Map-based Cloning And Functional Analyses Of The Albino Lethal Gene AL1 And Grain Shape Gene GS9 In Rice(Oryza Sativa L.)

Rice is the most important food crop in China,and thus it is essential to keep the high yield to meet the requirement from both national and consumer's levels.There are main factors determining the final yield.The photosynthesis in leaves has a great influence on plant growth and production,which is mainly determined by chloroplast development.After maturation,the rice yield is finally determined by three factors,panicle number,grain number and grain size.Among them,the grain size is very important to not only the yield but also grain quality.Up to now,there are more and more useful genes cloned.But,it is also necessary to clone and mine more novel genes/alleles to full illustrate the mechanism of plant growth as well as the formation of final yield and grain qualities.In our previous works,a series of chromosome segment substitution lines(CSSL)had been developed between the japonica and indica cultivars.Among them,several lines with novel traits were selected.In present study,two advanced CSSLs,one with albino phenotype and another showing long grain shape,were used,and the target genes were isolated by map-based cloning.Subsequently,several experiments were carried out to illustrate their functions.1.Cloning and functional analyses of the albino lethal gene AL1.The plastid ribosome is essential for chloroplast biogenesis as well as seedling formation.Up to now,all the plastid ribosomal proteins(PRP)have been identified in higher plants.However,their assembly in the chloroplast ribosome,especially in rice remains unclear.From the CSSL population,we identified a novel CSSL N150,and in its self progeny,there were also some albino lethal seedlings.These mutants displayed an albino phenotype at the seedling stage and did not survive past the three-leaf stage.No other apparent differences in plant morphology were observed in the mutant.The albino phenotype was associated with decreased chlorophyll content and abnormal chloroplast morphology.Besides,the albino seedlings contained some proplastid-like structure,and there was no any thylakoid membrane or lamellar structure in the plastid of the albino seedlings.Genetic analysis showed that the proportion of green to albino seedlings was about 3:1,which was consistent with the genetic principle of single Mendelian factor.Based on the whole genome re-sequencing analysis,the albino seedlings from the CSSL line N150 was identified to have an introgressed homozygous segment of about 3.74 Mb on chromosome 1 from the donor parent.The albino lethal phenotype was found to closely link with the introgressed segment,thus the gene responsible for the albino phenotype was named as all(albino lethal on chromosome 1).Thirty molecular markers located the introgressed region were developed and used to analyze the 1417 albino individuals.Finally,the target gene,AL1,was mapped to a physical region of about 42.2 kb between two molecular markers CAPS 107 and CAPS113.In the 42.2 kb region,there are 7 open reading frames(ORF).Expression analyses showed that the ORF1 and ORF7 genes were not expressed in rice seedling,and there was not significant difference on the expression levels of other five genes between the wild type and aZ1 mutant.Sequence analysis implied that there was a spontaneous single base mutation(C/T)in ORF2 coding region,which resulted in an amino acid substitution,from leucine in AL1 to phenylalanine in all,ORF2 encodes the plastid ribosomal L12 protein(PRPL12,L12),which affects chloroplast development.Transgenic complementation test confirmed that the AL1/L12 gene from wild type Nipponbare could recover the normal green phenotype of the albino mutant.Besides,the results from RNAi test showed that there could generate seedlings with varying degrees of leaf color and complete albino phenotype by suppressing the expression of wild type AL1/L12 gene in Nipponbare.These results indicated that the mutation of the PRPL12(ORF2)caused the albino phenotype in all mutant.The expression level of AL1 was significant higher in leaves than that of other tissues,such as the leaf sheath and root.The AL1 expression is light induced,and remains at a relatively stable level in different light conditions.Interestingly,the all transcript level in albino mutant was similar to that of AL1 gene in wild type,but the all protein level in all mutant was much lower than AL1 protein level in the wild type.The results showed that either AL1::GFP fusion protein or all::GFP fusion protein were localized in rice chloroplast,indicated that the all mutation did not alter the subcellular localization of the target protein.The analyses from bioinformatics showed that the 50S ribosomal protein L12 had high sequence similarity in different species,and the phylogenetic tree could be roughly divided into two distinct groups,which are located in the chloroplast and cytoplasm,respectively.AL1 protein contains 185 amino acid residues,the point mutation in all is located in the conserved domain of AL1/L12 protein,which is in the region for the binding between L12 and L10(another PRP).Yeast two hybrid analysis indicated that AL1/L12 could interact with L10,while the interaction between mutated all and L10 proteins disappeared.We also found that the stability of mutated all protein in yeast decreased significantly.These data indicated that the point mutation in all protein resulted in not only the interaction with L10 but also its instability.The data from RNA analyses showed that there was obvious influence of all mutation on chloroplast ribosome RNA accumulation,but not for the cytoplasmic rRNA.It indicated that the assembly of large and small subunits of chloroplast ribosome decreased significantly.Quantitative RT-PCR and RNA-seq analyses revealed that al.mutation affects the expression of chloroplast biogenesis related genes.The transcripts of chloroplast development related genes in all mutant significantly increased,while those of photosynthetic and chlorophyll synthesis related genes decreased significantly.There is no obvious difference for the RpoA protein level of PEP subunit between all mutant and wild type.While the accumulation of chloroplast encoded proteins Petb,PsaB and PsbA decreased significantly,implying that the impact of all mutation on the accumulation of most chloroplast photosynthesis related proteins.All the above results provide useful information to understand the specific functions of L12 protein in rice.2.Fine mapping and genetic effects of the grain shape gene GS9 in riceIn rice,the grain shape is not only a key determinant for grain yield,but also greatly affects the grain qualities.Among the advanced backcross populations with the indica cultivar Qingluzhanll as donor and Nipponbare as receptor parent,the N138 line shows an elongated grain phenotype when compared with its backcross parent Nipponbare.The grain shape from N13 8 mature plant is narrower in width and longer in length than Nipponbare.The segregation ratio of normal and narrow grain phenotype among the F2 population from N138 and Nipponbare was consistent with the inheritance principle of a single Mendelian factor,indicating that the grain shape trait in N138 line was controlled by a single locus.Meanwhile,the genetic analysis also showed that the grain shape was similar between the individuals with homozygous and heterozygous genotype,implying the allele from Nipponbare was complete dominant to that from N138 line.The data from whole genome re-sequencing analysis revealed that N138 line contained six introgressed fragments from the donor parent.Linkage analysis was carried out using 28 recessive individual and the molecular markers corresponding to these six fragments,and the results indicated that the the narrow phenotype of grain shape was closely linked with the introgressed fragment on chromosome 9.Thus,we called the target gene related to this grain shape as GS9(Grain shape gene on chromosome 9).The interval size of the introgressed fragment is estimated as about 5.9 Mb.For fine mapping the target gene,we developed 10 molecular markers in this region to analyze 789 recessive individuals screened from the F2 and F3 population between N138 and Nipponbare.Finally,the target gene GS9 was limited to a physical range of about 58.9 kb between two molecular markers IN0916 and IN0919.The grain width of the key exchange individuals was narrow as expected.Based on the sequenced genome information from Nipponbare,there were 9 ORFs in the limited region,which were not allelic to the grain shape related genes that has been reported.Form backcross population from N138 and Nipponbare,a near isogenic line,named as NIL(gs9),was identified to contain only a small segment within the GS9 locus from the donor.The spikelet hull from NIL(gs9)shows significantly narrower and longer than that of Nipponbare.During grain filling,the shape of kernels was investigated carefully,and the results implied that there was obvious difference of grain size from the 7 days after following.Cytological evidence suggested that the different size of spikelet hull might result from the change in the cell number.Meanwhile,the measurements from spikelet hull transverse sections showed that the cell number of NIL(gs9)was significant less than that of Nipponbare.There was no significant difference in longitudinal cell density of the central lemma between NIL(gs9)and Nipponbare,indicating the longer spikelet hull of NIL(gs9)may also due to the increase of cell number.Therefore,it could be concluded that the GS9 locus controlled the cell number by regulating the expression of cell cycle genes,and then affected the grain size.The grain qualities were carefully analyzed and compared between Nipponbare and NIL(gs9),and the results showed that there was no significant difference of the cooking and eating qualities,including apparent amylose content,gel consistency,RVA profile and total protein content between the NIL(gs9)and Nipponbare.It was also identical of the milling qualities(brown rice rate,milled rice rate and head milled rice rate)between these two lines.As expected,the length of milled rice became longer,the width was narrower,and the ratio of length to width was significant higher in NIL(gs9)than those of Nipponbare.Interestingly,the chalkiness of NIL(gs9)was much lower than that of Nipponbare.Besides,the data from field trials revealed that the plant phenotype of Nipponbare and NIL(gs9)was similar,and the agronomic traits and yield were also comparable between the two lines.Therefore,the data from quality and yield analyses implied that the gs9 allele could improve the apparent quality with the maintaining of the other qualities and yield.The data from RNA-seq analysis revealed that there were many differential expressed genes(DEGs)in NIL(gs9)relative to Nipponbare.Quantitative RT-PCR analyses showed that the GS9 allele affected the cell cycle related gene expression,which might be the reason for the regulation of cell number and size by the GS9 locus.Moreover,the expression levels of cloned genes involving in grain size were compared,and it was indicated that the function of GS9 locus might be related to the brassinosteroid synthesis and signaling pathway.Taken together,all the above results will lay the foundations for cloning the GS9 gene and further functional analyses.