Fine Mapping Of The Main Effect Of QTL PA7-1 And PA7-2 On The Low Hydrocyanic Acid Content Trait And Its Candidate Gene Of Sorghum-sudangrass Hybrid

As an important forage crop,sorghum-sudangrass hybrid has strong good heterosis,palatability,high nutritional value and can be moved and used for many times.However,due to the hydrocyanic acid in its young stems and leaves,it is easy for livestock to be poisoned after eating too much.Therefore,it is an important breeding target to breed a sorghum-sudangrass hybrid with low hydrocyanic acid.Based on the major QTL PA7-1 associated with low hydrocyanic acid in sorghum-sudangrass hybrid identified by our previous research,we used 1200 F2 individuals from a cross scattered ear sorghum×red shell sudangrass to locate QTLs for low hydrocyanic acid and a new major QTL named PA7-2 was detected.Then using BSA-SSR technology and the target QTL flanking markers associated with low hydrocyanic acid,the QIRs population was established by selecting 1200 F2 individuals,which were used to obtain F3 population by self-crossing.Then 130 recombinants with low hydrocyanic acid content were selected from F3 population to construct the fine mapping population.In this study,we focused on the fine mapping for two major QTLs PA7-1 and PA7-2,and developed the candidate genes and conducted functional analysis.The main research results are as following:1.The DNA of ten low and ten high hydrocyanic acid plants from 1200 F2 individuals of a cross scattered ear sorghum×red shell sudangrass was used to establish an extreme gene pool,which mixed with equal amounts of DNA.Using their parents as control,we selected 11 suitable SSR primers and used them to amplify 1200 individuals and their parents,and obtained a total of 253 polymorphic SSR loci.2.The 253 markers were used to construct a linkage group map based on the F2 population of sorghum-sudangrass hybrid,with a covering genome length of 211.5 cM and an average map distance between markers of 0.84 cM.Based on this linkage group,four QTLs related to the hydrocyanic acid content were detected,of which two major QTLs,PA7-1 and PA7-2,were identified with genetic contribution rates of 57.4%and 47.1%,respectively.3.The 1200 F2 individuals were screened by the QTL flanking SSR markers,and two QIRs populations from PA7-1 and PA7-2 were established,which contained 379 and 121 recombinant strains,respectively.Then the F2:3 population was obtained and used for QTL mapping,and the stability of the main QTL PA7-1 and PA7-2 was verified.4.For narrowing down PA7-1 and PA7-2 interval,the fine mapping population with 130 recombinants were used to fine mapping,respectively.Finally,PA7-1 was determined between markers sorbi4G4-120 and sorbi4G4-680,which contained eight markers;and PA7-2 was finally determined between Sobic.8g1-600 and XM00242-400,which contained six markers.5.Eight SSR marker fragments in the PA7-1 regions and six markers in the PA7-2 regions were recovered,purified,sequenced and sequence alignment,the high-resolution physical maps of the PA7-1 and PA7-2 target region was established,respectively.Finely,PA7-1 region was located in the 203.6 kb genome region of sorghum chromosome 4,which contained 18 candidate genes,and PA7-2 region was located on the sorghum cloned BAC 88M4 gene AY661656.1 and sorghum chromosome 8,which was reduced to 18.4 kb and 25.5 kb contained 5 candidate genes.6.According to the RT-PCR expression level analysis,three genes XM₀21458168.1,XP₀21313843.1 and AY661656.1 associated with low hydrocyanic acid at three different development stages of seedling,tillering and elongation stage in male parent red shell sudangrass and F2 plants with low hydrocyanic acid showed obvious expression.It indicates that the three genes are important candidate genes which can regulate low hydrocyanic acid content of sorghum-sudangrass hybrid.