BACKGROUND
maize rough dwarf disease  is a viral disease that results in substantial yield losses in europe, east asia, and south america  <cit> .
mrdd was discovered in  <dig> in china  and has posed a grave threat to maize production during the last two decades, especially in the yellow-huai-hai river plain  <cit> .
between  <dig> and  <dig>  mrdd has affected over three million hm <dig> of crops each year.
yield losses are generally over 30% in affected areas and can reach 100% in regions of severe infection  <cit> .
the virus that causes mrdd belongs to the genus fijivirus in the family reoviridae, but virus strains vary between continents.
mrdd is caused by maize rough dwarf virus in europe, mal de río cuarto virus in south america, and rice black-streaked dwarf virus in east asia  <cit> .
these viruses are transmitted in a persistent manner by planthopper insect vectors  <cit> .
mrdd symptoms include stunting, dark-green leaves, waxy enations on abaxial surfaces of leaves and sheaths, malformed tassels and upper leaves, suppressed flowering, and a lack of ears .
current methods for controlling mrdd include pesticides, shifting the date when seeds are planted , and improving field management  <cit> .
these methods limit the planthopper population and reduce, to some extent, mrdd severity, but always with high risk and low efficiency.
the identification of mrdd-resistant strains, however, likely represents the most cost-effective and environmentally friendly way to minimize yield losses.
it is therefore important to develop and deploy resistant hybrids by mapping and cloning genes and quantitative trait loci  that confer resistance to mrdd.
under natural-infection conditions, the maize germplasm displays variable resistance to mrdd  <cit> .
the major source of resistance is derived from us hybrid p <dig>  evaluation of  <dig> inbred lines and  <dig> hybrids suggests that mrdd resistance is a quantitative trait  <cit> .
wang et al.
reported that maize resistance to mrdd is a quantitative trait controlled by many genes, each with a small effect  <cit> .
in argentina, a partially resistant line yielded moderate heritability of resistance to the mal de río cuarto virus, ranging from  <dig>  to  <dig>   <cit> .
using an f2: <dig> qtl-mapping strategy, two qtls were identified on bins  <dig>  and  <dig> / <dig> that together explained  <dig> % of the phenotypic variance  <cit> .
a major qtl on chromosome  <dig> for mrdd resistance was identified in the chinese maize inbred line, x <dig>  based on  <dig> gene-derived single nucleotide polymorphisms   <cit> .
using an f <dig> population derived from the highly resistant line  <dig> and the susceptible line ye <dig>  luan  found at least three qtls within chromosome bins  <dig> ,  <dig> , and  <dig>  that confer mrdd resistance  <cit> .
in this study, we applied trait-marker association to  <dig> heterogeneous inbred families  and qtl analysis to segregating population derived from hifs to identify regions of the maize genome that affect resistance to mrdd.
we then fine-mapped the major qtl by subjecting self-pollinated backcrossed families to recombinant-derived progeny testing.
finally, an f <dig> recombinant inbred line  population was used to validate the effect of this qtl.
these results provide valuable information concerning maize resistance to mrdd, and markers developed within the qmrdd <dig> region may prove useful in resistance breeding programs.
RESULTS
evaluation of hifs in resistance to mrdd
the  <dig> hifs developed from a maize hybrid cl <dig> were evaluated for their resistance to mrdd in taian for three years  and another two locations  in  <dig>  of them,  <dig> displayed consistent resistance regardless of the year or location; while  <dig> showed high susceptibility in every location across three years .
these  <dig> hifs were therefore used for subsequent trait-marker analysis .
trait-marker association analysis in hifs
genotyping  <dig>  snps on the  <dig> hifs generated  <dig>  successful calls based on a cluster-separation score of ≥ <dig>  and <50% missing data.
of these  <dig>  snps,  <dig>  were polymorphic with a minor allelic frequency >  <dig>  among the  <dig> hifs.
importantly,  <dig> snps co-segregated with mrdd resistance.
after projecting onto the maize accessioned golden path ,  <dig> co-segregating snps were found to cluster within six chromosomal regions .
the remaining two snps likely resulted from random error and were excluded from further analysis.
the six genomic regions represent candidate mrdd-resistant loci.
location corresponds to maize b <dig> reference genome apg v <dig>
qtl analysis of maize resistance to mrdd
based on both genotypes and phenotypes, four hifs, nt <dig> , nt <dig> , nt <dig> , and nt <dig> , were selected to prepare segregating populations.
the f <dig> hybrid between nt <dig> and nt <dig> was backcrossed twice to the susceptible nt <dig> to generate bc2f <dig> populations.
the f <dig> hybrid between nt <dig> and nt <dig> was backcrossed to the susceptible nt <dig> and then self-pollinated to generate bc1f <dig> populations .
the totally  <dig> bc2f <dig> and  <dig> bc1f <dig> families were then evaluated for mrdd susceptibility at four experimental stations: taian, feicheng, heze, and jining.
the disease-severity index  values were estimated for bc <dig> individuals based on their bc2f <dig> or bc1f <dig> families.
the resultant dsi values displayed continuous distributions, ranging from  <dig> –100% in taian ,  <dig> –100% in feicheng ,  <dig> –100% in taian , and  <dig> –100% in jining , respectively , implying the quantitative nature of maize resistance to mrdd.
intriguingly, dsi distributions skewed toward the susceptible parent, suggesting that a major dominant resistance qtl was absent.
in taian, for example, bc2f <dig> individuals were extremely susceptible to mrdd, and bc1f <dig> individuals exhibited a segregation bias towards susceptibility .
in addition, location had a large effect on dsi.
a population planted in jining generally had a higher dsi than the same bc2f <dig> family in taian.
there was no mrdd outbreak in heze, so nearly every family appeared mrdd resistant .
data from this location were therefore excluded from further analysis.
as such, environmental conditions could fluctuate wildly in mrdd resistance .
within the six candidate regions,  <dig> simple-sequence repeat  primer pairs were retrieved from the public database, and additional  <dig> primer pairs were developed.
these  <dig> ssr markers were used to screen for polymorphisms between the parental lines, resulting in the identification of  <dig> polymorphic ssr markers .
each bc <dig> individual was then genotyped using these  <dig> ssr markers to distinguish heterozygous from homozygous genotypes at the six candidate regions.
the phenotype of a given bc <dig> individual was represented by dsi values calculated from its bc1f <dig> or bc2f <dig> progeny.
mean dsi value was calculated individually for either heterozygous or homozygous bc <dig> genotypes at each of the six candidate regions.
based on dsi values from bc1f <dig> progeny at taian and jining, three ssr markers within bins  <dig> / <dig>  bnlg <dig>  umc <dig>  and umc <dig>  were significantly associated with mrdd resistance, as difference in dsi was significant between heterozygous and homozygous bc <dig> genotypes .
another two ssr markers within bin  <dig> , c5- <dig> and c5- <dig>  were marginally associated with mrdd resistance in taian  .
for bins  <dig> / <dig>  the mean difference in dsi between homozygous and heterozygous bc <dig> genotypes differed by  <dig> % and  <dig> % in taian and jining, respectively.
at the other five chromosomal regions, however, differences in dsi between these two bc <dig> genotypes were less than  <dig> %.
based on dsi values from bc2f <dig> progeny at taian and feicheng, three ssr markers on bins  <dig> / <dig> and two ssr markers on bin  <dig>  were marginally associated with mrdd resistance .
the homozygous and heterozygous bc <dig> genotypes exhibited very similar dsi values .
taken together, the bins  <dig> / <dig> region was most likely to contain a major mrdd resistance qtl.
*markers used for validation in bc1f <dig> and bc2f <dig> segregating populations.
#markers used for validation in a ril population.
location corresponds to maize b <dig> reference genome apgv <dig>
the bc1f <dig> population was selected to map the resistance qtl since it showed stronger segregation in mrdd resistance than the bc2f <dig> population.
based on b <dig> reference sequence,  <dig> pcr-based markers were synthesized within bins  <dig> / <dig> and nine of them were polymorphic between the parental lines, nt <dig> and nt <dig>  besides three previous ssr markers, these nine markers were also used to genotype the  <dig> bc <dig> individuals.
based on genotypic data and the dsi values from bc1f <dig> families, we performed linkage mapping to define the resistance qtl .
a resistance qtl was detected within a 15-mb region flanked by the markers m103- <dig> and umc <dig> .
this resistance qtl, designated qmrdd <dig>  explained  <dig> % and  <dig> % of total phenotypic variation in jining and taian, respectively .
lod: logarithm of odds, sra: source of resistance allele, r2: percentage of the phenotypic variance explained by a qtl.
fine-mapping of qmrdd1
to fine-map qmrdd <dig>  the flanking markers umc <dig> and m103- <dig> were used to screen recombinants from bc1f <dig> families in taian.
fifteen bc1f <dig> recombinants were identified and self-pollinated to generate bc1f <dig> families.
of  <dig>  bc1f <dig> individuals,  <dig> recombinants were screened and self-pollinated to generate bc1f <dig> progeny.
to resolve recombinants associated with bc1f <dig> progeny,  <dig> ssr primer pairs were designed within the qmrdd <dig> region,  <dig> of which were polymorphic.
finally,  <dig> ssr markers  that were evenly distributed  throughout the qmrdd <dig> region were used to resolve the  <dig> recombinants, resulting in  <dig> types .
recombinant-derived bc1f <dig> progeny were selected and planted in three locations.
in taian, we grew  <dig>  bc1f <dig> individuals derived from  <dig> recombinants that included all  <dig> types.
in feicheng, we grew  <dig>  individuals derived from  <dig> recombinants that included  <dig> types.
finally, in jining we grew  <dig>  bc1f <dig> individuals derived from  <dig> recombinants that included  <dig> types.
self-pollinated bc1f <dig> progeny had three genotypes within the heterozygous portion of the qmrdd <dig> locus: homozygous nt <dig>  homozygous nt <dig>  and heterozygous.
dsis for these three genotypes were separately calculated for each bc1f <dig> family.
for each of the  <dig> recombination types, the genotype matched the phenotype.
types i–vii  had the homozygous nt <dig> sequence upstream of the recombination breakpoint, and heterozygous sequences downstream.
types ii–vii were highly susceptible to mrdd regardless of the genotypes, whereas types i exhibited a significant difference in mrdd resistance between the three genotypes.
this indicated that qmrdd <dig> is located downstream of m103- <dig> and upstream of bnlg <dig>  types viii–xiii had heterozygous sequences upstream of the recombination breakpoint and homozygous nt <dig> sequences downstream.
all types showed a significant difference in mrdd resistance between the three genotypes, regardless of the experimental location.
this clearly indicated that qmrdd <dig> is located within the heterozygous region.
types xiv and xv also showed segregation of the mrdd resistance trait and thus restricted qmrdd <dig> into the heterozygous region upstream of the breakpoint.
the remaining types  were resistant to mrdd regardless of the genotype or experimental location.
this implied that qmrdd <dig> is located within the homozygous nt <dig> region but not in the heterozygous region.
only types xiii exhibited a phenotype that varied with experimental location.
a significant difference  in mrdd resistance between genotypes of types xiii bc1f <dig> progeny was detected in taian and jining but not in feicheng .
this discrepancy may have resulted from the small number of bc1f <dig> progeny  in feicheng.
as such, the resistance phenotype for types xiii was considered to segregate, placing qmrdd <dig> within the heterozygous region upstream of m106- <dig>  recombination breakpoints associated with types i and xiv were closest to qmrdd <dig>  allowing us to fine-map qmrdd <dig> into the region between m103- <dig> and m105- <dig>  a physical distance of  <dig>  mb .
genetic model of qmrdd <dig> resistance to mrdd
the genetic effect of qmrdd <dig> was investigated in bc1f <dig> families .
plants homozygous for the nt <dig> qmrdd <dig> region showed a significantly lower dsi than the other two genotypes .
in taian, resistance to mrdd was evaluated for  <dig>  bc1f <dig> plants.
plants homozygous for nt <dig> qmrdd <dig> alleles had a dsi of  <dig> %, which was very similar to heterozygous plants .
in contrast, plants homozygous for nt <dig> qmrdd <dig> alleles had a significantly lower dsi .
in jining,  <dig>  bc1f <dig> plants were evaluated; although dsis were generally higher, a similar genetic pattern was observed.
dsis were  <dig> % and  <dig> % for plants carrying homozygous nt <dig> or heterozygous qmrdd <dig> alleles, respectively.
in contrast, plants carrying homozygous nt <dig> qmrdd <dig> alleles had a dsi of  <dig> %.
of  <dig>  bc1f <dig> plants grown in feicheng, plants carrying qmrdd <dig> regions that were homozygous for nt <dig>  heterozygous, or homozygous for nt <dig> had dsis of  <dig> %,  <dig> %, and  <dig> %, respectively.
at all three test facilities there was no difference  in dsi between heterozygous and homozygous nt <dig> genotypes at the qmrdd <dig> locus, indicating that the qmrdd <dig> qtl acts in a recessive manner to confer resistance to mrdd.
moreover, the strong genetic effect of homozygous nt <dig> qmrdd <dig> alleles, which reduce dsi by  <dig> – <dig> %, suggested that this qtl represents a viable tool for enhancing maize resistance to mrdd.
validation of qmrdd <dig> in f <dig> rils
we next determined whether the qmrdd <dig> qtl is present in other mrdd-resistant lines of maize.
resistance was measured for  <dig> f <dig> rils derived from the cross between x <dig> and huangc.
these studies were conducted in three locations: taian, feicheng, and jining.
ssr markers developed for the qmrdd <dig> region were used to screen parental lines for polymorphisms.
marker m103- <dig>  which is located within the  <dig> -mb region of qmrdd <dig>  was selected to genotype the ril population.
of  <dig> rils,  <dig> and  <dig> were homozygous for x <dig> and huangc sequences at m103- <dig>  respectively.
only three rils were heterozygous at the m103- <dig> locus and thus were excluded from subsequent analyses.
rils homozygous for the x <dig> genotype had dsis of  <dig> %,  <dig> %, and  <dig> % in taian, jining, and feicheng, respectively.
in contrast, rils homozygous for the huangc genotype had dsis of  <dig> %,  <dig> %, and  <dig> % in taian, jining, and feicheng, respectively.
as such, there was a clear difference in mrdd resistance  between two homozygous genotypes .
these findings suggested that the qmrdd <dig> qtl could reduce dsi by  <dig> – <dig> % in the ril population, which agrees with our previous fine-mapping result.
discussion
accurate phenotypic evaluation is critical for marker-trait association analyses, especially for quantitative traits  <cit> .
because large-scale inoculation of plants with mrdd is unfeasible and uniform infection is unreliable, we relied on natural infection processes.
plants were raised in the cities of jining, feicheng, taian, and heze within shandong province, where mrdd is prevalent.
because of poor performance in  <dig>  i.e., the lack of planthoppers, heze was eliminated as a testing site during subsequent analyses.
subsequent fine-mapping tests were also performed in taian, jining, and feicheng to avoid mrdd escape.
the environment significantly influenced mrdd development, as the disease was more serious in jining compared with taian and feicheng.
this may have resulted from different numbers of planthoppers at these three test sites.
fortunately, the qmrdd <dig> locus had a stable genetic effect across these different environments, implying that the natural infection method was a valid approach and that our scoring system was appropriate for qtl analysis of mrdd resistance.
viral resistance can be influenced by both genetic background and the environment  <cit> .
hifs derived from the same cross share similar genetic backgrounds, making them ideal for analyzing quantitative traits.
to identify the major qtl involved in mrdd resistance,  <dig> hifs developed from our breeding program were selected for this study.
for segregating populations prepared from two hifs with very different levels of mrdd resistance, a continuous distribution of resistance was observed rather than two distinct classes.
this may have resulted from residual genetic-background differences or environmental conditions.
whole-genome snp analysis revealed that  <dig> % of  <dig>  called snps differed between susceptible  and resistant  plant lines.
a smaller difference  was observed between nt <dig> and nt <dig>  which are susceptible and resistant lines, respectively.
here we focused on the major qtl for mrdd resistance, but other chromosomal regions may also be involved.
a region in chromosome  <dig>  for example, showed a marginally significant correlation with mrdd resistance in both mapping populations.
to generate populations for fine mapping we self-pollinated rather than backcrossed because most bc2f <dig> families were highly susceptible to mrdd, since different genotypes at qmrdd <dig> had similar phenotypes in bc2f <dig> plants in  <dig>
the recombinant-derived progeny test is an efficient and powerful method for fine-mapping qtls within a backcrossed population when a susceptible inbred line is used as the recurrent parent.
this method can be used to accurately phenotype recombinants by analyzing trait-marker associations in progeny  <cit> .
here we expand the application of this method to include self-pollinated progeny.
compared with backcrossed progeny, progeny generated by self-pollination can capture the effect of all three genotypes and create more recombinants for fine-mapping.
however, not all recombinants produced by self-pollination can be used for fine-mapping, as segregating genotypes within the targeted region are not created when recombinants that are homozygous at both flanking markers are self-pollinated.
crosses that involve heterozygous plants represent an effective way of solving this problem.
by applying the recombinant-derived progeny test to self-pollinated progeny, qmrdd <dig> was fine-mapped to a  <dig> -mb region.
this region decreased dsi by  <dig> – <dig> %.
forty-three additional recombinants were identified from  <dig>  bc1f <dig> for further fine-mapping .
compared with previous reports, this represents significant progress towards cloning and applying qmrdd <dig>  consistent fine-mapping results between test sites suggest that the recombinant-derived progeny-test represents a powerful solution for fine-mapping a dominant qtl in backcrossed progeny or a partially dominant or recessive qtl in self-pollinated progeny.
moreover, mapping results from different years  indicated that the genetic effect of qtl-qmrdd <dig> is heritable.
finally, qmrdd <dig> decreased dsi by  <dig> – <dig> % in an f <dig> ril population composed of  <dig> lines, suggesting that qmrdd <dig> confers a stable genetic effect in diverse genetic backgrounds.
the major qtl mapped in the current study overlaps with resistance regions reported by di renzo  <cit>  and shi  <cit> , implying the same qtl functions across different mapping populations.
however, this qtl has not been detected in the research conducted by luan  <cit> .
this may be due to a different scoring system used by luan who adopted four indexes, including shorten superior internode, waxy enation, tassel type, and disease severity of mrdd, rather than an overall evaluation of mrdd symptom.
most important agronomic traits are quantitative in nature and polygenic.
compared with monogenic or oligogenic traits, therefore, these polygenic traits are extremely difficult for breeders and pathologists to manage  <cit> .
isolation of qtls, especially major qtls, may simplify the analysis of quantitative traits and provide important resources for trait improvements.
before qtls can be applied routinely to breeding programs, a number of challenges must be addressed.
these include improving diagnostic assays to detect qtls and identifying genetic markers for marker-assisted selection  <cit> .
in the present study, we used a reliable scoring system and developed a number of high-density markers within and around the qmrdd <dig> region.
these tools can be used for widespread marker-assisted selection to improve maize resistance to mrdd.
there are two categories of viral resistance in plants—passive resistance and positive resistance.
passive resistance is conferred by recessive plant factors, which are essential for the virus to complete the infection cycle.
these typically involve protein forms that cannot be recognized by specific viral components.
in contrast, positive resistance is conferred by dominant plant factors that trigger defense mechanisms in response to viral invasion  <cit> .
the qmrdd <dig> qtl conferred mrdd resistance only in plants homozygous for nt <dig> alleles, indicating that it involves a recessive gene involved in passive resistance.
this information will facilitate identification of a candidate gene for qmrdd <dig>
CONCLUSIONS
breeding resistant maize hybrids is the most cost-effective way to minimize yield losses from mrdd.
we have mapped qmrdd <dig> to a  <dig> -mb region and showed that it acts in a recessive manner across different genetic backgrounds.
the discovery and fine-mapping of this major qtl involved in mrdd resistance lays the foundation for positional cloning of qmrdd <dig> and moves us closer to genetically controlling mrdd infestation during maize production.

 maize rough dwarf disease is a viral disease that results in substantial yield losses in europe, east asia, and south america. mrdd was discovered in in china and has posed a grave threat to maize production during the last two decades, especially in the yellow-huai-hai river plain. between and mrdd has affected over three million hm of crops each year. yield losses are generally over 30% in affected areas and can reach 100% in regions of severe infection. the virus that causes mrdd belongs to the genus fijivirus in the family reoviridae, but virus strains vary between continents. mrdd is caused by maize rough dwarf virus in europe, mal de río cuarto virus in south america, and rice black-streaked dwarf virus in east asia. these viruses are transmitted in a persistent manner by planthopper insect vectors. mrdd symptoms include stunting, dark-green leaves, waxy enations on abaxial surfaces of leaves and sheaths, malformed tassels and upper leaves, suppressed flowering, and a lack of ears. current methods for controlling mrdd include pesticides, shifting the date when seeds are planted, and improving field management. these methods limit the planthopper population and reduce, to some extent, mrdd severity, but always with high risk and low efficiency. the identification of mrdd-resistant strains, however, likely represents the most cost-effective and environmentally friendly way to minimize yield losses. it is therefore important to develop and deploy resistant hybrids by mapping and cloning genes and quantitative trait loci that confer resistance to mrdd. under natural-infection conditions, the maize germplasm displays variable resistance to mrdd. the major source of resistance is derived from us hybrid p evaluation of inbred lines and hybrids suggests that mrdd resistance is a quantitative trait. wang et al. reported that maize resistance to mrdd is a quantitative trait controlled by many genes, each with a small effect. in argentina, a partially resistant line yielded moderate heritability of resistance to the mal de río cuarto virus, ranging from to. using an f2: qtl-mapping strategy, two qtls were identified on bins and / that together explained % of the phenotypic variance. a major qtl on chromosome for mrdd resistance was identified in the chinese maize inbred line, x based on gene-derived single nucleotide polymorphisms. using an f population derived from the highly resistant line and the susceptible line ye luan found at least three qtls within chromosome bins,, and that confer mrdd resistance. in this study, we applied trait-marker association to heterogeneous inbred families and qtl analysis to segregating population derived from hifs to identify regions of the maize genome that affect resistance to mrdd. we then fine-mapped the major qtl by subjecting self-pollinated backcrossed families to recombinant-derived progeny testing. finally, an f recombinant inbred line population was used to validate the effect of this qtl. these results provide valuable information concerning maize resistance to mrdd, and markers developed within the qmrdd region may prove useful in resistance breeding programs.  evaluation of hifs in resistance to mrdd the hifs developed from a maize hybrid cl were evaluated for their resistance to mrdd in taian for three years and another two locations in of them, displayed consistent resistance regardless of the year or location; while showed high susceptibility in every location across three years. these hifs were therefore used for subsequent trait-marker analysis. trait-marker association analysis in hifs genotyping snps on the hifs generated successful calls based on a cluster-separation score of ≥ and <50% missing data. of these snps, were polymorphic with a minor allelic frequency >  among the hifs. importantly, snps co-segregated with mrdd resistance. after projecting onto the maize accessioned golden path, co-segregating snps were found to cluster within six chromosomal regions. the remaining two snps likely resulted from random error and were excluded from further analysis. the six genomic regions represent candidate mrdd-resistant loci. location corresponds to maize b reference genome apg v qtl analysis of maize resistance to mrdd based on both genotypes and phenotypes, four hifs, nt, nt, nt, and nt, were selected to prepare segregating populations. the f hybrid between nt and nt was backcrossed twice to the susceptible nt to generate bc2f populations. the f hybrid between nt and nt was backcrossed to the susceptible nt and then self-pollinated to generate bc1f populations. the totally bc2f and bc1f families were then evaluated for mrdd susceptibility at four experimental stations: taian, feicheng, heze, and jining. the disease-severity index values were estimated for bc individuals based on their bc2f or bc1f families. the resultant dsi values displayed continuous distributions, ranging from –100% in taian, –100% in feicheng, –100% in taian, and –100% in jining, respectively, implying the quantitative nature of maize resistance to mrdd. intriguingly, dsi distributions skewed toward the susceptible parent, suggesting that a major dominant resistance qtl was absent. in taian, for example, bc2f individuals were extremely susceptible to mrdd, and bc1f individuals exhibited a segregation bias towards susceptibility. in addition, location had a large effect on dsi. a population planted in jining generally had a higher dsi than the same bc2f family in taian. there was no mrdd outbreak in heze, so nearly every family appeared mrdd resistant. data from this location were therefore excluded from further analysis. as such, environmental conditions could fluctuate wildly in mrdd resistance. within the six candidate regions, simple-sequence repeat primer pairs were retrieved from the public database, and additional primer pairs were developed. these ssr markers were used to screen for polymorphisms between the parental lines, resulting in the identification of polymorphic ssr markers. each bc individual was then genotyped using these ssr markers to distinguish heterozygous from homozygous genotypes at the six candidate regions. the phenotype of a given bc individual was represented by dsi values calculated from its bc1f or bc2f progeny. mean dsi value was calculated individually for either heterozygous or homozygous bc genotypes at each of the six candidate regions. based on dsi values from bc1f progeny at taian and jining, three ssr markers within bins / bnlg umc and umc were significantly associated with mrdd resistance, as difference in dsi was significant between heterozygous and homozygous bc genotypes. another two ssr markers within bin, c5- and c5- were marginally associated with mrdd resistance in taian. for bins / the mean difference in dsi between homozygous and heterozygous bc genotypes differed by % and % in taian and jining, respectively. at the other five chromosomal regions, however, differences in dsi between these two bc genotypes were less than %. based on dsi values from bc2f progeny at taian and feicheng, three ssr markers on bins / and two ssr markers on bin were marginally associated with mrdd resistance. the homozygous and heterozygous bc genotypes exhibited very similar dsi values. taken together, the bins / region was most likely to contain a major mrdd resistance qtl. *markers used for validation in bc1f and bc2f segregating populations. #markers used for validation in a ril population. location corresponds to maize b reference genome apgv the bc1f population was selected to map the resistance qtl since it showed stronger segregation in mrdd resistance than the bc2f population. based on b reference sequence, pcr-based markers were synthesized within bins / and nine of them were polymorphic between the parental lines, nt and nt besides three previous ssr markers, these nine markers were also used to genotype the bc individuals. based on genotypic data and the dsi values from bc1f families, we performed linkage mapping to define the resistance qtl. a resistance qtl was detected within a 15-mb region flanked by the markers m103- and umc. this resistance qtl, designated qmrdd explained % and % of total phenotypic variation in jining and taian, respectively. lod: logarithm of odds, sra: source of resistance allele, r2: percentage of the phenotypic variance explained by a qtl. fine-mapping of qmrdd1 to fine-map qmrdd the flanking markers umc and m103- were used to screen recombinants from bc1f families in taian. fifteen bc1f recombinants were identified and self-pollinated to generate bc1f families. of bc1f individuals, recombinants were screened and self-pollinated to generate bc1f progeny. to resolve recombinants associated with bc1f progeny, ssr primer pairs were designed within the qmrdd region, of which were polymorphic. finally, ssr markers that were evenly distributed throughout the qmrdd region were used to resolve the recombinants, resulting in types. recombinant-derived bc1f progeny were selected and planted in three locations. in taian, we grew bc1f individuals derived from recombinants that included all types. in feicheng, we grew individuals derived from recombinants that included types. finally, in jining we grew bc1f individuals derived from recombinants that included types. self-pollinated bc1f progeny had three genotypes within the heterozygous portion of the qmrdd locus: homozygous nt homozygous nt and heterozygous. dsis for these three genotypes were separately calculated for each bc1f family. for each of the recombination types, the genotype matched the phenotype. types i–vii had the homozygous nt sequence upstream of the recombination breakpoint, and heterozygous sequences downstream. types ii–vii were highly susceptible to mrdd regardless of the genotypes, whereas types i exhibited a significant difference in mrdd resistance between the three genotypes. this indicated that qmrdd is located downstream of m103- and upstream of bnlg types viii–xiii had heterozygous sequences upstream of the recombination breakpoint and homozygous nt sequences downstream. all types showed a significant difference in mrdd resistance between the three genotypes, regardless of the experimental location. this clearly indicated that qmrdd is located within the heterozygous region. types xiv and xv also showed segregation of the mrdd resistance trait and thus restricted qmrdd into the heterozygous region upstream of the breakpoint. the remaining types were resistant to mrdd regardless of the genotype or experimental location. this implied that qmrdd is located within the homozygous nt region but not in the heterozygous region. only types xiii exhibited a phenotype that varied with experimental location. a significant difference in mrdd resistance between genotypes of types xiii bc1f progeny was detected in taian and jining but not in feicheng. this discrepancy may have resulted from the small number of bc1f progeny in feicheng. as such, the resistance phenotype for types xiii was considered to segregate, placing qmrdd within the heterozygous region upstream of m106- recombination breakpoints associated with types i and xiv were closest to qmrdd allowing us to fine-map qmrdd into the region between m103- and m105- a physical distance of mb. genetic model of qmrdd resistance to mrdd the genetic effect of qmrdd was investigated in bc1f families. plants homozygous for the nt qmrdd region showed a significantly lower dsi than the other two genotypes. in taian, resistance to mrdd was evaluated for bc1f plants. plants homozygous for nt qmrdd alleles had a dsi of %, which was very similar to heterozygous plants. in contrast, plants homozygous for nt qmrdd alleles had a significantly lower dsi. in jining, bc1f plants were evaluated; although dsis were generally higher, a similar genetic pattern was observed. dsis were % and % for plants carrying homozygous nt or heterozygous qmrdd alleles, respectively. in contrast, plants carrying homozygous nt qmrdd alleles had a dsi of %. of bc1f plants grown in feicheng, plants carrying qmrdd regions that were homozygous for nt heterozygous, or homozygous for nt had dsis of %, %, and %, respectively. at all three test facilities there was no difference in dsi between heterozygous and homozygous nt genotypes at the qmrdd locus, indicating that the qmrdd qtl acts in a recessive manner to confer resistance to mrdd. moreover, the strong genetic effect of homozygous nt qmrdd alleles, which reduce dsi by – %, suggested that this qtl represents a viable tool for enhancing maize resistance to mrdd. validation of qmrdd in f rils we next determined whether the qmrdd qtl is present in other mrdd-resistant lines of maize. resistance was measured for f rils derived from the cross between x and huangc. these studies were conducted in three locations: taian, feicheng, and jining. ssr markers developed for the qmrdd region were used to screen parental lines for polymorphisms. marker m103- which is located within the -mb region of qmrdd was selected to genotype the ril population. of rils, and were homozygous for x and huangc sequences at m103- respectively. only three rils were heterozygous at the m103- locus and thus were excluded from subsequent analyses. rils homozygous for the x genotype had dsis of %, %, and % in taian, jining, and feicheng, respectively. in contrast, rils homozygous for the huangc genotype had dsis of %, %, and % in taian, jining, and feicheng, respectively. as such, there was a clear difference in mrdd resistance between two homozygous genotypes. these findings suggested that the qmrdd qtl could reduce dsi by – % in the ril population, which agrees with our previous fine-mapping result. discussion accurate phenotypic evaluation is critical for marker-trait association analyses, especially for quantitative traits. because large-scale inoculation of plants with mrdd is unfeasible and uniform infection is unreliable, we relied on natural infection processes. plants were raised in the cities of jining, feicheng, taian, and heze within shandong province, where mrdd is prevalent. because of poor performance in i.e., the lack of planthoppers, heze was eliminated as a testing site during subsequent analyses. subsequent fine-mapping tests were also performed in taian, jining, and feicheng to avoid mrdd escape. the environment significantly influenced mrdd development, as the disease was more serious in jining compared with taian and feicheng. this may have resulted from different numbers of planthoppers at these three test sites. fortunately, the qmrdd locus had a stable genetic effect across these different environments, implying that the natural infection method was a valid approach and that our scoring system was appropriate for qtl analysis of mrdd resistance. viral resistance can be influenced by both genetic background and the environment. hifs derived from the same cross share similar genetic backgrounds, making them ideal for analyzing quantitative traits. to identify the major qtl involved in mrdd resistance, hifs developed from our breeding program were selected for this study. for segregating populations prepared from two hifs with very different levels of mrdd resistance, a continuous distribution of resistance was observed rather than two distinct classes. this may have resulted from residual genetic-background differences or environmental conditions. whole-genome snp analysis revealed that % of called snps differed between susceptible and resistant plant lines. a smaller difference was observed between nt and nt which are susceptible and resistant lines, respectively. here we focused on the major qtl for mrdd resistance, but other chromosomal regions may also be involved. a region in chromosome for example, showed a marginally significant correlation with mrdd resistance in both mapping populations. to generate populations for fine mapping we self-pollinated rather than backcrossed because most bc2f families were highly susceptible to mrdd, since different genotypes at qmrdd had similar phenotypes in bc2f plants in the recombinant-derived progeny test is an efficient and powerful method for fine-mapping qtls within a backcrossed population when a susceptible inbred line is used as the recurrent parent. this method can be used to accurately phenotype recombinants by analyzing trait-marker associations in progeny. here we expand the application of this method to include self-pollinated progeny. compared with backcrossed progeny, progeny generated by self-pollination can capture the effect of all three genotypes and create more recombinants for fine-mapping. however, not all recombinants produced by self-pollination can be used for fine-mapping, as segregating genotypes within the targeted region are not created when recombinants that are homozygous at both flanking markers are self-pollinated. crosses that involve heterozygous plants represent an effective way of solving this problem. by applying the recombinant-derived progeny test to self-pollinated progeny, qmrdd was fine-mapped to a -mb region. this region decreased dsi by – %. forty-three additional recombinants were identified from bc1f for further fine-mapping. compared with previous reports, this represents significant progress towards cloning and applying qmrdd consistent fine-mapping results between test sites suggest that the recombinant-derived progeny-test represents a powerful solution for fine-mapping a dominant qtl in backcrossed progeny or a partially dominant or recessive qtl in self-pollinated progeny. moreover, mapping results from different years indicated that the genetic effect of qtl-qmrdd is heritable. finally, qmrdd decreased dsi by – % in an f ril population composed of lines, suggesting that qmrdd confers a stable genetic effect in diverse genetic backgrounds. the major qtl mapped in the current study overlaps with resistance regions reported by di renzo and shi, implying the same qtl functions across different mapping populations. however, this qtl has not been detected in the research conducted by luan. this may be due to a different scoring system used by luan who adopted four indexes, including shorten superior internode, waxy enation, tassel type, and disease severity of mrdd, rather than an overall evaluation of mrdd symptom. most important agronomic traits are quantitative in nature and polygenic. compared with monogenic or oligogenic traits, therefore, these polygenic traits are extremely difficult for breeders and pathologists to manage. isolation of qtls, especially major qtls, may simplify the analysis of quantitative traits and provide important resources for trait improvements. before qtls can be applied routinely to breeding programs, a number of challenges must be addressed. these include improving diagnostic assays to detect qtls and identifying genetic markers for marker-assisted selection. in the present study, we used a reliable scoring system and developed a number of high-density markers within and around the qmrdd region. these tools can be used for widespread marker-assisted selection to improve maize resistance to mrdd. there are two categories of viral resistance in plants—passive resistance and positive resistance. passive resistance is conferred by recessive plant factors, which are essential for the virus to complete the infection cycle. these typically involve protein forms that cannot be recognized by specific viral components. in contrast, positive resistance is conferred by dominant plant factors that trigger defense mechanisms in response to viral invasion. the qmrdd qtl conferred mrdd resistance only in plants homozygous for nt alleles, indicating that it involves a recessive gene involved in passive resistance. this information will facilitate identification of a candidate gene for qmrdd  breeding resistant maize hybrids is the most cost-effective way to minimize yield losses from mrdd. we have mapped qmrdd to a -mb region and showed that it acts in a recessive manner across different genetic backgrounds. the discovery and fine-mapping of this major qtl involved in mrdd resistance lays the foundation for positional cloning of qmrdd and moves us closer to genetically controlling mrdd infestation during maize production. 
the primary approach used to minimize yield losses from these viruses is to breed and deploy resistant maize hybrids. maize rough dwarf disease is a devastating viral disease that results in considerable yield losses worldwide. we performed trait-marker association analysis on the hifs and found six chromosomal regions which were putatively associated with mrdd resistance. three major strains of virus cause mrdd, including maize rough dwarf virus in europe, mal de río cuarto virus in south america, and rice black-streaked dwarf virus in east asia. the genetic effect of qmrdd was validated using another f recombinant inbred line population in which mrdd resistance was segregating and two genotypes at the qmrdd locus differed significantly in dsi values. these viral pathogens belong to the genus fijivirus in the family reoviridae. resistance against mrdd is a complex trait that involves a number of quantitative trait loci. we then conducted qtl analysis and detected a major resistance qtl, qmrdd on chromosome by applying recombinant-derived progeny testing to self-pollinated backcrossed families, we fine-mapped the qmrdd locus into a -mb region flanked by markers m103- and m105- the qmrdd locus acted in a recessive manner to reduce the disease-severity index by – %. of the heterogeneous inbred families, showed consistent responses to mrdd across different years and locations, in which were resistant and were susceptible. the qmrdd locus is a major resistance qtl, acting in a recessive manner to increase maize resistance to mrdd.
 maize rough dwarf disease is a devastating viral disease that results in considerable yield losses worldwide. three major strains of virus cause mrdd, including maize rough dwarf virus in europe, mal de río cuarto virus in south america, and rice black-streaked dwarf virus in east asia. these viral pathogens belong to the genus fijivirus in the family reoviridae. resistance against mrdd is a complex trait that involves a number of quantitative trait loci. the primary approach used to minimize yield losses from these viruses is to breed and deploy resistant maize hybrids.  of the heterogeneous inbred families, showed consistent responses to mrdd across different years and locations, in which were resistant and were susceptible. we performed trait-marker association analysis on the hifs and found six chromosomal regions which were putatively associated with mrdd resistance. we then conducted qtl analysis and detected a major resistance qtl, qmrdd on chromosome by applying recombinant-derived progeny testing to self-pollinated backcrossed families, we fine-mapped the qmrdd locus into a -mb region flanked by markers m103- and m105- the qmrdd locus acted in a recessive manner to reduce the disease-severity index by – %. the genetic effect of qmrdd was validated using another f recombinant inbred line population in which mrdd resistance was segregating and two genotypes at the qmrdd locus differed significantly in dsi values.  the qmrdd locus is a major resistance qtl, acting in a recessive manner to increase maize resistance to mrdd. we mapped qmrdd to a -mb region, which will enable the introgression of qmrdd1-based resistance into elite maize hybrids and reduce mrdd-related crop losses. maizemrddqtlfine-mappingrecombinant-derived progeny test 