| Anthocyanin phenotypes,with strong visual detection,is not essential for development,and non-toxic,has long been considered as marker traits in purity identification of hybrid seeds and a useful reporter system for both transient expression studies and transformation indicating.As an example of many dispersed genes controlling a single trait,pigmentation plays an important role in studies of gene interaction.As a pigment trait,coleoptile purple line,which clearly appears in both opposite side of Coleoptile in some rice materials,is useful in identifying the purity of rice hybrid seeds quickly,exactly and cheaply because it expresses at budding period and can performance a inheritance behavior of F1 generations expressing purple line but its two parents don't.And also these coleoptile purple line involved genes can be employed in developing pigment reporter gene system for those characteristics above.To effectively advance its using in those expected fields,follow efforts have been done:comparing the modality and tissue structure between wild and mutant by anatomical and cytological observation; candidate genes guessing and detection of mutation locus by fine mapping and sequencing;the organ speciality of genes expression and the interrelation of the involved genes by RT-PCR and Q-PCR; transformation of OsAi gene;and breeding the hybrid combinations whose purity can be easily identified;and validation the reliability of the purity obtained from the purple line method.And the main conclusions are as follows:1.there is no difference but pigmentation between purple line and no-purple line materialsIn wild R25,two purple lines lei in the coleoptile symmetrically.The cells in the pigment region are smaller and denser than the other parenchyma cells which results in the constrictions in the coleoptile.Except none anthocyanin staining on the corresponding region in YR25 there is no difference between the wild and mutant parents after anatomical and cytological examination.2.fine mapping of two purple line related genes and candidates screeningUsing the F2 generation ofⅡ-32A×R25,we restricted the OsAi gene between 8563749bp and 8614989b on chromosome 11.There is a bHLH factor gene OsbHLH14 in this region.Because the orthologs(ZmB1 and EGL3) of OsbHLH14 in Maize and Arabidopsis are involved in pigment pathway, we commend OsbHLH14 as the candidate of OsAi.By the other F2 generation of ZhongjiuA×R25,we found that the chromogen gene C riding on chromosome 6,between RM5754 and RM19665.One maize PI/C1 orthoglog OsC1,which is a MYB transcription factor,is waiting there and we speculate that OsC1 is the candidate of gene C.3.the difference of OsAi and OsC1 genes between their wild and mutant type and the OsAi mRNA sequence3.1 the difference of OsAi betweenⅡ-32B and YⅡ-32BFrom the start to stop code of OsAi gene,we detected 24 mutant loci,in mutant type YⅡ-32B, including 3 amino acid substitution mutations.It is too much of the DNA mutation to determinate which mutantion resulted in function losing.However,we found that OsAi failed to express in YⅡ-32B coleoptile which indicate that no purple line in YⅡ-32B is resulted from the failed transcription promotion of OsAi gene.3.2 the difference of OsC1 between R25 and YR25We detected a 10bp deletion in the third extron of OsC1gene in mutant YR.25.The deletion occurred in the MYB domain and result in shift mutation in amino acid sequence and translation stop ahead.Such a tremendous change must prevent pigment structure gene downstream from expressing and make no purple line in coleptile.3.3 the gene structure and the mRNA sequence of OsAiIncluding 280bp 3'UTR,1284bp ORF and 46bp 5'UTR,the mRNA sequence is 1610bp long.By aligning with the DNA sequence,we found there are 5 extrons in OsAi gene.In the predicted fourth extron,a hidden intron was discovered.And thus,the factual extrons are one more than the prediction. Additional,the factual ORF extend 35bp towards the predicted 3'UTR.4.Phylogenetie analysis of OsAi and OsC1There are orthologs of OsAi in many plants such as maize(ZmB1 and ZmRs),sorghum(SbB1-1 and SbB1-1),Vitis vinifera(VvMYCA1) and Arabidopsis(GL3 and EGL3) and so on.And another bHLH factor gene OsB1,which controls the purple pericarp in rice,plays as a paralog of OsAi.On the phylogenetic tree,OsAi and OsB1,ZmB1 and ZmRs,SbB1-1 and SbB1-1,these six genes fell into the same grass bHLH clade.The OsC1 and its orthologs,ZmC1,ZmPL3,Sb350P03.14 and TaMYB3,fell into a grass MYB branch.Dislike OsAi gene,OsC1 has no paralog in rice.5.the organ specificity of OsAi and OsC1 expression and the interrelation between these two genesIn this research,OsAi gene only expressed in coleoptile and OsC1 gene expressed in coleoptile and leaf.And we found that the OsAi gene can expresses near normally in mutant of YR25 and OsC1 gene can expresses near normally in mutant of YⅡ-32B.It shows that the relation between OsAi and OsC1 is not an up-downstream but a cooperation relationship.6.Function complementation vector of OsAi gene was constructed and transgenic plants have been detected.An～4.9kb genomic DNA fragment containing the entire OsAi coding region,a 1504-bp upstream sequence,and a 1052-bp downstream sequence was cloned into the binary vector pCAMBIA1301 to generate p1301OsAi construct.We have detected four positive transformants from the differentiated plants and the function validation is underway.7.hybrid seeds(YⅡ-32A,2081A×Luhui 17) purity identification can be conducted by employing purple line methodBy backcross breeding and hybridization breeding,we made two CMS lines YⅡ-32A and 2081A respectively.No of them was pigmented in coleoptile but in stigma.Pollinating by restorer line Luhui 17 which performance no pigmentation in all organs,we got the hybrid seeds of YⅡ-32A and 2081A×Luhui 17.Both of the two combinations seeds express coleoptile purple line when germinated. F1 displaying purple line but CMS and restorer lines having no purple line in coleoptile presents a possibility of purity identifying by purple line,8.the purity identification results by purple line is reliableAfter being validated by blind test,molecular marker detection and field planting examining, purple line method is very reliable in purity identification of hybrid seeds,In additional,this method is the most time and money saving way compared to other used methods.So we concluded that purple line method can satisfy farthest the 'quick,exact and cheap' requirement from hybrid seeds purity identification in rice.
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