BACKGROUND
marker assisted selection  is an efficient approach for plant breeding, especially for woody perennials such as apple.
generally, mas is dependent on high-density genetic linkage maps.
before  <dig>  a number of genetic maps were constructed using different types of markers, such as random amplified polymorphic dnas , amplified fragment length polymorphisms , sequence characterized amplified regions , and simple sequence repeats .
a saturated reference map for apple using  <dig> molecular markers  was published in  <dig>  <cit> .
genetic maps of ‘braeburn’ and ‘telamon’ were constructed in  <dig> using 257 f <dig> mapping populations.
the maps of ‘braeburn’ consisted of  <dig> aflps and  <dig> ssrs, while the ‘telamon’ maps comprised  <dig> aflps and  <dig> ssrs  <cit> .
genetic maps of apple rootstock ‘m9’ × ‘r5’ have been produced containing  <dig> newly developed ssr marker loci  <cit> .
the whole genome sequence of apple was released in  <dig>  making the development of massive snps and the construction of high-density genetic maps of apple possible  <cit> .
for example, an snp-based linkage map constructed using m <dig> malus mapping population comprised  <dig> snp markers,  <dig> ssr markers, and the s-loci, the average distance between markers was increased to  <dig>  cm  <cit> .
the consensus map of ‘honeycrisp’ was developed using three f <dig> populations via an snp chip containing  <dig> snp markers with an average distance of  <dig>  cm between consecutive markers  <cit> .
there was no doubt that these high-density maps could be used in breeding programs more efficiently; however, qtl analysis mostly relied on polymorphisms between the parents of mapping populations.
many markers have different genotypes or linkage relationships among different hybrid populations, therefore it is not easy to transfer the qtl mapping results acquired from one population to another .
thus, there is the need to develop new high-density and standard genetic linkage maps of apple for further research.
snps are widely distributed throughout the whole genomes.
in pear,  <dig> snps were detected, comprising approximately  <dig>  % of the whole genome  <cit> .
snps were more frequently detected in grape, reaching 7 %  <cit> , and the amount of snps obtained in citrus was  <dig> million  <cit> .
in apple, the density of snps was about  <dig>  per kb  <cit> .
except whole genome sequencing, next-generation sequencing  was also a strategy for exploring snps  <cit> .
radseq, one of the ngs technologies, was first described by miller et al.
in  <dig>  <cit> .
compared with snp arrays, radseq was more rapid, cost effective, and independent of any genome information .
recently, several genetic linkage maps were constructed via radseq and were used for genetic mapping and population evolution in different species, such as pear  <cit> , barley  <cit> , and lupinus angustifolius l.  <cit> .
in this decade, apple consumers care about fruit quality traits, both the appearance and flavor, more than ever.
improving quality traits has been an endless pursuit for apple breeders, and most efforts on qtl mapping have been made on fruit size, firmness, sugar contents, and acidity .
the involvement of major gene segregation was previously proposed to explain the variations in the fruit acidity of several hybrid populations .
a major gene controlling fruit acidity in apple, the ma locus, was first mapped to lg <dig>  <cit> .
in the population ‘fiesta’ × ‘discovery’ and ‘telamon’ × ‘braeburn’, qtls of fruit acidity were also mapped to lg <dig>  <cit> , which confirmed the previous results.
recently, the ma region located on lg <dig> has been delimited within 150 kb in the golden delicious genome  <cit> .
although there are a few sequence gaps, it is a big step for breeders in uncovering the candidate gene.
another major qtl associated with fruit acidity was, however, detected on lg <dig> , which is worthy of future research.
the inheritance of fruit sugar contents has been proven to be quantitative  <cit> .
qtls for sugar content in apple are dispersed on different linkage groups in different hybrid populations.
in ‘fiesta’ × ‘discovery’, five qtls for sugar content were mapped to lg <dig>   <dig>   <dig>   <dig>  and  <dig> using integrated maps, however, in ‘telamon’ × ‘braeburn’, two qtls linked to total soluble solids were located on lg <dig> and  <dig> in both parents  <cit> .
in the usda-nifa funded rosbreed project, qtls linked to sugars were detected on apple chromosomes  <dig>   <dig>  and  <dig> using almost  <dig> individuals  <cit> .
fruit size and fruit weight are typical quantitative traits, and qtls linked to these traits always have low rates for the explanation of variance.
in ‘fiesta’ × ‘discovery’, qtls associated with fruit weight were detected on eight linkage groups, and the highest explanation of variance among these qtls was 13 %  <cit> .
in ‘telamon’ × ‘braeburn’, fruit weight was attributed to genomic regions dispersed on five linkage groups, lg <dig>   <dig>   <dig>   <dig>  and  <dig>  <cit> .
however, only lg <dig> and lg <dig> were common to the two hybrid populations.
later, by combining the data for fruit weight from two mapping populations ‘royal gala’ × ‘braeburn’ and ‘starkrimson’ × ‘granny smith’, the qtls were detected on eight linkage groups  <cit> .
although fruit firmness is also quantitatively inherited, the identification of qtls on chromosome  <dig> leads to the discovery of a candidate gene, md-pg <dig>  the qtl cluster on lg <dig> associated with the fruit texture parameters was co-located with the md-pg1ssr marker developed from the sequence 3 kb upstream of the md-pg <dig> start codon  <cit> .
it is suggested that md-pg <dig> plays an important role impacting fruit firmness in  <dig> apple cultivars  <cit> .
our goal in this study was to increase understanding of the genetic control of fruit quality traits in apple, utilizing qtl mapping of several traits associated with quality and subsequent identification of candidate genes, that might provide new insights into the regulation of fruit quality.
although qtl analysis has been a hotspot in apple quality trait research to date, the qtls detected previously were always within long genomic regions, which are difficult to use in breeding programs and further research.
in this work, we set out to remedy this problem by constructing a high-quality genetic map using a radseq strategy, which we could use as the basis for qtl mapping and candidate gene analysis.
methods
plant materials
the hybrid population, including  <dig> seedlings used in this experiment, was generated from the cross between ‘jonathan’ as the maternal parent and ‘golden delicious’ as the pollen parent in  <dig>  the seedlings were all grown on their own roots and planted at a density of  <dig>  m × 2 m at the changli institute of pomology .
the orchard was subjected to conventional field managements and pest control.
at the first apple harvest in  <dig>  the plants were 6 years old.
development of snp markers using radseq
from the segregation population,  <dig> individuals were selected randomly to construct the genetic map.
genomic dna of the 318 f <dig> individuals and two parents, ‘jonathan’ and ‘golden delicious’, was extracted from young leaves using a genomic dna isolation kit  and then processed into radseq libraries, following the protocol described by baird and the colleagues  <cit> .
briefly, the genomic dna was digested with two restriction endonuleases, ecori and hindiii, respectively.
p <dig> adapter, a modified solexa© adapter , was ligated to the samples.
after p <dig> adapter ligation, samples were pooled and randomly sheared with a bioruptor , and dna fragments of the desired length, approximately 500 bp, were gel purified.
to complete the construction of dna libraries, p <dig> adapter, a divergent modified solexa© adapter , was ligated to the obtained dna fragments at 18 °c.
then the purified and quantified samples were used for pcr amplification.
finally, samples were gel purified again, isolated dna fragments  <dig> to 650 bp, and diluted to  <dig> nm.
the prepared dna libraries were sequenced on an illumina hiseq <dig> platform at bgi-shenzhen , using the pe <dig>  strategy.
sequence data were analyzed using custom perl scripts designed by bgi-shenzhen  which were reported before  <cit> .
rad markers were developed using the clean data after removing the adapters, index sequences, and low-quality reads.
reads from each individual were clustered into tag reads by sequence similarity  and clusters with <  <dig> or >  <dig> reads were discarded.
all the snps had total support reads ≥  <dig>  and for heterozygous snps the inferior base depth was ≥  <dig>  based on the genotypes of the parents, monomorphic markers were removed.
finally, three types of markers were obtained: lm × ll, nn × np and hk × hk.
map construction and marker nomenclature
markers used for map construction were filtered using the following criteria: a) for each marker, individuals that lacked genotyping data were less than  <dig> ; and b) all markers were tested by chi-square test .
the segregation ratio of lm × ll and nn × np markers was expected to be 1: <dig>  while that of the hk × hk markers was 1:2: <dig>  genetic linkage maps were generated using joinmap version  <dig>   <cit> .
a logarithm of the odds  score of  <dig>  was set to distinguish linkage groups.
the regression mapping was used as the mapping algorithm, and the genetic distances were calculated based on kosambi’s mapping function.
both parental and consensus maps were constructed, and markers that appeared in the ‘suspect linkage’ table and individuals carrying several double recombination events were removed.
all mapped markers were named after aligning to the reference genome sequence of apple.
the new names include three or four alphabet characters.
the first character, ‘h/e’, indicates restriction enzyme, ‘h’ represents hindiii and ‘e’ represents ecori.
the second letter, ‘m/u/n’, indicates the alignment to the reference genome sequence, ‘m’ represents multiple alignments, ‘u’ represents unique alignment and ‘n’ represents no alignment results.
the following letter, ‘c’ or ‘m’ or ‘lg’, show the linkage groups numbered in accordance with the chromosome numbers of the genome data base.
when the chromosome number of a snp-containing sequence was the same as the number of linkage groups that the snp was mapped to, the third alphabet character of the marker name will be ‘c’, eg.
huc <dig> , where ‘c’ is the abbreviation for chromosome.
the two-digit number prior to the period represents the chromosome number, and the numerals following the period indicate the exact physical position of the snp on the chromosome of the apple genome database .
if the linkage group number of the mapped snp did not correspond to the chromosome number in the genome, the characters following the second letter are formatted as lgn_numerals, such as eulg02_ <dig> and hmlg09_ <dig>  where ‘lg’ is the abbreviation for linkage group.
the following ‘n’, a two-digit number, represents the linkage group number and the numerals following the underscore ‘_’ indicate the number given serially within the linkage group .
therefore, when the second letter in the nomination is ‘n’, the name of that marker will uniquely follow this format, eg.
hnlg02_ <dig> .
in the case where an snp-containing sequence aligned with an unanchored contig in the genome database, the characters after the second letter in the nomination are mx.numerals, such as hum <dig> .
<dig>  where ‘m’ means chromosome  <dig>  the ‘x’ consists of two numerals flanking the first period, representing the contig code in the apple genome database.
the numerals following the second period indicate the exact position of the snp on that contig .
phenotyping
because of the alternate bearing of the segregating population, fruit from  <dig>   <dig>   <dig>  and  <dig> seedlings were harvested in  <dig>   <dig>   <dig>  and  <dig>  respectively.
ripening apples were harvested from each fruit-bearing individual and six apples from each plant were used for phenotyping of fruit quality traits.
average fruit weight was measured using an electronic balance and recorded in grams for  <dig> successive years.
fruit firmness was determined on four directions of each fruit using a desik-gy- <dig> penetrometer  in  <dig> and  <dig>  contents of fruit soluble sugar  and organic acids  were analyzed by high-performance liquid chromatography  in  <dig>  <cit> .
samples were prepared by grinding 5 g fresh fruit flesh from a mixture of six apples in 10 ml redistilled water.
the homogenate was centrifuged at  <dig>  rpm for 10 min at room temperature after 30 min in 75 °c water.
the pomace was extracted again with 8 ml redistilled water.
then, the combined supernatant was diluted up to 25 ml and filtered through  <dig>  μm millipore filter.
the resultant supernatant was used for hplc analysis.
sugar analyses were performed on a lc-10atvp chromatograph  with an asahipak nh2p- <dig> 4e column   attached to a rid-10a refractive index detector .
the mobile phase was acetonitrile/water , with a flow rate of 1 ml min− <dig> at 40 °c.
organic acids analyses were carried out at 30 °c at a flow rate of  <dig>  ml min− <dig> with  <dig>  m k2hpo4 · 3h2o, ph  <dig>  as the mobile phase using a waters  <dig> chromatograph, and a waters  <dig> ultraviolet detector .
the column used here was a reversed-phase c <dig> column  .
the distributions of traits analyses were carried out using the shapiro-wilk test.
qtl analysis
qtl analysis was performed using mapqtl version  <dig>   <cit>  software based on the parental maps and phenotype data from  <dig>   <dig>   <dig>  and  <dig> seedlings in  <dig>   <dig>   <dig>  and  <dig>  respectively.
qtls were detected using interval mapping initially, and the mapping algorithm was a mixed model.
then multiple qtl mapping  was performed to detect additional qtls that might be masked by the major qtls.
after a  <dig> permutation test, a lod threshold of  <dig>  was set to find significant qtls at the 95 % confidence level.
the ranges above the lod threshold of  <dig>  were identified as qtl intervals.
markers located at or flanked with the peak lod value of a qtl were recognized as qtl-associated markers.
candidate gene mining in silico
the corresponding regions of qtls on the physical map were identified by mapping the associated markers.
the genes within the qtl region, together with the functional annotations information, were available on the genome database for rosaceae  website   <cit> .
possible candidate genes related to a specific trait were predicted based on their biological functions.
more attention was paid to functional genes, transcription factors, regulatory genes, and unknown genes.
validation of candidate genes related to fruit acidity
because three qtls involved in the segregation of fruit malic acid, citric acid, and total acid contents mapped to an overlapped genomic region on chromosome  <dig>  six candidate genes, including three functional genes directly linked to fruit acidity, and three transcription factors were chosen for validation.
fruit of two cultivars, ‘fuji’ and ‘jonathan’, with low and high fruit acidity levels, respectively, were sampled at  <dig>   <dig>   <dig>   <dig>   <dig> , and  <dig>  days after full bloom .
each sample was collected from the flesh of six apples.
additionally, fruit acidity  and gene expression were analyzed.
total rna was extracted following the modified cetyltrimethylammonium bromide method described by zhang et al.
<cit> .
extracts were digested using dnasei  at 37 °c for 30 min.
after determining the concentration using a spectrophotometer , 1 μg rna of each sample was reverse transcribed into cdna with the m-mlv kit .
then, the gene expression was analyzed, using the cdna samples, by an ab <dig> real-time pcr system and sybr green fluorescence dye   <cit> .
the six analyzed candidate genes were mdp <dig>  mdp <dig>  mdp <dig>  mdp <dig>  mdp <dig>  and mdp <dig>  among these genes, mdp <dig> and mdp <dig> failed to produce specific primers owing to their high homology with mdp <dig> and mdp <dig>  respectively.
the primers for the rest of the genes were designed using primer express  <dig>  software .
both β-actin and 18s-rrna were analyzed as reference genes.
because of the high expression level of 18s-rrna, only β-actin, which showed a similar expression level with our genes, was used to calculate the relative quantitative expression of genes.
all primer sequences are listed in additional file 1: table s <dig>
RESULTS
marker development
data from multiple illumina/solexa sequence channels was assorted by the appropriate 4–8 bp nucleotide multiplex identifier assigned to each sample.
the raw data were modified by following two steps: first, the adapter and index sequences in reads were deleted, and then, the reads that contained more than 50 % low-quality bases  were removed.
after filtering, the average q20s of the samples were about 96 % , indicating the high quality of the data.
finally,  <dig>  gb of sequencing data were generated using ecori to digest, including  <dig>  gb of ‘jonathan’,  <dig>  gb of ‘golden delicious’, and  <dig>  gb of the 318 f <dig> seedlings.
the gc content of the parents was ~37 %, while the average of the hybrid population was  <dig>  %.
however, hindiii digestion produced  <dig>  gb of sequencing data, consisting of  <dig>  gb,  <dig>  gb, and  <dig>  gb of ‘jonathan’, ‘golden delicious’, and the  <dig> hybrid seedlings, respectively.
the gc content for both the parents and the progenies were almost 40 %.
the average coverage levels of the f <dig> progeny were  <dig>   and  <dig>  .
for ‘jonathan’, the coverage levels were  <dig>   and  <dig>  , while they were  <dig>   and  <dig>   for ‘golden delicious’ .table  <dig> quality evaluation of restriction-site associated dna sequencing data in  <dig> hybrid seedlings and their parents
data of progeny were collected from all  <dig> individuals derived from ‘jonathan’ × ‘golden delicious’.
the size of apple whole genome referenced the estimated genome size  <dig>  mb by velasco et al.
<cit>
based on the rad tags, a high fidelity snp dataset was generated.
for each individual, two types of snp were detected, one was the heterozygous loci itself, and the other was a homozygous loci but polymorphic amongst hybrid seedlings.
using ecori to digest,  <dig> and  <dig> snps were developed from ‘jonathan’ and ‘golden delicious’, respectively.
among these snps,  <dig>  and  <dig>  were homozygous, while  <dig>  and  <dig>  were heterozygous loci.
there were, in total,  <dig>  snps detected amongst the hybrid seedlings, including  <dig>  homozygous and  <dig>  heterozygous loci.
in contrast,  <dig>  snps were obtained through hindiii digestion of ‘jonathan’, including  <dig>  homozygous and  <dig> heterozygous loci, representing  <dig>  % and  <dig>  % of the total, respectively.
in ‘golden delicious’, the total number of snps amounted to  <dig> ,  <dig>  % of the total were homozygous and the rest,  <dig>  %, were heterozygous loci.
the number of snps generated from the progeny was  <dig>  when subjected to hindiii digestion, more than twice the amount from ecori digestion, which indicated that the apple genome was rich in hindiii restriction sites.
meanwhile, the percentage of hindiii-digested homozygous snp loci was higher, at  <dig>  %, compared with heterozygous loci that only accounted for  <dig>  % of the total .table  <dig> features of markers developed from restriction-site associated dna sequencing in f <dig> progeny and parents
the f <dig> progeny includes  <dig> individuals generated from ‘jonathan’ × ‘golden delicious’.
homo homozygous, hete heterozygous
map construction and marker alignment
snp markers developed from radseq cannot all be used in map construction.
because of the use of the f <dig> mapping population, homozygous markers with polymorphisms between the two parents were removed owing to non-segregation in the f <dig> progeny.
high missing values affected the map orders and reduced the map accuracy  <cit> .
to find appropriate missing values, groupings were calculated at different rates of missing data  with a lod threshold of  <dig> .
with the  <dig> and 25 % missing values, the number of generated groupings was inconsistent with  <dig>  the chromosome number of the diploid apple.
however, we obtained  <dig> groupings with the  <dig> or 20 % missing values.
there were  <dig> valid markers with 15 % missing values and  <dig> available markers with 20 % missing values, thus the 20 % missing value was chosen .
of the  <dig> markers,  <dig> did not belong to a linkage group and, therefore, were removed.
furthermore,  <dig> markers were discarded after further analysis by the chi-square test .
during the map construction,  <dig> markers were excluded because of their suspected or weak linkages with other markers, or their estimated positions changed.
during map construction,  <dig> seedlings were deleted from the mapping population, including  <dig> that showed several double recombination events and three with limited sequencing data, which caused more than 50 % missing genotyping data.
finally, the newly constructed consensus map contained  <dig> individuals and  <dig> markers  .
markers mapped on each linkage group range from  <dig> to  <dig>  the total length of the consensus map was  <dig>  cm with the average distance about  <dig>  cm between markers.
using the ‘create maternal and paternal node’ function, maps of the two parents were constructed.
of the  <dig> markers,  <dig> were mapped to ‘jonathan’ maps with a length of  <dig>  cm, and the average distance between markers was  <dig>  cm;  <dig> were mapped to ‘golden delicious’ maps with a length of  <dig>  cm, and the average distance between markers was  <dig>  cm .table  <dig> features of genetic linkage maps
the new maps were constructed using  <dig> snps and  <dig> hybrid seedlings derived from ‘jonathan’ × ‘golden delicious’.
marker amount and total length of each linkage group of parental and consensus maps were showed
all the markers on the genetic map were aligned to the reference genome of ‘golden delicious’  <cit>  and 98 % of that were successfully matched.
there were  <dig> c-type,  <dig> lg-type, and 189 m-type markers, respectively.
however, there were  <dig> n-type markers that failed to match the current reference genome sequence.
the distributions of four types of markers on each linkage group were calculated .
among the  <dig> linkage groups,  <dig> lg-type markers, accounting for  <dig>  %, were mapped on lg <dig>  while there were only  <dig> lg-type markers  on lg <dig>  lg-type markers from more than three different rad tags that co-located on the linkage groups were defined as lg-type clusters.
markers within an lg-type cluster on lg <dig>  lg <dig>  lg <dig>  lg <dig>  and lg <dig>  and two on lg <dig> of ‘jonathan’ were mapped to the same chromosome, which deviated from their linkage groups.
for example, the  <dig> snp markers in the lg-type cluster located from  <dig>  to  <dig>  cm on lg <dig> of ‘jonathan’ were almost mapped to the genomic region from  <dig>  to  <dig>  mb on chromosome  <dig> of the reference genome.
four lg-type clusters were found in the linkage maps of ‘golden delicious’ .fig.
<dig> distributions of different types of markers on each linkage group of consensus genetic maps.
c-type markers: markers had consistent location of linkage map and reference genome of ‘golden delicious’.
lg-type markers: markers had inconsistent location of linkage map and reference genome of ‘golden delicious’.
m-type markers: markers aligned to unanchored chromosome of reference genome of ‘golden delicious’.
n-type markers: markers cannot align to reference genome of ‘golden delicious’
j jonathan, g golden delicious, and chr: chromosome.
the ‘anchored chromosome’ showed the uniquely aligned chromosome numbers of markers in an lg-cluster on the reference genome of ‘golden delicious’
frequency distributions of fruit quality traits
fruit weight is a typical quantitative trait controlled by polygenes.
data on fruit weight were collected over four successive years from  <dig> to  <dig>  the value of fruit weight varied continuously but had a non-normal distribution according to the shapiro-wilk test .
the population mean value of fruit weight was  <dig>  ±  <dig>  g ,  <dig>  ±  <dig>  g ,  <dig>  ±  <dig>  g , and  <dig>  ±  <dig>  g .
the population mean value varied with year, indicating the significant impacts of environmental factors.table  <dig> an overview of population features of phenotyping data for each trait
fruit weight were measured over 4 years, fruit firmness were determined over 2 years, whereas fruit acidity and sugar content were analyzed only in  <dig>  each trait values were obtained from six fruits per n seedlings .
distributions were calculated using shapiro-wilk test
fruit firmness was measured in  <dig> and  <dig>  the mean value was  <dig>  ±  <dig>  kg cm− <dig>  while the minimum was  <dig>  kg cm− <dig> and the maximum was  <dig>  kg cm− <dig> in  <dig>  the average of fruit firmness in  <dig> was  <dig>  ±  <dig>  kg cm− <dig>  which approximately equaled the value in  <dig>  the data ranged from  <dig>  to  <dig>  kg cm− <dig>  the frequency distribution of fruit firmness revealed a gaussian distribution in  <dig> .
fructose was the dominant soluble sugar in apple fruit.
the content of fructose ranged from  <dig>  to  <dig>  mg g− <dig>  with an average of  <dig>  ±  <dig>  mg g− <dig> in the population.
the highest degree of variability was detected in fruit sucrose content .
the fruit glucose content  was lower than fructose and sucrose contents across the population.
total sugar content  was derived by summing the contents of glucose, sucrose, and fructose.
based on the shapiro-wilk test, all of the sugar contents conformed to the law of normal distribution except for that of glucose .
fruit acidity was calculated based on the content of six types of organic acids.
malic acid, the main contributor of fruit acidity in apple, varied between  <dig>  and  <dig>  mg g− <dig>  with a population mean value of  <dig>  ±  <dig>  mg g− <dig> and had a normal distribution.
the contents of tartaric acid, citric acid, and oxalic acid were far lower than that of malic acid.
only the content of oxalic acid fit a normal distribution, while the contents of acetic acid and succinic acid showed biased distributions .
qtls identified for fruit quality traits
a total of  <dig> significant qtls were detected using the interval mapping method.
of the  <dig> qtls, six contributed to fruit acidity, two were related to fruit sugar contents, two were associated with fruit firmness, and the remaining two were linked to fruit weight .
all these qtls were confirmed by mqm mapping, and no additional qtls that might be masked by other qtls were identified .table  <dig> qtls for fruit quality traits detected by interval mapping
the phenotyping data were collected from the mapping population derived from ‘jonathan’ × ‘golden delicious’ in  <dig> to  <dig>  qtl intervals were the ranges that above the lod threshold of  <dig>  at a 95 % confidence level.
markers located at or flanked with the peak lod value of a qtl were recognized as qtl-associated markers.
j jonathan, g golden delicious
of the six qtls for fruit acidity, a major qtl  for malic acid, with a lod score of  <dig>  and a  <dig>  % variance explanation, was mapped on lg <dig> of ‘jonathan’.
the markers emc <dig>  and emc <dig>  were associated with qtlma.j <dig>  three qtls were identified as being linked to citric acid, one of them, qtlc.j <dig> , was identified near emc <dig>  on lg <dig> of jonathan.
the second qtl for citric acid content was qtlc.j <dig>  on lg <dig> of ‘jonathan’, associated with marker huc <dig> .
the third, qtlc.g <dig> with a lod score of  <dig> , was mapped to lg <dig> of ‘golden delicious’, which explained  <dig>  % of the phenotypic variance.
the marker hum <dig> .
<dig> was located at the peak of qtlc.g <dig>  additionally, qtlta.j <dig> , which was linked to total acidity, was detected on lg <dig> of ‘jonathan’.
furthermore, qtla.j <dig>  on lg <dig> of ‘jonathan’ was related to acetic acid.
among the qtls for fruit acidity, qtlma.j <dig>  qtlc.j <dig>  and qtlta.j <dig> overlapped near the marker emc <dig>  on lg <dig> of ‘jonathan’, suggesting that this genomic region contained important genetic information related to fruit acidity.
based on the consensus map, hum <dig> .
<dig> associated with the qtl linked to citric acid on lg <dig> of ‘golden delicious’ was located  <dig>  cm downstream of the marker emc <dig> .
two qtls related to fruit sugar contents were detected.
qtlf.j <dig> , which contributed to fruit fructose content, was mapped to lg <dig> of ‘jonathan’, and the nearest marker was huc <dig> .
while qtls.g <dig> , which was linked to sucrose, was mapped to lg <dig> of ‘golden delicious’, and the nearest marker was huc <dig> .
the distance between the two markers, huc <dig>  and huc <dig> , was about 145 kb on the reference genome of apple.
in  <dig>  two qtls of fruit weight were identified on genetic maps of ‘jonathan’.
one qtl, qtlfw.j <dig>  was mapped on lg <dig>  which explained  <dig>  % of variance with a peak lod score of  <dig> .
the other, qtlfw.j <dig>  was mapped on lg <dig>  with a lod score of  <dig>  and an  <dig>  % explanation of variance.
there were two qtls related to fruit firmness located on lg <dig> of ‘jonathan’, qtlff.j11a  was associated with euc <dig>  and huc <dig> , while qtlff.j11b  was between marker hmc <dig>  and euc <dig>  .
candidate genes involved in fruit quality traits
based on the alignment of the qtl regions with the physical positions on the apple genome pseudo-chromosomes in silico, five qtls with a region < 5 cm, having the highest lod value and variance explanation among the three traits, had their corresponding positions identified, which were suitable for searching for candidate genes directly  <cit> .
the five regions were  <dig> - <dig>  mb of chromosome  <dig> ,  <dig> – <dig>  mb of chromosome  <dig> ,  <dig> - <dig>  mb of chromosome  <dig> ,  <dig> – <dig>  mb of chromosome  <dig>  and  <dig> – <dig>  mb of chromosome  <dig> .
initially  <dig>   <dig>   <dig>   <dig>  and  <dig> genes and their annotation within the five genomic regions, respectively, were obtained from the apple genome database.
according to their functional predictions,  <dig>   <dig>   <dig>   <dig>  and  <dig> genes, respectively, were good candidates .
analysis of fruit acidity and expressions of candidate genes
to understand the potential relationship between candidate genes and fruit acidity, the expression profiles of candidate genes and fruit acidity were analyzed during different fruit development stages of a high acidity cultivar, ‘jonathan’, and a low acidity cultivar, ‘fuji’.
the contents of malic, citric, and total acid were determined to indicate fruit acidity and the regularity of the data were confirmed.
generally, in young fruit of both ‘jonathan’ and ‘fuji’, high fruit acidity was detected at  <dig> dafb, and then after  <dig> dafb, the fruit acidity declined significantly.
however, the acidity of ‘jonathan’ was always higher than that of ‘fuji’ .
among the analyzed genes, mdp <dig> was not detectable in fruit tissue.
mdp <dig>  expressed extensively in flesh tissue at  <dig> dafb and  <dig> dafb in both ‘jonathan’ and ‘fuji’, and the expression level was higher in ‘jonathan’.
after  <dig> dafb, gene expression of mdp <dig> was relatively low in both cultivars.
the expression level of mdp <dig>  was reduced significantly during  <dig> dafb to  <dig> dafb and then, increased gradually from  <dig> dafb to  <dig> dafb in fruit of both ‘jonathan’ and ‘fuji’.
the expression of mdp <dig> was higher in ‘jonathan’ than in ‘fuji’ throughout the sampling seasons.
the change pattern of expression level of mdp <dig> was coordinate with that of fruit acidity.
the expression of mdp <dig>  increased significantly from  <dig> dafb to fruit ripening in fruit of both ‘jonathan’ and ‘fuji’ .
correlation analysis indicated that only the relative expression level of mdp <dig> was significantly correlated with the total acid content  .
these results suggested that transcription factor mdmyb <dig> and functional gene mdme may be involved in the segregation of fruit acidity.fig.
<dig> changes of acid contents and expressions of candidate genes during fruit development in ‘jonathan’ and ‘fuji’.
‘jonathan’ was chosen as a high acidity apple cultivar, while ‘fuji’ as a low acidity cultivar.
expressions of three filtered candidate genes for fruit acidity were determined .
since qtl on lg <dig> was linked to malic acid, citric acid and total acidity, d-f showed their dynamic changes during different periods of fruit development
fig.
<dig> regression between relative expression of mdp <dig> and total acid content in ‘jonathan’ and ‘fuji’
discussion
this was the first time that large numbers of snp markers were developed using radseq in apple.
for a species with high heterozygocity, more than one restriction enzyme may be required to reveal more molecular diversity  <cit> .
in this work, a total of  <dig> valid snp markers were generated through ecori digestion for map construction.
while the three times more snps,  <dig>  were detected via hindiii digestion.
besides radseq, snp markers developed using expressed sequenced tags  database, bac-end sequences, and chips have been reported in apple .
by comparison with radseq, est-based snps have low coverage of the genome but useful for bridging functional and structural genomics  <cit> .
bac-end sequence-based snps were also limited in marker abundance and depended on bac library which was not easy to construct  <cit> .
snp arrays and chips needed genomic information in advance and were always custom designed or commercialized, which means it was costly for screening a big population .
altogather, it is suggested that radseq was a rapid, high efficient and less cost method for molecular breeding using larger population and larger amount of markers.
in total,  <dig> markers approach to  <dig> percent of mapped markers were mapped to unexpected positions when aligned to the apple genome sequence.
this was due at least in part to non-unique sequences aligning by partial homology between non-homologous chromosomes in the apple genome, such as chromosome 1/ <dig>  2/15/ <dig>  3/ <dig>  4/ <dig>  5/ <dig>  6/ <dig>  9/ <dig>  and 13/ <dig>  <cit> .
for example, marker hulg03_ <dig> was mapped to lg <dig> according to its linkage relationship to the neighboring markers, but the best sequence match in the genome was on chromosome  <dig>  this type of event had been reported previously.
the primer sequences of ssr marker hi04a <dig> aligned to chromosome  <dig>  but it mapped to lg <dig> in the ‘fiesta’ genetic map, while nzmseb <dig> z mapped to lg <dig> of the m <dig> map, contrary to the best sequence match on chromosome  <dig> of the ‘golden delicious’  <cit> .
another possible reason for mapping markers on unexpected linkage groups was chromosome structural variations, which has also been reported in the apple genome  <cit> .
some clustered lg-type markers on genetic maps reminded us there may be some chromosome fragment rearrangement events occurred in both parents.
in the region from  <dig>  to  <dig>  cm of lg <dig> of the ‘jonathan’ map, in total of  <dig> snp makers, including hulg09_ <dig>  hulg09_ <dig>  and hulg09_ <dig> etc., were uniquely matched to chromosome  <dig>  this implied a probability of a large chromosome fragment rearrangement between chromosome  <dig> and  <dig> in ‘jonathan’ genome.
additionally, other nine regions were noted in both parents with  <dig> to  <dig> markers .
similarly, in capsicum, a chromosomal translocation event between chromosome  <dig> and  <dig>  between wild and cultivated capsicum, respectively, had been reported based on an est-based linkage map  <cit> .
the validation and biological significance of chromosome structural variations are often analyzed using fluorescence in situ hybridization  <cit> , and we have already been working on it and the results could be expected soon.
the qtls for fruit acidity detected on lg <dig> or chromosome  <dig> were suggested that there was a major gene affecting fruit acidity in this region.
in our previous study using an ssr genetic linkage map of the same population ‘jonathan’ × ‘golden delicious’, a qtl for malic acid was mapped on lg <dig> associated with the marker ch05a02y explaining  <dig>  % of population variance  <cit> .
because of the low density of the ssr genetic map, the qtl interval was about  <dig>  cm.
here, the qtls qtlma.j <dig>  qtlc.j <dig>  and qtlta.j <dig> linked to citric acid, malic acid, and total acidity, respectively, were mapped to a similar genomic region on lg <dig> of ‘jonathan’, with the highest lod score of  <dig>  and more than 39 % variance explanation.
the qtl interval was around 4 cm for malic acid, and the marker emc <dig> , nearest to qtlma.j <dig>  was ~ <dig>  mb apart from ch05a02y on the apple genome  <cit> .
in addition, a qtl on lg <dig> for fruit acidity had also been detected near the aflp marker e31m38- <dig> using the population ‘fiesta’ × ‘discovery’  <cit> .
it is known that the regulation of apple fruit acidity involves several key enzymes, such as mdpepc, mdcymdh, and mdcyme  <cit> .
based on the results of genomic selection for fruit acidity in apple, the snp marker ss <dig> on lg <dig>  had the largest effect was co-localized with a ring finger and chy zinc finger domain-containing protein gene   <cit> .
in our qtl region on chromosome  <dig>  two genes encoding nadp-dependent malic enzyme were found.
the malic enzyme could influence the malic acid content by catalysing the conversion of malic acid to pyruvic acid  <cit> .
candidate genes for fruit acidity also included three myb transcription factors and a citrate-binding protein.
qtl for total soluble solids in apple had never been mapped to lg <dig> before, however, a qtl on lg <dig> was detected recently by gas chromatography–mass spectrometry for sugar composition  <cit> .
two major qtls, qtlf.j <dig> and qtls.g <dig>  linked to fruit sugar contents were identified on lg <dig> in this paper by hplc analysis.
the distance between markers huc <dig>  and huc <dig>  associated with qtlf.j <dig> and qtls.g <dig>  respectively, was about 145 kb on the genome sequence.
two qtls, qtlff.j11a and qtlff.11b, for fruit firmness were mapped to lg <dig>  in ‘fiesta’ × ‘discovery’, 10 years ago, a qtl for fruit firmness was also mapped on lg <dig> despite that fact that the linked aflp markers cannot be anchored to the genome  <cit> .
later, using a genomic selection approach, one of the three largest snp effects on fruit firmness was also located on lg <dig>  but no known candidate genes were found at this genomic region  <cit> .
during fruit ripening, changes in ethylene and the cell wall structure affected fruit firmness, therefore the genes involved in cell wall composition, modification, ethylene metabolic or signaling pathways were considered to be candidates .
in the qtl regions of qtlff.j11a and qtlff.11b,  <dig> genes were potential candidates for fruit firmness.
several qtls related to fruit weight were detected on lg <dig> and lg <dig>  in the ‘co-op17’ × ‘co-op16’ population, qtls for fruit weight on lg <dig> and lg <dig> were closely linked to markers ctg <dig> and ch01b <dig>  with qtl intervals extended over  <dig>  and  <dig>  cm, respectively  <cit> .
using the same population ‘jonathan’ × ‘golden delicious’ but an ssr genetic map, two minor qtls related to fruit weight were mapped on lg <dig> of ‘jonathan’ map, flanked with marker hi02a <dig> and lg <dig> of ‘golden delicious’ map, linked to marker wbgcas <dig>  <cit> .
because of the incompatible physical and genetic positions of hi02a <dig> and wbgcas <dig>  <cit> , the genomic regions of these two qtls could not be estimated accurately.
combined the results of predecessors with our experiment, hmc <dig>  associated with qtlfw.j <dig> for fruit weight on lg <dig>  was on the opposite end from the location of ctg <dig>  and euc <dig>  linked to qtlfw.j <dig>  was about 7 mb away from ch01b <dig> on the apple genome.
the qtl intervals were narrowed down apparently to  <dig>  cm for qtlfw.j <dig> and 2 cm for qtlfw.j <dig>  fruit weight is facilitated by cell division at the early growth stage and cell expansion during the later growth stage.
according to previous research, some genes, such as e <dig> ubiquitin ligase regulating cell proliferation, cdkb <dig> controlling cell division, and auxin response factors etc.
may be potential candidates .
summarized the qtl mapping results in our study, we found that for traits such as fruit firmness and fruit weight, qtls were detected in only one of the successive years of measurement in this study.
this may be because that the minor qtl loci are not year-stably detected except for in a larger population  <cit> .
moreover, environmental factors have a marked influence on fruit weight, which could have led to differences in qtl mapping results  <cit> .
however, we have combined our obtained qtls for fruit quality traits with previously reported qtls and most of them showed a good agreement.
meanwhile candidate genes were searched in qtl regions.
all these results could confirm that the qtls identified in this study were reliable.
candidate genes searching based on qtl regions was universal in current breeding programs .
it is suggested that candidate genes could perform an important role in molecular breeding.
on one hand, functional genes related to a specific trait could be transformed as dna markers using in qtl analysis and mas.
for example, pg <dig> and aco <dig> were tightly linked to qtls mapped on lg <dig> for fruit firmness in apple  <cit> ; lar <dig> was associated with qtl cluster located on lg <dig> for polyphenolic composition in apple fruit  <cit> .
a mutation, transition g to a, at base  <dig> in the open reading frame of an almt  gene leads to a premature stop codon is responsible for the low acidity of apple fruit  <cit> , which could be used in earlier stage selection of breeding process.
on the other hand, the expression level of trait related genes also can help for selection.
assisted by the expression levels of mxha <dig>  mxfit <dig>  mxirt <dig>  mxcs <dig> and mxrd <dig>   <dig> iron deficiency tolerant lines were preliminarily selected out of the  <dig> hybrids between malus xiaojinensis and m. baccata  <cit> .
in our manuscript, candidate genes were searched in qtl regions related to three fruit quality traits.
the expression level of functional genes and transcription factors obtained in qtl on lg <dig> for fruit acidity were analyzed.
the results showed that changes in the expression of mdmyb <dig>  were in agreement with the pattern of malic acid content in apple, and the relative expression of mdme  was significantly correlated with fruit acidity, suggesting their possible involvement in affecting fruit acidity.
in summary, it is supposed that the dedicated studies of candidate genes could provide a new insight into the genetic control of fruit quality traits, and help the further research of qtls that were not year stable.
now, studies of the other acquired candidate genes in this paper are still in progress.
the applications of our candidate genes in future breeding projects could be expected.
CONCLUSIONS
this study provides insights into map construction and map based molecular breeding method in apple.
radseq, first time performed on apple, was a rapid and efficient strategy for snp maker development and genotyping in big mapping populations.
a main contribution was that we constructed a new standard genetic map of apple with  <dig> snp markers and  <dig> individuals derived from a cross between ‘jonathan’ and ‘golden delicious’.
this may improves the accuracy of genetic maps greatly.
based on the map,  <dig> reliable qtls responsible for fruit quality traits were detected.
these qtl regions were narrowed down substantially and easy to find their corresponding positions on the genome sequence due to the associated markers.
subsequently, candidate genes were effectively predicted in the qtl regions less than 5 cm, and some of them were validated using real time pcr.
briefly, a high-density genetic linkage map was generated in apple.
the availability of such maps may facilitate a variety of genomic studies in apple, including qtl analysis, candidate genes searching and chromosomal variation.
availability of supporting data
the data sets supporting the results of this article are included within the article and its additional files.
additional files
additional file 1:
table s <dig>  primer sequences for candidate genes analyzed by real-time pcr.
table s <dig>  numbers of linkage groups and estimated markers by different rates of missing data with lod =  <dig> .
additional file 2: table s <dig>  list of genotyping data of  <dig> snp markers among 297 f <dig> seedlings.
additional file 3: figure s <dig>  genetic linkage maps generated using  <dig> hybrid seedlings derived from ‘jonathan’ × ‘golden delicious’.
the  <dig> linkage groups represented the  <dig> chromosomes of malus × domestica.
the common markers between each parental map and consensus map were showed by the read lines.
lg: linkage groups; j: jonathan; g: golden delicious.
additional file 4: figure s <dig>  frequency distribution diagrams of fruit quality traits in an f <dig> population derived from ‘jonathan’ × ‘golden delicious’.
fruit weight were measured from year  <dig> to  <dig> showed in a-d, fruit firmness were determined in  <dig> and  <dig> showed in e and f, whereas sugar content consist of three composition  and fruit acidity including six kinds of organic acid and total acidity  were analyzed only in  <dig>  each trait values were obtained from six fruits per n seedlings .
additional file 5: figure s <dig>  qtls for fruit quality traits identified by multiple qtl mapping .
the results were consistent with that obtained by interval mapping.
j: jonathan, g: golden delicious.
additional file 6: table s <dig>  candidate genes searched from the regions of five significant qtls .
these five qtls were associated with three important fruit quality traits, two of fruit firmness, two of fruit weight and one of fruit acidity.
the gene function annotation information of whole apple genome was downloaded from gdr website .
candidate genes which have been used for expression analysis were in bold fonts.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
rs handled the genetic map construction and qtl analysis, carried out searching and validation of candidate genes and drafted the manuscript.
ysc and hl participated in the collection of phenotyping data.
fqy, ybz and dmc helped to develop the segregating populations.
yw and tw participated in the design of the study.
xzz and zhh conceived of the study, and lead the whole project.
all authors read and approved the final manuscript.
authors’ information
not applicable.
availability of data and materials
not applicable.
the authors thank key laboratory of biology and genetic improvement of horticultural crops , ministry of agriculture, p. r. china for providing the test platform and instruments.
also, thank to bgi-shenzhen  for rad sequencing service and bioinformatics support.
funding
this work was supported by the  <dig> project , the hi-tech research and development  program of china ; national special funds for scientific research on public causes  project 200903044; and modern agricultural industry technology system  .

 marker assisted selection is an efficient approach for plant breeding, especially for woody perennials such as apple. generally, mas is dependent on high-density genetic linkage maps. before a number of genetic maps were constructed using different types of markers, such as random amplified polymorphic dnas, amplified fragment length polymorphisms, sequence characterized amplified regions, and simple sequence repeats. a saturated reference map for apple using molecular markers was published in. genetic maps of ‘braeburn’ and ‘telamon’ were constructed in using 257 f mapping populations. the maps of ‘braeburn’ consisted of aflps and ssrs, while the ‘telamon’ maps comprised aflps and ssrs. genetic maps of apple rootstock ‘m9’ × ‘r5’ have been produced containing newly developed ssr marker loci. the whole genome sequence of apple was released in making the development of massive snps and the construction of high-density genetic maps of apple possible. for example, an snp-based linkage map constructed using m malus mapping population comprised snp markers, ssr markers, and the s-loci, the average distance between markers was increased to  cm. the consensus map of ‘honeycrisp’ was developed using three f populations via an snp chip containing snp markers with an average distance of  cm between consecutive markers. there was no doubt that these high-density maps could be used in breeding programs more efficiently; however, qtl analysis mostly relied on polymorphisms between the parents of mapping populations. many markers have different genotypes or linkage relationships among different hybrid populations, therefore it is not easy to transfer the qtl mapping results acquired from one population to another. thus, there is the need to develop new high-density and standard genetic linkage maps of apple for further research. snps are widely distributed throughout the whole genomes. in pear, snps were detected, comprising approximately  % of the whole genome. snps were more frequently detected in grape, reaching 7 %, and the amount of snps obtained in citrus was million. in apple, the density of snps was about per kb. except whole genome sequencing, next-generation sequencing was also a strategy for exploring snps. radseq, one of the ngs technologies, was first described by miller et al. in. compared with snp arrays, radseq was more rapid, cost effective, and independent of any genome information. recently, several genetic linkage maps were constructed via radseq and were used for genetic mapping and population evolution in different species, such as pear, barley, and lupinus angustifolius l.. in this decade, apple consumers care about fruit quality traits, both the appearance and flavor, more than ever. improving quality traits has been an endless pursuit for apple breeders, and most efforts on qtl mapping have been made on fruit size, firmness, sugar contents, and acidity. the involvement of major gene segregation was previously proposed to explain the variations in the fruit acidity of several hybrid populations. a major gene controlling fruit acidity in apple, the ma locus, was first mapped to lg. in the population ‘fiesta’ × ‘discovery’ and ‘telamon’ × ‘braeburn’, qtls of fruit acidity were also mapped to lg, which confirmed the previous results. recently, the ma region located on lg has been delimited within 150 kb in the golden delicious genome. although there are a few sequence gaps, it is a big step for breeders in uncovering the candidate gene. another major qtl associated with fruit acidity was, however, detected on lg, which is worthy of future research. the inheritance of fruit sugar contents has been proven to be quantitative. qtls for sugar content in apple are dispersed on different linkage groups in different hybrid populations. in ‘fiesta’ × ‘discovery’, five qtls for sugar content were mapped to lg and using integrated maps, however, in ‘telamon’ × ‘braeburn’, two qtls linked to total soluble solids were located on lg and in both parents. in the usda-nifa funded rosbreed project, qtls linked to sugars were detected on apple chromosomes and using almost individuals. fruit size and fruit weight are typical quantitative traits, and qtls linked to these traits always have low rates for the explanation of variance. in ‘fiesta’ × ‘discovery’, qtls associated with fruit weight were detected on eight linkage groups, and the highest explanation of variance among these qtls was 13 %. in ‘telamon’ × ‘braeburn’, fruit weight was attributed to genomic regions dispersed on five linkage groups, lg and. however, only lg and lg were common to the two hybrid populations. later, by combining the data for fruit weight from two mapping populations ‘royal gala’ × ‘braeburn’ and ‘starkrimson’ × ‘granny smith’, the qtls were detected on eight linkage groups. although fruit firmness is also quantitatively inherited, the identification of qtls on chromosome leads to the discovery of a candidate gene, md-pg the qtl cluster on lg associated with the fruit texture parameters was co-located with the md-pg1ssr marker developed from the sequence 3 kb upstream of the md-pg start codon. it is suggested that md-pg plays an important role impacting fruit firmness in apple cultivars. our goal in this study was to increase understanding of the genetic control of fruit quality traits in apple, utilizing qtl mapping of several traits associated with quality and subsequent identification of candidate genes, that might provide new insights into the regulation of fruit quality. although qtl analysis has been a hotspot in apple quality trait research to date, the qtls detected previously were always within long genomic regions, which are difficult to use in breeding programs and further research. in this work, we set out to remedy this problem by constructing a high-quality genetic map using a radseq strategy, which we could use as the basis for qtl mapping and candidate gene analysis. methods plant materials the hybrid population, including seedlings used in this experiment, was generated from the cross between ‘jonathan’ as the maternal parent and ‘golden delicious’ as the pollen parent in the seedlings were all grown on their own roots and planted at a density of  m × 2 m at the changli institute of pomology. the orchard was subjected to conventional field managements and pest control. at the first apple harvest in the plants were 6 years old. development of snp markers using radseq from the segregation population, individuals were selected randomly to construct the genetic map. genomic dna of the 318 f individuals and two parents, ‘jonathan’ and ‘golden delicious’, was extracted from young leaves using a genomic dna isolation kit and then processed into radseq libraries, following the protocol described by baird and the colleagues. briefly, the genomic dna was digested with two restriction endonuleases, ecori and hindiii, respectively. p adapter, a modified solexa© adapter, was ligated to the samples. after p adapter ligation, samples were pooled and randomly sheared with a bioruptor, and dna fragments of the desired length, approximately 500 bp, were gel purified. to complete the construction of dna libraries, p adapter, a divergent modified solexa© adapter, was ligated to the obtained dna fragments at 18 °c. then the purified and quantified samples were used for pcr amplification. finally, samples were gel purified again, isolated dna fragments to 650 bp, and diluted to nm. the prepared dna libraries were sequenced on an illumina hiseq platform at bgi-shenzhen, using the pe strategy. sequence data were analyzed using custom perl scripts designed by bgi-shenzhen which were reported before. rad markers were developed using the clean data after removing the adapters, index sequences, and low-quality reads. reads from each individual were clustered into tag reads by sequence similarity and clusters with <  or >  reads were discarded. all the snps had total support reads ≥  and for heterozygous snps the inferior base depth was ≥  based on the genotypes of the parents, monomorphic markers were removed. finally, three types of markers were obtained: lm × ll, nn × np and hk × hk. map construction and marker nomenclature markers used for map construction were filtered using the following criteria: a) for each marker, individuals that lacked genotyping data were less than; and b) all markers were tested by chi-square test. the segregation ratio of lm × ll and nn × np markers was expected to be 1: while that of the hk × hk markers was 1:2: genetic linkage maps were generated using joinmap version. a logarithm of the odds score of was set to distinguish linkage groups. the regression mapping was used as the mapping algorithm, and the genetic distances were calculated based on kosambi’s mapping function. both parental and consensus maps were constructed, and markers that appeared in the ‘suspect linkage’ table and individuals carrying several double recombination events were removed. all mapped markers were named after aligning to the reference genome sequence of apple. the new names include three or four alphabet characters. the first character, ‘h/e’, indicates restriction enzyme, ‘h’ represents hindiii and ‘e’ represents ecori. the second letter, ‘m/u/n’, indicates the alignment to the reference genome sequence, ‘m’ represents multiple alignments, ‘u’ represents unique alignment and ‘n’ represents no alignment results. the following letter, ‘c’ or ‘m’ or ‘lg’, show the linkage groups numbered in accordance with the chromosome numbers of the genome data base. when the chromosome number of a snp-containing sequence was the same as the number of linkage groups that the snp was mapped to, the third alphabet character of the marker name will be ‘c’, eg. huc, where ‘c’ is the abbreviation for chromosome. the two-digit number prior to the period represents the chromosome number, and the numerals following the period indicate the exact physical position of the snp on the chromosome of the apple genome database. if the linkage group number of the mapped snp did not correspond to the chromosome number in the genome, the characters following the second letter are formatted as lgn_numerals, such as eulg02_ and hmlg09_ where ‘lg’ is the abbreviation for linkage group. the following ‘n’, a two-digit number, represents the linkage group number and the numerals following the underscore ‘_’ indicate the number given serially within the linkage group. therefore, when the second letter in the nomination is ‘n’, the name of that marker will uniquely follow this format, eg. hnlg02_. in the case where an snp-containing sequence aligned with an unanchored contig in the genome database, the characters after the second letter in the nomination are mx.numerals, such as hum. where ‘m’ means chromosome the ‘x’ consists of two numerals flanking the first period, representing the contig code in the apple genome database. the numerals following the second period indicate the exact position of the snp on that contig. phenotyping because of the alternate bearing of the segregating population, fruit from and seedlings were harvested in and respectively. ripening apples were harvested from each fruit-bearing individual and six apples from each plant were used for phenotyping of fruit quality traits. average fruit weight was measured using an electronic balance and recorded in grams for successive years. fruit firmness was determined on four directions of each fruit using a desik-gy- penetrometer in and contents of fruit soluble sugar and organic acids were analyzed by high-performance liquid chromatography in. samples were prepared by grinding 5 g fresh fruit flesh from a mixture of six apples in 10 ml redistilled water. the homogenate was centrifuged at  rpm for 10 min at room temperature after 30 min in 75 °c water. the pomace was extracted again with 8 ml redistilled water. then, the combined supernatant was diluted up to 25 ml and filtered through  μm millipore filter. the resultant supernatant was used for hplc analysis. sugar analyses were performed on a lc-10atvp chromatograph with an asahipak nh2p- 4e column attached to a rid-10a refractive index detector. the mobile phase was acetonitrile/water, with a flow rate of 1 ml min− at 40 °c. organic acids analyses were carried out at 30 °c at a flow rate of  ml min− with  m k2hpo4 · 3h2o, ph  as the mobile phase using a waters chromatograph, and a waters ultraviolet detector. the column used here was a reversed-phase c column. the distributions of traits analyses were carried out using the shapiro-wilk test. qtl analysis qtl analysis was performed using mapqtl version software based on the parental maps and phenotype data from and seedlings in and respectively. qtls were detected using interval mapping initially, and the mapping algorithm was a mixed model. then multiple qtl mapping was performed to detect additional qtls that might be masked by the major qtls. after a permutation test, a lod threshold of was set to find significant qtls at the 95 % confidence level. the ranges above the lod threshold of were identified as qtl intervals. markers located at or flanked with the peak lod value of a qtl were recognized as qtl-associated markers. candidate gene mining in silico the corresponding regions of qtls on the physical map were identified by mapping the associated markers. the genes within the qtl region, together with the functional annotations information, were available on the genome database for rosaceae website. possible candidate genes related to a specific trait were predicted based on their biological functions. more attention was paid to functional genes, transcription factors, regulatory genes, and unknown genes. validation of candidate genes related to fruit acidity because three qtls involved in the segregation of fruit malic acid, citric acid, and total acid contents mapped to an overlapped genomic region on chromosome six candidate genes, including three functional genes directly linked to fruit acidity, and three transcription factors were chosen for validation. fruit of two cultivars, ‘fuji’ and ‘jonathan’, with low and high fruit acidity levels, respectively, were sampled at, and days after full bloom. each sample was collected from the flesh of six apples. additionally, fruit acidity and gene expression were analyzed. total rna was extracted following the modified cetyltrimethylammonium bromide method described by zhang et al.. extracts were digested using dnasei at 37 °c for 30 min. after determining the concentration using a spectrophotometer, 1 μg rna of each sample was reverse transcribed into cdna with the m-mlv kit. then, the gene expression was analyzed, using the cdna samples, by an ab real-time pcr system and sybr green fluorescence dye. the six analyzed candidate genes were mdp mdp mdp mdp mdp and mdp among these genes, mdp and mdp failed to produce specific primers owing to their high homology with mdp and mdp respectively. the primers for the rest of the genes were designed using primer express software. both β-actin and 18s-rrna were analyzed as reference genes. because of the high expression level of 18s-rrna, only β-actin, which showed a similar expression level with our genes, was used to calculate the relative quantitative expression of genes. all primer sequences are listed in additional file 1: table s  marker development data from multiple illumina/solexa sequence channels was assorted by the appropriate 4–8 bp nucleotide multiplex identifier assigned to each sample. the raw data were modified by following two steps: first, the adapter and index sequences in reads were deleted, and then, the reads that contained more than 50 % low-quality bases were removed. after filtering, the average q20s of the samples were about 96 %, indicating the high quality of the data. finally, gb of sequencing data were generated using ecori to digest, including gb of ‘jonathan’, gb of ‘golden delicious’, and gb of the 318 f seedlings. the gc content of the parents was ~37 %, while the average of the hybrid population was  %. however, hindiii digestion produced gb of sequencing data, consisting of gb, gb, and gb of ‘jonathan’, ‘golden delicious’, and the hybrid seedlings, respectively. the gc content for both the parents and the progenies were almost 40 %. the average coverage levels of the f progeny were and. for ‘jonathan’, the coverage levels were and, while they were and for ‘golden delicious’.table quality evaluation of restriction-site associated dna sequencing data in hybrid seedlings and their parents data of progeny were collected from all individuals derived from ‘jonathan’ × ‘golden delicious’. the size of apple whole genome referenced the estimated genome size  mb by velasco et al. based on the rad tags, a high fidelity snp dataset was generated. for each individual, two types of snp were detected, one was the heterozygous loci itself, and the other was a homozygous loci but polymorphic amongst hybrid seedlings. using ecori to digest, and snps were developed from ‘jonathan’ and ‘golden delicious’, respectively. among these snps, and were homozygous, while and were heterozygous loci. there were, in total, snps detected amongst the hybrid seedlings, including homozygous and heterozygous loci. in contrast, snps were obtained through hindiii digestion of ‘jonathan’, including homozygous and heterozygous loci, representing  % and  % of the total, respectively. in ‘golden delicious’, the total number of snps amounted to,  % of the total were homozygous and the rest,  %, were heterozygous loci. the number of snps generated from the progeny was when subjected to hindiii digestion, more than twice the amount from ecori digestion, which indicated that the apple genome was rich in hindiii restriction sites. meanwhile, the percentage of hindiii-digested homozygous snp loci was higher, at  %, compared with heterozygous loci that only accounted for  % of the total.table features of markers developed from restriction-site associated dna sequencing in f progeny and parents the f progeny includes individuals generated from ‘jonathan’ × ‘golden delicious’. homo homozygous, hete heterozygous map construction and marker alignment snp markers developed from radseq cannot all be used in map construction. because of the use of the f mapping population, homozygous markers with polymorphisms between the two parents were removed owing to non-segregation in the f progeny. high missing values affected the map orders and reduced the map accuracy. to find appropriate missing values, groupings were calculated at different rates of missing data with a lod threshold of. with the and 25 % missing values, the number of generated groupings was inconsistent with the chromosome number of the diploid apple. however, we obtained groupings with the or 20 % missing values. there were valid markers with 15 % missing values and available markers with 20 % missing values, thus the 20 % missing value was chosen. of the markers, did not belong to a linkage group and, therefore, were removed. furthermore, markers were discarded after further analysis by the chi-square test. during the map construction, markers were excluded because of their suspected or weak linkages with other markers, or their estimated positions changed. during map construction, seedlings were deleted from the mapping population, including that showed several double recombination events and three with limited sequencing data, which caused more than 50 % missing genotyping data. finally, the newly constructed consensus map contained individuals and markers. markers mapped on each linkage group range from to the total length of the consensus map was  cm with the average distance about  cm between markers. using the ‘create maternal and paternal node’ function, maps of the two parents were constructed. of the markers, were mapped to ‘jonathan’ maps with a length of  cm, and the average distance between markers was  cm; were mapped to ‘golden delicious’ maps with a length of  cm, and the average distance between markers was  cm.table features of genetic linkage maps the new maps were constructed using snps and hybrid seedlings derived from ‘jonathan’ × ‘golden delicious’. marker amount and total length of each linkage group of parental and consensus maps were showed all the markers on the genetic map were aligned to the reference genome of ‘golden delicious’ and 98 % of that were successfully matched. there were c-type, lg-type, and 189 m-type markers, respectively. however, there were n-type markers that failed to match the current reference genome sequence. the distributions of four types of markers on each linkage group were calculated. among the linkage groups, lg-type markers, accounting for  %, were mapped on lg while there were only lg-type markers on lg lg-type markers from more than three different rad tags that co-located on the linkage groups were defined as lg-type clusters. markers within an lg-type cluster on lg lg lg lg and lg and two on lg of ‘jonathan’ were mapped to the same chromosome, which deviated from their linkage groups. for example, the snp markers in the lg-type cluster located from to  cm on lg of ‘jonathan’ were almost mapped to the genomic region from to  mb on chromosome of the reference genome. four lg-type clusters were found in the linkage maps of ‘golden delicious’.fig. distributions of different types of markers on each linkage group of consensus genetic maps. c-type markers: markers had consistent location of linkage map and reference genome of ‘golden delicious’. lg-type markers: markers had inconsistent location of linkage map and reference genome of ‘golden delicious’. m-type markers: markers aligned to unanchored chromosome of reference genome of ‘golden delicious’. n-type markers: markers cannot align to reference genome of ‘golden delicious’ j jonathan, g golden delicious, and chr: chromosome. the ‘anchored chromosome’ showed the uniquely aligned chromosome numbers of markers in an lg-cluster on the reference genome of ‘golden delicious’ frequency distributions of fruit quality traits fruit weight is a typical quantitative trait controlled by polygenes. data on fruit weight were collected over four successive years from to the value of fruit weight varied continuously but had a non-normal distribution according to the shapiro-wilk test. the population mean value of fruit weight was  ±   g,  ±   g,  ±   g, and  ±   g. the population mean value varied with year, indicating the significant impacts of environmental factors.table an overview of population features of phenotyping data for each trait fruit weight were measured over 4 years, fruit firmness were determined over 2 years, whereas fruit acidity and sugar content were analyzed only in each trait values were obtained from six fruits per n seedlings. distributions were calculated using shapiro-wilk test fruit firmness was measured in and the mean value was  ±   kg cm− while the minimum was  kg cm− and the maximum was  kg cm− in the average of fruit firmness in was  ±   kg cm− which approximately equaled the value in the data ranged from to  kg cm− the frequency distribution of fruit firmness revealed a gaussian distribution in. fructose was the dominant soluble sugar in apple fruit. the content of fructose ranged from to  mg g− with an average of  ±   mg g− in the population. the highest degree of variability was detected in fruit sucrose content. the fruit glucose content was lower than fructose and sucrose contents across the population. total sugar content was derived by summing the contents of glucose, sucrose, and fructose. based on the shapiro-wilk test, all of the sugar contents conformed to the law of normal distribution except for that of glucose. fruit acidity was calculated based on the content of six types of organic acids. malic acid, the main contributor of fruit acidity in apple, varied between and  mg g− with a population mean value of  ±   mg g− and had a normal distribution. the contents of tartaric acid, citric acid, and oxalic acid were far lower than that of malic acid. only the content of oxalic acid fit a normal distribution, while the contents of acetic acid and succinic acid showed biased distributions. qtls identified for fruit quality traits a total of significant qtls were detected using the interval mapping method. of the qtls, six contributed to fruit acidity, two were related to fruit sugar contents, two were associated with fruit firmness, and the remaining two were linked to fruit weight. all these qtls were confirmed by mqm mapping, and no additional qtls that might be masked by other qtls were identified.table qtls for fruit quality traits detected by interval mapping the phenotyping data were collected from the mapping population derived from ‘jonathan’ × ‘golden delicious’ in to qtl intervals were the ranges that above the lod threshold of at a 95 % confidence level. markers located at or flanked with the peak lod value of a qtl were recognized as qtl-associated markers. j jonathan, g golden delicious of the six qtls for fruit acidity, a major qtl for malic acid, with a lod score of and a  % variance explanation, was mapped on lg of ‘jonathan’. the markers emc and emc were associated with qtlma.j three qtls were identified as being linked to citric acid, one of them, qtlc.j, was identified near emc on lg of jonathan. the second qtl for citric acid content was qtlc.j on lg of ‘jonathan’, associated with marker huc. the third, qtlc.g with a lod score of, was mapped to lg of ‘golden delicious’, which explained  % of the phenotypic variance. the marker hum. was located at the peak of qtlc.g additionally, qtlta.j, which was linked to total acidity, was detected on lg of ‘jonathan’. furthermore, qtla.j on lg of ‘jonathan’ was related to acetic acid. among the qtls for fruit acidity, qtlma.j qtlc.j and qtlta.j overlapped near the marker emc on lg of ‘jonathan’, suggesting that this genomic region contained important genetic information related to fruit acidity. based on the consensus map, hum. associated with the qtl linked to citric acid on lg of ‘golden delicious’ was located  cm downstream of the marker emc. two qtls related to fruit sugar contents were detected. qtlf.j, which contributed to fruit fructose content, was mapped to lg of ‘jonathan’, and the nearest marker was huc. while qtls.g, which was linked to sucrose, was mapped to lg of ‘golden delicious’, and the nearest marker was huc. the distance between the two markers, huc and huc, was about 145 kb on the reference genome of apple. in two qtls of fruit weight were identified on genetic maps of ‘jonathan’. one qtl, qtlfw.j was mapped on lg which explained  % of variance with a peak lod score of. the other, qtlfw.j was mapped on lg with a lod score of and an  % explanation of variance. there were two qtls related to fruit firmness located on lg of ‘jonathan’, qtlff.j11a was associated with euc and huc, while qtlff.j11b was between marker hmc and euc. candidate genes involved in fruit quality traits based on the alignment of the qtl regions with the physical positions on the apple genome pseudo-chromosomes in silico, five qtls with a region < 5 cm, having the highest lod value and variance explanation among the three traits, had their corresponding positions identified, which were suitable for searching for candidate genes directly. the five regions were -  mb of chromosome, –  mb of chromosome, -  mb of chromosome, –  mb of chromosome and –  mb of chromosome. initially and genes and their annotation within the five genomic regions, respectively, were obtained from the apple genome database. according to their functional predictions, and genes, respectively, were good candidates. analysis of fruit acidity and expressions of candidate genes to understand the potential relationship between candidate genes and fruit acidity, the expression profiles of candidate genes and fruit acidity were analyzed during different fruit development stages of a high acidity cultivar, ‘jonathan’, and a low acidity cultivar, ‘fuji’. the contents of malic, citric, and total acid were determined to indicate fruit acidity and the regularity of the data were confirmed. generally, in young fruit of both ‘jonathan’ and ‘fuji’, high fruit acidity was detected at dafb, and then after dafb, the fruit acidity declined significantly. however, the acidity of ‘jonathan’ was always higher than that of ‘fuji’. among the analyzed genes, mdp was not detectable in fruit tissue. mdp expressed extensively in flesh tissue at dafb and dafb in both ‘jonathan’ and ‘fuji’, and the expression level was higher in ‘jonathan’. after dafb, gene expression of mdp was relatively low in both cultivars. the expression level of mdp was reduced significantly during dafb to dafb and then, increased gradually from dafb to dafb in fruit of both ‘jonathan’ and ‘fuji’. the expression of mdp was higher in ‘jonathan’ than in ‘fuji’ throughout the sampling seasons. the change pattern of expression level of mdp was coordinate with that of fruit acidity. the expression of mdp increased significantly from dafb to fruit ripening in fruit of both ‘jonathan’ and ‘fuji’. correlation analysis indicated that only the relative expression level of mdp was significantly correlated with the total acid content. these results suggested that transcription factor mdmyb and functional gene mdme may be involved in the segregation of fruit acidity.fig. changes of acid contents and expressions of candidate genes during fruit development in ‘jonathan’ and ‘fuji’. ‘jonathan’ was chosen as a high acidity apple cultivar, while ‘fuji’ as a low acidity cultivar. expressions of three filtered candidate genes for fruit acidity were determined. since qtl on lg was linked to malic acid, citric acid and total acidity, d-f showed their dynamic changes during different periods of fruit development fig. regression between relative expression of mdp and total acid content in ‘jonathan’ and ‘fuji’ discussion this was the first time that large numbers of snp markers were developed using radseq in apple. for a species with high heterozygocity, more than one restriction enzyme may be required to reveal more molecular diversity. in this work, a total of valid snp markers were generated through ecori digestion for map construction. while the three times more snps, were detected via hindiii digestion. besides radseq, snp markers developed using expressed sequenced tags database, bac-end sequences, and chips have been reported in apple. by comparison with radseq, est-based snps have low coverage of the genome but useful for bridging functional and structural genomics. bac-end sequence-based snps were also limited in marker abundance and depended on bac library which was not easy to construct. snp arrays and chips needed genomic information in advance and were always custom designed or commercialized, which means it was costly for screening a big population. altogather, it is suggested that radseq was a rapid, high efficient and less cost method for molecular breeding using larger population and larger amount of markers. in total, markers approach to percent of mapped markers were mapped to unexpected positions when aligned to the apple genome sequence. this was due at least in part to non-unique sequences aligning by partial homology between non-homologous chromosomes in the apple genome, such as chromosome 1/ 2/15/ 3/ 4/ 5/ 6/ 9/ and 13/. for example, marker hulg03_ was mapped to lg according to its linkage relationship to the neighboring markers, but the best sequence match in the genome was on chromosome this type of event had been reported previously. the primer sequences of ssr marker hi04a aligned to chromosome but it mapped to lg in the ‘fiesta’ genetic map, while nzmseb z mapped to lg of the m map, contrary to the best sequence match on chromosome of the ‘golden delicious’. another possible reason for mapping markers on unexpected linkage groups was chromosome structural variations, which has also been reported in the apple genome. some clustered lg-type markers on genetic maps reminded us there may be some chromosome fragment rearrangement events occurred in both parents. in the region from to  cm of lg of the ‘jonathan’ map, in total of snp makers, including hulg09_ hulg09_ and hulg09_ etc., were uniquely matched to chromosome this implied a probability of a large chromosome fragment rearrangement between chromosome and in ‘jonathan’ genome. additionally, other nine regions were noted in both parents with to markers. similarly, in capsicum, a chromosomal translocation event between chromosome and between wild and cultivated capsicum, respectively, had been reported based on an est-based linkage map. the validation and biological significance of chromosome structural variations are often analyzed using fluorescence in situ hybridization, and we have already been working on it and the results could be expected soon. the qtls for fruit acidity detected on lg or chromosome were suggested that there was a major gene affecting fruit acidity in this region. in our previous study using an ssr genetic linkage map of the same population ‘jonathan’ × ‘golden delicious’, a qtl for malic acid was mapped on lg associated with the marker ch05a02y explaining  % of population variance. because of the low density of the ssr genetic map, the qtl interval was about  cm. here, the qtls qtlma.j qtlc.j and qtlta.j linked to citric acid, malic acid, and total acidity, respectively, were mapped to a similar genomic region on lg of ‘jonathan’, with the highest lod score of and more than 39 % variance explanation. the qtl interval was around 4 cm for malic acid, and the marker emc, nearest to qtlma.j was ~  mb apart from ch05a02y on the apple genome. in addition, a qtl on lg for fruit acidity had also been detected near the aflp marker e31m38- using the population ‘fiesta’ × ‘discovery’. it is known that the regulation of apple fruit acidity involves several key enzymes, such as mdpepc, mdcymdh, and mdcyme. based on the results of genomic selection for fruit acidity in apple, the snp marker ss on lg had the largest effect was co-localized with a ring finger and chy zinc finger domain-containing protein gene. in our qtl region on chromosome two genes encoding nadp-dependent malic enzyme were found. the malic enzyme could influence the malic acid content by catalysing the conversion of malic acid to pyruvic acid. candidate genes for fruit acidity also included three myb transcription factors and a citrate-binding protein. qtl for total soluble solids in apple had never been mapped to lg before, however, a qtl on lg was detected recently by gas chromatography–mass spectrometry for sugar composition. two major qtls, qtlf.j and qtls.g linked to fruit sugar contents were identified on lg in this paper by hplc analysis. the distance between markers huc and huc associated with qtlf.j and qtls.g respectively, was about 145 kb on the genome sequence. two qtls, qtlff.j11a and qtlff.11b, for fruit firmness were mapped to lg in ‘fiesta’ × ‘discovery’, 10 years ago, a qtl for fruit firmness was also mapped on lg despite that fact that the linked aflp markers cannot be anchored to the genome. later, using a genomic selection approach, one of the three largest snp effects on fruit firmness was also located on lg but no known candidate genes were found at this genomic region. during fruit ripening, changes in ethylene and the cell wall structure affected fruit firmness, therefore the genes involved in cell wall composition, modification, ethylene metabolic or signaling pathways were considered to be candidates. in the qtl regions of qtlff.j11a and qtlff.11b, genes were potential candidates for fruit firmness. several qtls related to fruit weight were detected on lg and lg in the ‘co-op17’ × ‘co-op16’ population, qtls for fruit weight on lg and lg were closely linked to markers ctg and ch01b with qtl intervals extended over and  cm, respectively. using the same population ‘jonathan’ × ‘golden delicious’ but an ssr genetic map, two minor qtls related to fruit weight were mapped on lg of ‘jonathan’ map, flanked with marker hi02a and lg of ‘golden delicious’ map, linked to marker wbgcas. because of the incompatible physical and genetic positions of hi02a and wbgcas, the genomic regions of these two qtls could not be estimated accurately. combined the results of predecessors with our experiment, hmc associated with qtlfw.j for fruit weight on lg was on the opposite end from the location of ctg and euc linked to qtlfw.j was about 7 mb away from ch01b on the apple genome. the qtl intervals were narrowed down apparently to  cm for qtlfw.j and 2 cm for qtlfw.j fruit weight is facilitated by cell division at the early growth stage and cell expansion during the later growth stage. according to previous research, some genes, such as e ubiquitin ligase regulating cell proliferation, cdkb controlling cell division, and auxin response factors etc. may be potential candidates. summarized the qtl mapping results in our study, we found that for traits such as fruit firmness and fruit weight, qtls were detected in only one of the successive years of measurement in this study. this may be because that the minor qtl loci are not year-stably detected except for in a larger population. moreover, environmental factors have a marked influence on fruit weight, which could have led to differences in qtl mapping results. however, we have combined our obtained qtls for fruit quality traits with previously reported qtls and most of them showed a good agreement. meanwhile candidate genes were searched in qtl regions. all these results could confirm that the qtls identified in this study were reliable. candidate genes searching based on qtl regions was universal in current breeding programs. it is suggested that candidate genes could perform an important role in molecular breeding. on one hand, functional genes related to a specific trait could be transformed as dna markers using in qtl analysis and mas. for example, pg and aco were tightly linked to qtls mapped on lg for fruit firmness in apple; lar was associated with qtl cluster located on lg for polyphenolic composition in apple fruit. a mutation, transition g to a, at base in the open reading frame of an almt gene leads to a premature stop codon is responsible for the low acidity of apple fruit, which could be used in earlier stage selection of breeding process. on the other hand, the expression level of trait related genes also can help for selection. assisted by the expression levels of mxha mxfit mxirt mxcs and mxrd iron deficiency tolerant lines were preliminarily selected out of the hybrids between malus xiaojinensis and m. baccata. in our manuscript, candidate genes were searched in qtl regions related to three fruit quality traits. the expression level of functional genes and transcription factors obtained in qtl on lg for fruit acidity were analyzed. the results showed that changes in the expression of mdmyb were in agreement with the pattern of malic acid content in apple, and the relative expression of mdme was significantly correlated with fruit acidity, suggesting their possible involvement in affecting fruit acidity. in summary, it is supposed that the dedicated studies of candidate genes could provide a new insight into the genetic control of fruit quality traits, and help the further research of qtls that were not year stable. now, studies of the other acquired candidate genes in this paper are still in progress. the applications of our candidate genes in future breeding projects could be expected.  this study provides insights into map construction and map based molecular breeding method in apple. radseq, first time performed on apple, was a rapid and efficient strategy for snp maker development and genotyping in big mapping populations. a main contribution was that we constructed a new standard genetic map of apple with snp markers and individuals derived from a cross between ‘jonathan’ and ‘golden delicious’. this may improves the accuracy of genetic maps greatly. based on the map, reliable qtls responsible for fruit quality traits were detected. these qtl regions were narrowed down substantially and easy to find their corresponding positions on the genome sequence due to the associated markers. subsequently, candidate genes were effectively predicted in the qtl regions less than 5 cm, and some of them were validated using real time pcr. briefly, a high-density genetic linkage map was generated in apple. the availability of such maps may facilitate a variety of genomic studies in apple, including qtl analysis, candidate genes searching and chromosomal variation. availability of supporting data the data sets supporting the results of this article are included within the article and its additional files. additional files additional file 1: table s primer sequences for candidate genes analyzed by real-time pcr. table s numbers of linkage groups and estimated markers by different rates of missing data with lod = . additional file 2: table s list of genotyping data of snp markers among 297 f seedlings. additional file 3: figure s genetic linkage maps generated using hybrid seedlings derived from ‘jonathan’ × ‘golden delicious’. the linkage groups represented the chromosomes of malus × domestica. the common markers between each parental map and consensus map were showed by the read lines. lg: linkage groups; j: jonathan; g: golden delicious. additional file 4: figure s frequency distribution diagrams of fruit quality traits in an f population derived from ‘jonathan’ × ‘golden delicious’. fruit weight were measured from year to showed in a-d, fruit firmness were determined in and showed in e and f, whereas sugar content consist of three composition and fruit acidity including six kinds of organic acid and total acidity were analyzed only in each trait values were obtained from six fruits per n seedlings. additional file 5: figure s qtls for fruit quality traits identified by multiple qtl mapping. the results were consistent with that obtained by interval mapping. j: jonathan, g: golden delicious. additional file 6: table s candidate genes searched from the regions of five significant qtls. these five qtls were associated with three important fruit quality traits, two of fruit firmness, two of fruit weight and one of fruit acidity. the gene function annotation information of whole apple genome was downloaded from gdr website. candidate genes which have been used for expression analysis were in bold fonts. competing interests the authors declare that they have no competing interests. authors’ contributions rs handled the genetic map construction and qtl analysis, carried out searching and validation of candidate genes and drafted the manuscript. ysc and hl participated in the collection of phenotyping data. fqy, ybz and dmc helped to develop the segregating populations. yw and tw participated in the design of the study. xzz and zhh conceived of the study, and lead the whole project. all authors read and approved the final manuscript. authors’ information not applicable. availability of data and materials not applicable. the authors thank key laboratory of biology and genetic improvement of horticultural crops, ministry of agriculture, p. r. china for providing the test platform and instruments. also, thank to bgi-shenzhen for rad sequencing service and bioinformatics support. funding this work was supported by the project, the hi-tech research and development program of china; national special funds for scientific research on public causes project 200903044; and modern agricultural industry technology system.
of the markers, were mapped to ‘jonathan’ with a length of cm and the average distance between markers was cm, were mapped to ‘golden delicious’ with a length of cm and the average distance between markers was cm. genetic map based quantitative trait locus analysis is an important method for studying important horticultural traits in apple. a high-density genetic map with snp markers from individuals was generated. we demonstrated the construction of a dense snp genetic map in apple using next generation sequencing and that the increased resolution enabled the detection of narrow interval qtls linked to the three fruit quality traits assessed. we conclude that application of radseq for genetic map construction improved the precision of qtl detection and should be utilized in future studies on the regulatory mechanisms of important fruit traits in apple. applecandidate genesfruit quality traitsgenetic mapsqtlradseqsnpissue-copyright-statement© the author 2015 phenotyping data of fruit quality traits were calculated in 2008- once qtls were mapped, candidate genes were searched for in the corresponding regions of the apple genome sequence underlying the qtls. twelve significant qtls linked to the control of fruit weight, fruit firmness, sugar content and fruit acidity were mapped to seven linkage groups. based on gene annotation, and genes related to fruit weight, fruit firmness and fruit acidity, respectively, were analyzed.among the candidate genes associated with control of fruit acidity, changes in the expression of mdp were in agreement with the pattern of changes in malic acid content in apple during ripening, and the relative expression of mdp was significantly correlated withfruit acidity. the candidate genes mdp and mdp were found to be related to fruit acidity regulation. the snp markers were developed by restriction site-associated dna sequencing. then some of the candidate genes were validated using real-time pcr. to facilitate molecular breeding studies of fruit quality traits in apple, we aim to construct a high density map which was efficient for qtl mapping and possible to search for candidate genes directly in mapped qtls regions.
 genetic map based quantitative trait locus analysis is an important method for studying important horticultural traits in apple. to facilitate molecular breeding studies of fruit quality traits in apple, we aim to construct a high density map which was efficient for qtl mapping and possible to search for candidate genes directly in mapped qtls regions. methods a total of f seedlings derived from ‘jonathan’ × ‘golden delicious’ was used for the map constructionand qtl analysis. the snp markers were developed by restriction site-associated dna sequencing. phenotyping data of fruit quality traits were calculated in 2008- once qtls were mapped, candidate genes were searched for in the corresponding regions of the apple genome sequence underlying the qtls. then some of the candidate genes were validated using real-time pcr.  a high-density genetic map with snp markers from individuals was generated. of the markers, were mapped to ‘jonathan’ with a length of cm and the average distance between markers was cm, were mapped to ‘golden delicious’ with a length of cm and the average distance between markers was cm. twelve significant qtls linked to the control of fruit weight, fruit firmness, sugar content and fruit acidity were mapped to seven linkage groups. based on gene annotation, and genes related to fruit weight, fruit firmness and fruit acidity, respectively, were analyzed.among the candidate genes associated with control of fruit acidity, changes in the expression of mdp were in agreement with the pattern of changes in malic acid content in apple during ripening, and the relative expression of mdp was significantly correlated withfruit acidity.  we demonstrated the construction of a dense snp genetic map in apple using next generation sequencing and that the increased resolution enabled the detection of narrow interval qtls linked to the three fruit quality traits assessed. the candidate genes mdp and mdp were found to be related to fruit acidity regulation. we conclude that application of radseq for genetic map construction improved the precision of qtl detection and should be utilized in future studies on the regulatory mechanisms of important fruit traits in apple. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords applecandidate genesfruit quality traitsgenetic mapsqtlradseqsnpissue-copyright-statement© the author 2015 