Screening Of Genes Related To Synthesis Of Colchicine And Construction Of Genetic Map In Hemerocallis Citrina Baroni

The long yellow daylily(Hemerocallis citrina Baroni)is widely cultivated in China as a functional perennial vegetable.However,there is colchicine in the fresh flower bud of H.citrina and it could cause toxic reaction after taken excessively,and the period of sexual reproduction is longer than the other annual vegetables.These conditions lead to the crossbreeding progress for good varieties and characters of H.citrina is slow.So,we selected the gene for colchicine biosynthesis and built the genetic linkage map of H.citrina.It could be beneficial to clone the gene for colchicine biosynthesis and shorten the crossbreeding process with marker assisted selection.In this study,the root,flower bud and leaf in florescence of H.citrina were used as material for RNA-Seq,the gene for colchicine biosynthesis and the stable reference genes for RT-qPCR were selected.The polymorphism EST-SSR markers were used for the construction of intraspecific and interspecific genetic linkage map of H.citrina.The main results as follows:1.The root,flower bud and leaf in florescence of H.citrina were used as material for RNA-Seq and obtained 92107 Unigenes.These Unigenes were compared to the database of NR,Swiss-Prot,GO,COG,KOG,KEGG,and Pfam using BLAST and got annotation information for 41796 Unigenes.There were 404 Unigenes' annotation information corresponded with the enzymes which are involved in the pathway of colchicine metabolism.Among them,126 Unigenes were DEGs.There were 19 and 24 DEGs had positive and negative correlation property to the actual content of colchicine in different organs,respectively.These 43 DEGs were maybe related to colchicine biosynthesis and correlation coefficients of 3 DEGs in them were significant at p<0.05 level.2.Six candidate reference genes(18S,60S,UBQ,AP4,ACT,and TUB)were developed from RNA-Seq.The stability of 6 reference genes were calculated by geNorm,NormFinder,BestKeeper,and RefFinder programs for three experimental conditions including different development stages of flower buds,different organs,and commercial flower buds of different landraces.The results showed that AP4,60S,and ACT was the most stable reference gene for flower buds at different development stages,different organs,and commercial flower buds of different landraces,respectively.For all three experimental conditions,TUB exhibited the most stable expression,and UBQ was the least stable reference gene.3.The native variety 'Dongzhuang' and landrace 'Chongli' of H.citrina was used as parent for crossbreeding and obtained 120 F1 progenies.There were 1076 EST-SSR markers were analyzed and 205 markers had polymorphism among parents and progenies.We build a intraspecific genetic linkage map of H.citrina contained 11 linkage groups with 199 EST-SSR markers.The map length was 2522.34 cM and the average distance between markers was 12.68 cM.The estimated length of genome was 2674.51 cM,the coverage ratio of map was 94.31%.4.The landrace 'Datong' was used as female parent to cross-fertilize with 9 different ornamental cultivars of Hemerocallis fulva.The cross combination 'Datong' x 'Yaolanqu'harvested the most F1 progenies of 55.There were 222 EST-SSR were used to build a genetic linkage map with 11 linkage groups.The map length was 2485.59 cM and the average distance between markers was 11.20 cM.The estimated length of genome was 2620.04 cM,the coverage ratio of map was 94.87%.5.There were 90 identical EST-SSR markers in the intraspecific and interspecific genetic linkage map,and they had similar order on the corresponding linkage groups.However,there were several markers on the different location of the corresponding linkage groups,maybe they implied that the structural variation of the chromosome in H.citrina and H.fulva.