Genetic Character And Gene Mapping Of An Albino Green-Revertible Mutant In Rice

Leaf color mutant was a familiar mutant type in nature.,which attracted much attention for a long time because of its visible phenotype and discernable character. Albino green-revertible mutant was a new leaf color mutant in rice.The mutation was usually controlled by a recessive nuclear gene and therefore can be used as a phenotypic marker for monitoring seed purity in hybrid rice production,especially in two-line hybrid rice in which seed purity usually affected by temperature.Meanwhile,it also can be used as an ideal material for research on chloroplast genetic and development,chlorophyll biosynthesize and regulation,signal transduction and photosynthesize mechanism.A new rice mutant with albino green-revertible trait was found from the pollen culture of W9461S/W6154S in 1994.The first three leaves were albino and converted to green until the fourth leave came out.After 10 progenies observation,this mutant trait was also stable expression,so it belongs to a stable genetic variety,and a new male sterility line with this albino green-revertible trait has been bred through backcrossing with Pei Ai 64S,named as B01S.In this study.,we used B01S as the research object,the phenotype trait,genetic character,ultrastructures have been systematically investigated.Furthermore,the mutant gene was mapped with SSR markers,and four predicted candidate genes related with the albino green-revertible trait were obtained.This result will provide theory foundation for cloning of the mutant gene and research on molecular mechanism of chloroplast genetic and development The brief results of this paper were summaried as follows:1 Phenotype of leaf color B01SThe phenotype of the green-revertible albino in B01S was different from other albino mutants reported.The leaf color mutant trait of B01S could be described as follows:the coleoptile and limb of the first leaf was albino when sprouting,the main stem and tillers was pale green.The second and third leaf was gradually totally albino.After the fourth leave came out,all the leaves reverted to green as normal plants.2 Genetic analysis of mutant B01SThe leaf color of F₁ populations from the cross between B01S and normal cultivars showed totally green.The segregation ratios of green to albino in the F₂ populations were fitted to 3:1.These results indicated that the albino green—revertible trait was controlled by a recessive nuclear gene and not affected by cytoplasm3 Ultrastructure of the chloroplast in the mutant B01SThe ultrastructure of chloroplast in B01S at 2-1ear-stage and 4-1ear-stage was observed with TEM..At 2-leaf-stage,the chloroplast structure couldn't be observed,only plenty of small vesicle-like structures and some osmium particles..At 4-leaf-stage,the mutant exhibited distinct chloroplast structure,also the number and size of the chloroplast was as normal as control,which occupy 80%of the cell.4 Gene mapping of the mutant B01S.SSR markers and BSA method were employed to map the albino green -revertible trait,among 325 markers on 12 chromsomes of fice,14polymorphism markers were obtained between parents and 2 DNA bulks.The mutant gene was preliminary located on chromosome 3 and linkage with RM411,RM2346,separated by 3.40cM,4.56cM,respectively.Then F₂,F₃,F₄ were used as large mapping population,the mutant gene was mapped within a 390 kb interval between RM7134 and RM6832 at last,and named as rgr(t).5.Candidate gene predictionThe target region was analyzed with Rice Genome Annotation-Release 5 on TIGR.In 390 kb interval between RM7134 and RM6832,it includes 4BAC which contain 70 putative genes,4 predicted candidate genes related with the mutant trait were selected in the target region.One encodes a putative chlorophyll a/b binding protein and other three encode putative cytochrome P450.6 RT-PCR gene expression analysisFour candidate genes were selected for Real Time RT-PCR to confirm the mutant gene.Results indicate that the gene LOC-Os03g39690 which encodes putative cytochrome P450 with 512 aa has significant experession difference between B01S and P64S,which has massive gene expression in P64S,but nearly no expression in B01S.So it was considered to be an important candidate gene in leaf color regulation.