Mapping,Cloning And Application Of A Major QTL Against Maize Rough Dwarf Disease

Maize is one of the most important crops in our country,which is essential for food and energy safety.Maize rough dwarf disease(MRDD)is a devastating viral disease that results in considerable yield losses worldwide.Discovery of resistance genes and development of resistant maize hybrids are considered to be the most efficient way to genetically control the MRDD infestation.A total of 50 F9 heterogeneous inbred families(HIFs),derived from two F5 sibling plants of a hybrid CL1165,were evaluated for their resistance to MRDD to identify resistance QTL.Thereafter,a sequential recombinant-derived progeny testing strategy was adopted to narrow down the resistance QTL.Finally,the identification of the candidate resistance gene and its application in maize breeding were also conducted.All data were listed as follows:1,According to phenotypic evaluation conducted over years of 2008,2009 and 2010 in three locations in Shandong province(Taian,Jining,and Feicheng),24 of these 50 HIFs showed consistent responses to MRDD across different years and locations,in which 9 were resistant and 15 were susceptible.2,Based on the genotypic data generated with Maize SNP50 Beadchip(56,110 SNPs),trait-marker association analysis on the 24 HIFs revealed six chromosomal regions on chrs.1,3,4,5,8,and 9,which were putatively associated with MRDD resistance.3,In 2011,two segregating populations were used to validate these six candidate regions,a BC1/BC1F2 population consisting of 211 families derived from NT409/NT411 and a BC1/BC2 population consisting of 385 families derived from NT399/NT401.A major resistance QTL,qMrddl,was identified and mapped into a 15-Mb region on chromosome 8.4,With the recombinant-derived progeny testing strategy,we fine-mapped the qMrdd1 locus into a 1.2-Mb region in 2012 after analysis of the 6,708 BC1F4 plants derived from 101 BC1F3recombinants.The qMrddl locus acted in a recessive manner to reduce the disease-severity index(DSI)by 24.2-39.3%.5,In 2013,the qMrddl locus was further narrowed into a 201,335-bp region by using 2,238 BC1F6 plants derived from 21 BC1F5 recombinants.6,The qMrddl locus was re-discovered in another F6 recombinant inbred line(RIL)population and narrowed down into a 306,554-bp region using recombination RILs.7,Combining mapping results from two approaches enables delimitation of qMrddl into a 201,335-bp region.The candidate gene was identified based on B73 reference sequence.Positive BAC clones covering the the candidate gene were screened from the BAC library of the resistant inbred line 1145 and sequenced in BGI for gene prediction.Analysis of sequences of the candidate gene between 1145 and B73 revealed that a Helitron transposon inserted into the resistant allele.8,The candidate gene has two transcripts as revealed in RACE analysis.These two transcripts have been used to generate both RNAi and over expression constructs.The TO transgenic plants with two overexpression constructs have been obtainned.Four proteins deduced from two transcripts of both the resistant and susceptible inbred lines were all predicted to locate in cytoplasm.Meanwhile,significant difference in protein structure was revealed through homology modeling.9,A diagnostic co-dominant marker was developed based on the Helitron insertion to distinguish the resistance allele from susceptible allele.Helitron transposon insertion was verified to correlate with MRDD resistance in a panel of 53 diverse lines.A collection of 739 inbred lines,334 landraces and 192 teosintes,were used to screen the PAV(presence/absence variation)of the Helitron insertion,in which only 18 inbred lines and 1 landrace have the Helitron insertion.The Helitron sequence and its insertion site were identical among these Helitron-containing lines,indicating the Helitron insertion must occur post-domestication of maize from teosinte.10,The QTL-qMrddl region(9.97Mb flanking by M103-4 and M113-6)was demonstrated to have no impact on agronomic traits,such as plant height,top internode length,days to anthesis(DTA),days to silking(DTS),and days to tasseling(DTT).The resistance qMrddl was introgressed into six elite but susceptible lines to MRDD in China through marker-assisted selection(MAS).