Interval Mapping And Molecular Marker Developing Of Red/Green Skin Trait In Oriental Pear

Pear(Pyrus L.)is an international staple fruit,and the third largest fruit tree besides apple and citrus fruits in our country,with planting area of more than 1.11 million hm2.China is the main origin of oriental pear varieties and has many kinds and cultivars,including some precious resources with red skin.Red skin pear was favored by consumers at home and abroad because of its bright-colored appearance and potential health value.And it also has the attention of the scientific research and production institutions gradually.Locating red/green skin trait loci of oriental pear has important implications for elucidating the genetic mechanism and guiding red skin pear breeding.This research aims to lay the foundation for the further study of molecular markers assisted red skin pear breeding and red skin trait.We used hybrid populations from Zhengzhou Fruit Research Institute of Chinese Academy of Agricultural Sciences to carry out oriental pear red skin trait phenotype and genetic rules analysis,oriental pear red/green skin trait locus interval mapping,oriental pear red/green skin trait molecular markers developing and validating,and red/green skin trait locus and related genes linkage group mapping.The detailed results were as follows:1.Red skin trait observation and genetic analysis of oriental pearUsing two hybrid population of ’Mantianhong’× ’Hongxiangsu’ and ’Yuluxiang’×’Mantianhong’ as the materials,red skin trait phenotype and genetic rules of oriental pear were studied using chromatic aberration analysis and artificial sentence in consecutive two years.The results showed that red color was scattered around fruit dots,color concentration was highest near the fruit dots and gradually reduced from fruit dots.There was no yellow or green pigment accumulation within the epidermal layer.Red parts and transparent parts distributed over there.Parental genotypes had great influences on progeny coloring.The progeny of ’Yuluxiang’×’mantianhong’ was more red and better coloring than that of ’Mantianhong’ × ’Hongxiangsu’.Climate conditions had also a great influence on oriental pear coloring.The climate in 2014 was more conducive for red pears to coloring than 2015.2.Interval mapping of red/green skin trait loci in oriental pearA red pool with mixed DNA of 28 red extreme phenotypes and a green pool with mixed DNA of 27 red extreme phenotypes of the hybridized combination between’Mantianhong’ and ’Hongxiangsu’,and 4 parents of ’Mantianhong’ and ’Hongxiangsu’with relative phenotype were sended to perform high-throughput sequencing.Sequences were then aligned to the reference sequence of ’Dangshansu’ and performed SNP-calling for bulked segregation analysis(BSA)strategy.The SNP-index of red pool and green pool,as well as corresponding Δ(SNP-index)and |Δ(SNP-index)| were computed out based on the QTL-seq method.The result showed that there were 545 SNPs both in the set of 2 progeny pools with |A(SNP-index)|≥0.6 and in the difference homozygous SNP set of 2 relative phenotype pools of 4 parents.The primary election region of clumped SNPs of the 545 SNPs was 2.97Mb.Also the manhaton plots of |Δ(SNP-index)| were drew with 200Kb window and 20Kb step size by slip window method.The locus related with red/green skin trait of oriental pear were found to be located between a 1.86Mb candidate region,Which contained 14 sub regions on 11 scaffords.Most scaffords in the primary election region and the candidate region were the same,which showed the accuracy of positioning in our study.3.Molecular marker development and validation of oriental pear red/green skin traitAccording to the results of BSA interval mapping,we analyzed the F1 population derived from a cross between ’Mantianhong’ and ’Hongxiangsu’ with genic-SSR primers within signal interval,InDel primers and SSR primers designed on the basis of candidate interval.The results showed that four primers were best to distinguish the red and green hybrids within this population,including In2130-12,In2130-16,In2130-19 and ZFRI130-16.These four primers were able to identify all the 117 red plants from the population that included 299 plants totally.A linkage group was constructed which spanned 19.2cM.In2130-16 was linked to the nearest locus that controlled red/green fruit skin color with the linkage distance of 2.5cM.In2130-12,In2130-19 and ZFRI130-16 were linked to the locus with the distance of 2.6cM.With expanding this population,the results indicated that In2130-12,In2130-16 and ZFRI130-16 could still distinguish 458 hybrids derived from a cross between ’Yuluxiang’ and ’Mantianhong’ very well.The coincidence rate of each one’s genotype and phenotype among 310 plants was more than 90%.But these three markers showed differences in identifying the half-sib selections of male parent ’Hongxiangsu’ and the selected cultivars.ZFRI130-16’s distinguish ability was the best among them,and its accuracy was 97.87%.There was only one mistake when ZFRI130-16 identified all the tested cultivars.So ZFRI130-16 marker could be used for molecular markers assisted selection breeding in hybrid population which related to red skinned pears.4.Mapping candidate region and related genes of pear red/green skin trait to linkage groupUsing the SSR and SNP genetic map drawed by Wu et al.in 2014 as reference map,we mapped the candidate region of pear red/green skin trait and anthocyanin pathway genes to linkage groups of the reference map by comparing the common scaffold and the co-marker.The result showed that scaffold 3.0 of the candidate region were mapped to the 15th linkage group,while other scaffolds related to the candidate region were all mapped to the 5th linkage group.The red skin trait gene Red and a related locus RPS of european pear were mapped to the 4th linkage group and the 3rd linkage group respectively.The red/green skin trait locus of oriental pear and the red skin trait loci were mapped to different linkage groups.This result showed that there were different molecular mechanisms of the red skin trait between oriental pear and european pear.Among the anthocyanin pathway genes,PcCHS and PpCHS1 were mapped to the 4th linkage group,near the red skin trait gene Red of european pear.Another two anthocyanin pathway genes PpDFR1 and PpCHS3 were mapped to the 15th linkage group.PpDFR1 was located in the scafford 3.0,which was the sub region of oriental pear red skin trait,and PpCHS3 was near the scafford 3.0.These anthocyanin pathway genes may have a relatively close relationship with the red skin trait of oriental pear and european pear respectively.