QTL Analysis For Traits Associated With Photosynthetic Functions And Map-based Cloning Of Virescent White Leaf Gene In Rice (Oryza Sativa L.)

Food shortage is one of the most serious global problems with the increasing of the worldwide population in this century. Rice is the most important food crop in the world and feeds over half of the global population. To meet the expanding food demands of the rapidly growing world population, increases in rice yield and production will be required. Photosynthesis and its related physiological traits play an important role in yield determination. Leaf coloration mutant is one of the most ideal materials for photosynthesis research. In the present study, three mapping populations were utilized for quantitative trait loci (QTL) detection controlling photosynthesis and its related physiological traits. Meanwhile, a virescent white leaf mutant (tentatively designated as vwl), a Indica variety named Yutu S, was used for photosynthetic physiological analysis. Fine mapping and map-based cloning of vwl gene, were carried out on the mutant using molecular marker developed based on the public sequence data. These results should be useful for marker-assisted selection in rice high photosynthetic efficiency breeding and utilization of vwl mutant in rice production. The main conclusions are as follows:1 QTL detection for traits associated with photosynthetic functions in rice, using recombinant inbred linesA mapping population of 81 F₁₁ lines (Recombinant Inbred Lines, RILs), derived from a cross between a japonica variety Kinmaze and an indica variety DV85 by single-seed descent method, was used to detect quantitative trait loci (QTL) for traits associated with photosynthetic functions. Total leaf nitrogen content (TLN), chlorophyll a/b ratio (Chl.a:b) and chlorophyll content (Chl) were measured in leaves of rice (Oryza sativa L.) at seven days after heading, and a total of six putative QTLs were detected with percentage of variance explained (PVE) running between 11.2 - 29.6%, and LOD of QTLs 2.66 - 4.81. Of those putative QTLs, three for TLN were detected on chromosomes 1, 2 and 11, with PVE of 17.3%, 15.3% and 13.7%, respectively; Two controlling Chl.a:b on chromosomes 3 and 4, PVE 13.8% and 29.6%; One controlling Chl on chromosome 1, PVE 11.2%. Four of those detected QTLs were newly reported in this study. Interestingly, the QTL controlling chlorophyll content, namely qCC-1 reported here, was detected in the region of the RFLP marker C122 on chromosome 1, where harbored NADH-glutamate synthase structure gene according to the previous study. Because the biosynthesis of chlorophyll begins with glutamate, qCC-1 would play a vital role in photosynthetic functions. Whereas, no QTL controlling chlorophyll content were detected 30 days after heading, suggesting that the effect of the QTL controlling chlorophyll content decreased during leaf senescence.2 QTL detection for traits associated with photosynthetic functions in rice, using backcross inbred linesA mapping population, the backcross inbred lines derived from a cross of Nipponbare/Kasalath//Nipponbare by the single seed descent method, was used to detect quantitative trait loci (QTL) for traits associated with photosynthetic functions. Total leaf nitrogen content (TLN), chlorophyll a/b ratio (Chl.a:b) and chlorophyll content (Chl) were measured in leaves of rice (Oryza sativa L.) at 7th day after heading, and a total of eight putative QTLs were detected with percentage of variance explained (PVE) running between 9.7～21% and LOD of QTLs 2.61～6.42. Of those putative QTLs, two for TLN were detected on chromosomes 1 and 6, with PVE of 9.7% and 10.8%, respectively; Three controlling Chl.a:b on chromosomes 2,3 and 12, PVE 10%,10.5% and 21%; Three controlling Chl on chromosome 1,4 and 8, PVE 13.7%,10.7% and 10%. More interestingly, the QTL controlling chlorophyll content, namely qCC-1 in this population, was also detected in the region of the RFLP marker C122 on chromosome 1. So qCC-1 would play a vital role in photosynthetic functions. Two QTLs controlling Chl were detected on chromosomes 4 and 8 at the 30th day after heading, however, no QTL were also detected in the vicinity of C122, further suggesting that the effect of the qCC-1 controlling Chl decreased during leaf senescence.3 QTL detection for photosynthesis and its related physiological traits in rice, using chromosome segment substitution linesSixty-five chromosome segment substitution lines (CSSLs), derived from a cross between an indica (IR24) and a japonica variety (Asominori) of rice (Oryza sativa L.), were utilized for quantitative trait loci (QTL) mapping for photosynthetic and related physiological traits. Net photosynthesis rate (PHO), stomatal conductance (SCO), transpiration rate (TR), intercellular CO₂ concentration (IC), leaf chlorophyll (CHL), and total leaf nitrogen content (TLN) were measured in flag leaves 7 d after heading. The CSSLs showed transgressive segregation for many of the traits, and significant correlations were observed for most of the traits. In total, ten QTLs were detected on chromosomes 1, 3, 4, 5, 7, 8, and 10 with a range of percentages of variance explained (PVE) stomatal conductance (qSCO-10) and intercellular CO₂ concentration (qIC-10) in the vicinity of C1166 on chromosome 10. No other overlapping loci associated with different traits were detected. These results suggested that the genetic mechanism of photosynthesis was very complex in rice.4 Map-based cloning of a virescent white leaf gene in riceThe virescent white leaf mutant (tentatively designated as vwl) was screened from a PA64S M₂ population mutagenized with ⁶⁰Co gamma rays. The mutant (named Yutu S, a Indica rice) leaves exhibit albino but finally turn to normal green after 3rd leaf stage. However, no difference in other agriculture traits was observed between the wild type and the mutant. Systematical research including photosynthetic pigment, chloroplast microscopic observation and gene cloning was carried out on the vwl mutant. The results are as follows:(1) The measurement of photosynthetic pigment in the leaves showed that the mutant had no neither chlorophyll a and chlorophyll b, norβ-carotenoid before the leaves turned to normal green, however, no difference in photosynthetic pigment was observed between the mutant and the wild-type leaves after the leaves turned to normal green. These results suggested that the deficient in the total photosynthetic pigment led to the albinism at the stage office seedling.(2) Transmission electron microscopic examination revealed that the mesophyll cells of the mutant contained some proplastid-like structures instead of the normal chloroplasts before the leaves turned to normal green, however, had plenty of normal chloroplasts which contain thylakoid membranes after the leaves turned to normal green.(3) The genetic analysis of the vwl mutant indicated that the phynotype of virescent white leaf was controlled by a recessive gene. (4) The vwl locus is primarily located between markers RM411 and RM6832, at the distances of 1.1 and 1.2 cM, respectively, on chromosome 3, using available SSR molecular markers and 672 mutant individuals of the F₂ population.(5) For fine mapping of the vwl gene, more PCR-based markers, such as SSR, CAPS and dCAPS, were designed on basis of public rice genome sequence data. Using these markers and over 2240 mutant plants of the F2 population, the high-resolution genetic map and BAC eontig of the vwl locus were constructed. Eventually, the vwl locus was narrowed to a 45-kb genomic DNA region between two markers, P45 and D8, and cosegregated with marker P50.(6) In the 45-kb target gene region, a gene (tentatively designated as OsPPR2) encoding putative pentatricopeptide repeat (PPR) protein was discovered at the expected position of vwl. PPR proteins belong to RNA-binding protein family, which have shown to influence stability or translation of specific chloroplast mRNA. We took OsPPR2 as the possible candidate gene of vwl. Sequencing the OsPPR2 and comparing the counterpart DNA region between the vwl mutant and the wild-type revealed that the mutant had 5-bp deletions within the coding region. The InDel marker I1 developed on basis of the deletion was used to survey over 2240 mutant plants of the F2 population and 20 different wild-type rice varieties. The results showed that the I1 was cosegregated with the vwl lucos, suggesting that the gene OsPPR2 is very likely VWL.