Preliminary Analysis Of The Genetics Of Carthamus Tinctorius L. Germplasm Accessions With SRAP Markers, ITS And Specific Sequences Of Chloroplast

Safflower (Carthamus tinctorius L.) which belongs to the genus Carthamus tinctorius ofAsteraceae, originated in Egypt, is a special oil crop and using as ways of edible, officinal,dye and feed.It has a strong adaptability on dryland, and is widely cultivated in Central Asia,West Asia and the Mediterranean region.In this study,11varieties from the Xinjiang area,15varieties from the Yunnan area and8varieties from central provinces of China that in total of34varieties of safflower are testmaterials. Using SRAP molecular markers, ribosomal sequences of ITS, cpDNA spacersequences as three methods to research on34varieties of Safflower for genetic diversityanalysis, use flow cytometry assay on different varieties to calculate their genome value. Itrevealed the genetic information of safflower genome and the relationship between thedifferent varieties and their genetic background. The results are as follows:1. There are25SRAP upstream primers,37downstream primers, in total of925primercombinations. The YH0003genome was amplified and screened with those combinations，resulted with346pairs of primer combinations which had clear spectrums. Randomlyselected40primer combinations to amplified with34varieties of safflower sample, observed572clear bands, in which212were polymorphic. Using the software SLT_NTsys2.10toanalyze bands, and resulted similarity coefficient (GS) changes in the range between0.5375and0.9411, and34safflower varieties could be classified into three groups, groupⅠ contains11species from the Xinjiang region; group Ⅱ included in the central provinces of9speciesand one varieties in Yunnan;8varieties from Yunnan province are included in group Ⅲ;another five Yunnan safflower varieties are individually divided from the groups.2. Using33different varieties of safflower genomic DNA as the template for ITS-PCRamplification and Sequencing.Using DNAman software to edit the sequences beforeBLASTN homology searching. The sequencing results showed that32cultivars ITS sequencelength of751bp,and only the XI AN safflower and YH5511respectively for750bp and741bp.The length of5.8S rRNA sequence stay on164bp;32varieties of ITS1sequence length is308bp, only YH5511length is298bp for lacking10bp;32varieties of ITS2sequence length is279bp, only the XI AN safflower length of278bp for lacking1bp. There were few Insertionand deletion mutations in the ITS sequence of safflower varieties, and this situationmaintained the high stability of the length of the sequences. Using software ClustalW2to dothe ITS sequence aligment,33safflower cultivars could be divided into three major groups atthe GS45.69, only three species were divided out of groups, groups Ⅱ can be further dividedinto two subgroups. The study also found that ITS sequence variation are faster and moretypes of mutations in safflower varieties, some only a single safflower cultivars, indicatingthat the mutations associated with the category.3. Using the RNA structure software to predict the second structure5.8s rRNA, ITS1andITS2RNA, and choose the structures in selection with the minimum free energy. The resultsshowed that5.8s rRNA second structures are highly conserved, only find out one variation in second structure of1species of the occurrence of a mutation in a short arm; ITS2secondstructure types are divided into4kinds of configuration, in which6species for theconfiguration Ⅰ,25varieties belong to configuration Ⅱ, configuration Ⅲ and configuration IVeach contains one variety. The main difference between configuration I and configuration Ⅱare the quantity and length of C armlet. Configuration Ⅲ in A, B, D, E conformation areslightly changed, but only have a few differences with configuration Ⅰ,Ⅱ; configuration IVchanges greatly, has plenty of differences with configuration Ⅰ,Ⅱ, Ⅲ in all regions. ITS1second structure is relatively conservative, mainly divides into3configurations and2of themonly contain one single variety. In the ITS1second structure, configuration Ⅲ changesgreatly.Its C, D, E structure is similar to the B, C, D structure of the ITS2configurationI,which are a short arm structure supporting a plurality of stem-loop. The structure is similarto the "clover" configuration and may has some conformational recognition to combine thesplicing complex. Although the ITS1configuration Ⅲ changes a lot with I and Ⅱ, but due tothe similar structure to ITS2, so may still can be identified to the splice complex.The ITS sequence of5.8s rRNA second structure was highly conserved, due to it is acoding sequence which was under a great selected pressure; and ITS1, ITS2sequences aresheared off during the40S precursor rRNA processing, the two sequences are folding into thesecond structure of RNA and the structures may have recognition regions to rRNA slicingcomplex, and these recognition regions are very conservative because of its function. We caninfer that the second structure also contains a rich recognition regions and geneticinformation.4. Choosing the chloroplast petN-psbM spacer sequence primers and psbM-trnD spacersequence primers to do PCR reaction with safflowers. PCR products were purified andsequenced, then analyzed the sequences with the ClustalW2software. Experimental resultsshow that safflower petN-psbM sequence length is655bp, the content of CG are changes29.6%to29.8%, which occurs a C-A base replacement at412bp location. The psbM-trnDsequence length of710bp, CG content of37.2%,100%conserved. There is few Differences insafflower genus of either chloroplast petN-psbM or psbM-trnD spacer region,which indicatingthat they are highly conserved. Using BLASTN to search safflower petN-psbM homologoussequences and downloaded the petN-psbM sequences of Centaurea, Chrysanthemum,Atractylodes plants, which receals a highest similarity of98.63%, and the lowest of89.97%,and build the phylogenetic tree of the Compositae plants.5. Measuring C-value by flow cytometry in9varieties of safflower samples, and resultedthat the largest value is4439Mbp, and2988Mbp for the minimum.