and susceptible families of the two sets of BC3 progenies(BC3Q and BC3M in chapter2).Analysis of genotypes between the most resistant and susceptible families of BC3Q progeny allowed delimiting the major QTL into an interval of 12 cM,flanked by the SSR marker umc1525 and umc1207.And according to the genotype analysis for the most resistant and susceptible families of BC3M progeny,we deduced that there was another resistant gene locating at right side of S130 and p5138120 in bin 2.09 except for the one located in the interval(umc1525/umc1207) by comparing the results of QTL mapping and fine mapping resulting from BC3Q progeny.4) Developing STS marker basing on the polymorphic sequences between inbred lines of maize:First,we analyzed all the loci between umc1525 and umc1207 of IBM2 2008 Neighbors Frame2 map,which were in the resistant QTL supporting interval for the head smut of maize.Then two related GSS of RFLP locus mmp195e,AZ916344 and AZ916345, were identified.They were all a part of ZmGsstuc11-12-04.4163.2,which might snythsize proteins having a region of leucine rich repeats(LRRs).Second,two primers,AZ44 and AZ45,were designed according to AZ916344 and AZ916345 fragment basing on ZmGsstuc11-12-04.4163.2 sequence.And the corresponding sequences in Qi319,Mo17 and Huangzao4 were got by PCR method and sequenced and analyzed.The sequences of AZ916344 among Qi319,Mo17 and Huangzao4 were nearly identical,while the corresponding sequences of AZ916345 among them had two In/Del loci.At last,two primers,MH-1and MH-2,were designed according to the two In/Del loci and tested by the genomic DNA of Qi319,Mo17 and Huangzao4,respectively.Polymorphic and specific bands were observed and two STS markers were developed.Further,they were all mapped at chromosome bin1.09 in maize.