Genetic Analysis And Map-based Cloning Of The Albino Mutant AL5942 And The Early Senescence Mutant D475 In Rice

1. Genetic analysis and fine mapping of the albino mutant al5942 in riceLeaf is the main organ for photosynthesis. Photosynthetic efficiency can be directly or indirectly affected by the mutation on leaf color, and then, the yield is also changed. The Rice leaf color mutant mechanism has a very important role on many aspects such as on the gene function, chlorophyll biosynthesis pathway, photosynthesis, and rice genetics breeding. In this study, we got an albino mutant al5942 by treating G46B with chemical mutagen EMS. Then, phenotypic identification, genetic analysis and gene mapping of the al5942 mutant were performed. The main results were as follows:(1) The morphology and agronomic traits of al5942 mutant. The mutant was planted under the natural conditon in Wen-jiang district, Chengdu city. When the mutant is in 2-5 leaf stage, its leaf present a large area of bleaching like white stripes along the veins.With the growth of the plant, the area of leaf albino became smaller, however, there’s still a phenomenon of albino around the veins and the tip of the leaf at the mature period. We found the result that the value is falling in plant height, number of spikelets per panicle. seed setting rate and 1000-grains weight compared with the wild type plant, the value decreased by 17.2%.14.8%,7.3% and 4.8%. respectively.(2) Measurement of the photosynthetic pigment in al5942 mutant. It is found that the content of the total chlorophyll, chlorophyll a, chlorophyll b and acrotenoids in the mutant were significantly lower than the wild type. At seedling stage their contents decreased by 54.04%,51.99%,60.00% and 32.50%, respectively. At heading stage the photosynthetic pigment increased, but their contents still decreased by 51.45%,50.77%,53.60% and 21.74% compared with the wild type.(3) Genetic analysis of al5942 mutant:The mutant was crossed with the normal parents Nipponbare and Minghui 63, respectively. As result, F1 generations were all normal green color, and F2 generations could be classified into normal green and the albino. The statistical result showed that the albino phenotype is controlled by a pair of recessive nuclear genes according to the chi-square test.(4) Analysis by Transmission electron microscopy (TEM):Observing the second leaf (from the top) of the four-week-old plants of the the mutant and its wild type using TEM. We observed that the chloroplast is abundant and membrane system is perfectly developed in wild type, but chloroplast is little and the grana stack in a state of losing and cluttering in the al5942.(5) Fine mapping of al5942 mutant. F2 generations from the hybrid combinations of al5942 mutant and normal parent Nipponbare or Minghui 63 were used as the mapping population. Eventually, the al5942 mutant gene was mapped between InDel markers B3 and B4, Twenty two genes were predicted in this 134.2-kb region. Because there is no report on these genes so far, it is speculated that the al5942 gene is a novel gene for rice albino mutation.2. Map-based cloning of the early senescence mutant D475 gene in riceSenescence is a key process of the growth and development of plants, and it is also a form of programmed cell death, so it has extremely important biological significance for plants to adapt diverse environments. A large number of studies have shown that early senescence will seriously affect rice production, so the in-depth study of early senescence mutants is extremely significant to understanding the plant senescence mechanism, and breeding anti-aging varieties to increase rice yield. In this study, we got an early senescence mutant d475 by treating Nipponbare with chemical mutagen EMS. Then, phenotypic identification and gene mapping of the d475 mutant were performed. The results were as follows:(1)The morphology and agronomic traits of d475 mutant. Under the natural condition, d475 mutant has no obvious changes at seedling stage compared with the wild type. But at the heading stage, the lower tip of leaf turned to yellowish-white, and gradually spread to leaf base, only to the whole plant withered old death, until the whole plant withered to death, compared to the d475, the wild type still presented green. Measuring the main agronomic traits of d475, it is found that the mutant are not significantly affected in the aspects of panicle length, number of productive panicles per plant and number of spikelets per panicle, but 1000-grain weight, seed setting rate and plant height significantly reduced by 31.8%,44.4% and 9.2% compared to wild type.(2) Measurement of photosynthetic pigment in d475 mutant. Measuring the pigment of mutant d475 related three important period senescence properties, each pigment content of the mutant having no obvious change compared with wild type before the mutant’s senescence (before heading stage). As a continuation of the aging process, the photosynthetic pigment in the mutant is rapid degradation, until terminal senescence (the end of mature) the amount of total chlorophyll, chlorophyll a, chlorophyll b, chlorophyll a/b and carotenoids in d475 reduced by 78.950%、80.00%、76.67%、13.49% and 62.50%, respectively.(3) Analysis by Transmission electron microscopy (TEM). Observing the ultrstructures of leave cells using TEM, no obvious difference in the chloroplast structure was observed between wild type and d475 before senescence. As the aging process started, we observed that the array of grana stacks became disordered. In addition, we further observed that plastoglobuli became obvious, and the cell organelles gradually degradation.(4) Fine mapping of d475 mutant. On the basis of Pingyu Wang’s result in our lab, we examined F2 seedlings of a cross between d475 and G46B for the early senescence phenotype. Eventually, the d475 mutant gene was located on chromosome 10. Nine genes were predicted in this 44.9-kb region. Sequence analysis of the D475 allele revealed two sites change, C-to-T substitution at the 147th sites, and a T-to-A substitution at the 287th sites. This gene was considered as the candidate gene of d475, and designated tentatively as D475 gene.