| Potato(Solanum tuberrosum L.)is the fourth important food crop in the world.Chips and French fries are very popular in potato processing industry.However,cold-induced sweetening(CIS)is a big problem for the industry.CIS means when harvested tubers are stored in low temperature to reduce sprouting,weight loss and pathogenesis,it will result in the accumulation of reducing sugars fructose and glucose.Then a nonenzymatic Maillard reaction between reducing sugars and α-amino acids of nitrogenous compounds during high temperature frying leads to a dark-colored and bitter products.More severely,acrylamide carcinogenic to human will be formed during the Millard reaction based on reducing sugars and asparagine in potato tubers.The efficient solution to the two problems caused by the Millard reaction is to understand the mechanism of CIS and find tightly linked markers for CIS.Thus,we performed Linkage mapping and association mapping in this study.The main results are as follows:1.Development of SSR markers based on potato genome sequence28609 simple sequence repeats(SSR)were detected in the whole potato genome(PGSC＿DM＿v4.03),including 9 repeats of dinucleotide,6 repeats of trinucleotide and 5repeats of tetranucleotide.The average number of SSR loci in each chromosome is 2384.There are 189 kinds of motif types for SSR in total.Dinucleotide and trinucleotide repeats constitute the main types of SSR about 15943 and 11053 loci,respectively.The SSR numbers differ in motif types.Motif types enriching AT and TA constitute the main types of dinucleotide repeats including 7902 and 6933 loci,respectively.For trinucleotide repeats,the loci number of each of AAT,ATA,ATT,TAT or TTA type is about 1000,taking up 40 % in total while for tetronucleotide repeats,the loci number of each of AAAT,AATA,TATT or TTTA type is about 100,taking up 26 % in total.According to the distribution of SSR loci,40 and 59 pairs of primers were designed for chromosome 5 and 6,of which 13 and 12 pairs of primers were mapped and generated32 and 30 polymorphic sites,respectively.The 13 and 12 SSR markers on the genetic maps are in accordance with those on the physical maps.It is an effective way to develop SSR markers based on genome sequence for higher density genetic maps.2.PopulationA diploid F1 population consisting of 178 offspring was obtained from the cross between ED25 sensitive to CIS and wild species S.berthaultii tolerant to CIS.ED25 is a hybrid from 2cultivars S.phureja、S.tuberosum and wild species S.vernei.The results of phenotypic evaluation showed that reducing sugar content werenormal distribution,that was suitable for QTL mapping.3.Construction of parental linkage mapsLinkage maps of both parents E and B were constructed by two-way pseudo-testcross,resulting in 12 linkage groups for each parent.For paternal maps,the total length is 1037.9 c M with average marker distance about 8.0 c M.And the longest marker distance is 35.8 c M on the top of chromosome 10.The paternal maps contain 129 SSR markers with average 11 for each chromosome.Specifically,chromosome 3 has 19 markers at the most while chromosome 1 has 4 markers at the least.Besides,18 candidate genes yielding 29 alleles are distributed on 8 chromosomes.For maternal maps,the total length is 1043.5 c M with average marker distance about 4.3 c M.And the longest marker distance is 27.5 c M at the bottom of chromosome 5.The paternal maps contain 245 SSR markers with average 20 for each chromosome.Specifically,chromosome 5 has 33 markers at the most while chromosome 9 has 7 markers at the least.Besides,29 candidate genes yielding 54 alleles are mapped in maternal maps,more than in paternal maps.Our results indicate that parent E is more heterozygous than parent B according to SSR marker numbers and map density.4.QTL mapping of cold-induced sweetening(CIS)related traitsThe content of tuber reducing sugar was evaluated after harvest(0 d)in 2environments and also at day 30 stored at 4℃in 6 environments and reconditioning for 20 days in 6 environmets.For tubers after harvest,7 CIS QTLs were detected,withthe most powerful QTL explaining 14.6% of the phenotypic variation and while the weakest QTL explaining 7.1%.For tubers stored at 4℃ for 30 days,21 CIS QTLs were detected with the most powerful QTL explaining 16.8% of the phenotypic variation and while the weakest QTL explaining 5.4%.QTLs CISED05-2,CISED11-1 and CISSb11-3 were detected in both tested environments.For tubers reconditioned in 6 environment,16 CIS QTLs were detected with the most powerful QTL explaining 12.9% of the phenotypic variation and while the weakest QTL explaining 6.5%.QTLs CISSb05-1 and CISED11-1were detected in 3 environments while CISED11-1 was detected in 2 environments.In order to understand the mechanisms of reducing sugar accumulation during cold storage and sugar consumption during recondition,QTLs were detected based on reducing sugar changes after and before cold storage(4℃ 30d-0 d)in 2 environments as well as after and before recondition(Rec-4℃ 30d)in 6 environments.For the former,8 CIS QTLs were found with the most powerful QTL explaining 12.5% of the phenotypic variation and while the weakest QTL explaining 6.7%.For the latter,12 CIS QTLs were found with the most powerful QTL explaining 11.3% of the phenotypic variation and while the weakest QTL explaining 2.6%.QTL CISSb10-1 was detected in 2 environments.Overlaps were observed between QTLs for different traits at different time points.QTL CISSb05-1 was found at 3 different temperature treatments: 0 d,4℃ 30 d and Rec as well as Rec-4℃ 30 d while CISSb11-3 was found at 3 temperature treatments: 0 d,4℃ 30 d and Rec.There are 2,5 and 2 QTLs only detected for 0 d,4℃ 30 d,respectively.Our results show that tuber reducing sugar content is a sophisticated trait controlled by multiple genes independent of temperature treatment.Overlaps of QTLs were detected at different temperature treatments indicating that the same QTLs function from harvested,during cold storage to recondition.At the same time,CIS QTLs specifically for one temperature treatment were also found,which had effects only at a specific temperature treatment.5.Interaction analysis of detected CIS QTLsWith QTL Network,we analyzed the additive and epistatic effects of those detected CIS QTLs.We found that 7 QTLs have additive effects and 6 pairs epistatic QTLs involving 8 QTLs.Additive QTLs were used to analyze relationships between additive effects of QTLs and reducing sugar content.Results show that the sugar content is decreasing as positive markers are increasing.Compared to progenies with no positive markers,the reducing sugar content of progenies with 1 positive marker reduced by41.0% after harvest,with 4 positive markers reduced by 29.8% after cold storage and with5 positive markersreduced by 39.4% after recondition.There are 6 pairs epistatic QTLs detected all in marental maps involving in 8 QTLs,which are distributed on 6 chromosomes.4 of 8 epistatic QTLs on the maternal maps interact with 2 other epistatic QTLs and the other 4 interact with only one epistatic QTL.The interaction analysis of detected CIS QTLs indicates that sugar content is affected by both additive and epistatic effects.More interestingly,additive effects are powerful and stable across environments and treatments.6.Co-localization of candidate gene markers and CIS QTLsCandidate gene approach is an effective way to identify genes for target traits.39 genes were mapped,with 18 genes generating 29 alleles mapped on paternal maps and 29 genes generating 54 alleles mapped on maternal maps.Co-localization between candidate gene markers and CIS QTLs was observed.There are 10 candidate genes as well as 2CAPS markers generating 18 alleles are co-localized with CIS QTLs.For those 10 candidate genes,InvGF and St Lin6 are invertase degradating sucrose in the pathway of sucrose degradation.GWD and Sb RFP1 function in starch degradation.AGPase and SSSⅠare key enzymes in the pathway of starch synthesis.G6 pt and Sut1 are responsible for transporting Glucose 6-phosphate and sucrose.Fk,namely,fructokinase,havs effects on glycolysis.Ppe and Aco function in Calvin cycle and TCA cycle.The co-localization of CIS QTLs and candidate genes or enzymes of multiple pathways indicates that reducing sugar content is a complex trait involving in many biochemical and physiological pathways.Our results lay a good foundation for establishing regulatory network.7.Coefficient analysis between candidate gene markers and reducing sugar contentA natural population was composed of 64 potato materials including parents for hybridization and advanced breeding clones.The content of tuber reducing sugar was measured after cold storage(4℃)for 30 days.76 candidate gene markers were used for identifying polymorphism.By coefficient analysis between candidate gene markers and reducing sugar content,we found that 4 genes markers and a CAPS marker are significantly correlated with reducing sugar content.The 4 genes are named GWD,G6 pdh,Sus4 and Stp L.Stp L has a negative relationship with reducing sugar content while the others have positive relationship with reducing sugar content.Additive effects were found in 4 markers: GWD,G6 pdh,Sus4 and GP250.As the number of genotype 1 is increased,the reducing sugar content is decreasing.GP250,located on the bottom of chromosome 11,is likely to co-localize with CIS QTLs.Sus4 catalyzes sucrose degradation and synthesis,and mainly for the former process.G6 pdh is the key enzyme in the pentose phosphate pathway.GWD,alpha-glucan water dikinase,functions in starch degradation,co-localized with a CIS QTL.The above results also show that reducing sugar content after cold storage is involved in a number of biochemical and physiological pathways,largely regulated by additive effects,which is accordance with linkage analysis.In all,our study provides theoretical and substantial bases to establish molecular assisted selection(MAS)for CIS breeding. |
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