Analysis Of The Fertility For A Semi-sterile Mutant W207-2 And Fine Mapping Of A Pollen Semi-sterile Gene (Pss1) In Rice (Oryza Sativa L.)

Male-sterile mutations provide an ideal source material for a range of genetic andmolecular biological studies of reproductive biology. In order to well apprehend themolecular mechanism of pollen development, all genes involved in this coursedevelopmental stage must be identified and characteried.Many studies on the hybrid semi-sterility between indica and japonica have beenreported and many hypothesizes have been put forward, but the molecular mechanism ofsemi-sterility has been unknown. It is difficult to study this problem due to this hybridsemi-sterility causing by indica-japonica interaction. The hybrid genetic foundation iscomplex, so hybrid semi-sterility in different varietal combinations may be differentsterility loci. If we can use a stable genetic semi-sterile cultivar to study the semi-sterilemolecular mechanism, it would be a reference for overcoming the semi-sterility inpractices.W207-2 is a stable mutant, and mutants are favorable materials for studying geneexpression and function analysis, at the same time, analyzing mutants is also the develop-ment aspect for researching the functional genomes. The entire rice genome has beensequenced and publicly considered to be the genome of the model plant in grass, at thesame time, there is synteny between rice and other grass genome. Therefore, there is animportant use for reference to other grass plants through disclosing the biology basis inmolecular levels.The main results were as follows:1. On the basis of analysis with reciprocal F₁ hybrids between the mutant line W207-2and its original type Nipponbare(O. japonica) and a wide compatibility variety Dular(O.indica), the semi-sterility was found to be controlled by recessive nuclear genes with nocytoplasmic effect. The anther of W207-2 was thinner and almost indehiscent, while that ofNipponbare was plumper and dehiscent. The anther indehiscence of W207-2 was found toreduce the numbers of pollen grains dropped onto stigma per spikelet. Thus the germinated pollen grains were reduced and induced the spikelet semi-sterility of W207-2. The pollensemi-sterility may be one of the causes that make the anther indehiscent.2. Analysis of the F₂ population of W207-2/Nipponbare suggested that the correlationbetween spikelet fertility and pollen fertility was significant (γ=0.9119) and the semi-sterility was independent of growth duration. Genetic analysis of 291 plants in W207-2/Nipponbare F₂ population and 137 plants in W207-2/W207-2/Nipponbare populationindicated that this semi-sterility was controlled by a recessive nuclear gene. W207-2 andsterile line Liuyan189A, Bing8979A, 9522A was hand-pollinated with the pollen ofW207-2 and Nipponbare, respectively, and the semi-sterility of W207-2 was found to bemainly caused by the pollen semi-sterility while the female gamete fertility was normal.3. The developmental process of microsporogenesis in anthers of Nipponbare andW207-2 were examined with light microscope. Some special abortive types were detectedin the sporogenous cells or microspore mother cells, and their structure was destroyed, theydeveloped into abnormal black clumps. The main reasons of pollen abortion of W207-2 are:(1) The meiosis of pollen mother cell in W207-2 was abnormally. (2) Tapetum cell delayeddisintegration. And the aberrant vascular bundle of connective tissue and abnormal theca ofanther was another cause of semi-sterility for W207-2. Whole-stain clearing method wasused for evaluating embryo sac fertility of Nipponbare and W207-2. The embryo sacfertility was normal, and the male gamete developmental abnormality of W207-2 was themain cause for the semi-sterility of W207-2.4. Contents of IAA, GA₄, iPA, ZR and ABA were measured by enzyme-linkedimmunosorbent assay (ELISA) in leaves, panicles and anthers of W207-2 and its originaltype Nipponbare during panicle development. IAA, GA₄ and iPA contents and the ratios ofIAA+GA₄+iPA to ABA+ZR in leaves, panicles and anthers of W207-2 were shown to belower than those of Nipponbare, while ZR and ABA contents in W207-2 higher than thosein Nipponbare. It is proposed that the changes in contents of these endogenesis hormone inW207-2 are abnormal, and IAA, GA₄ and iPA deficiency and ABA as well as ZRaccumulation might be related to the generation of semi-sterility in rice.5. Twenty semi-sterility and twenty fertility plants were random selected from the F₂population of W207-2/Dular according to pollen fertility, then genomic DNA from themwas pooled to make up the fertile and semi-sterile DNA bulks, respectively. A total of 565SSR markers throughout the rice genome were surveyed for their polymorphisms betweenthe fertile and semi-sterile bulks and between the two parents. Five SSR markers including RM178 on chromosome 5, RM118 on chromosome 7, RMS06, RM152 and RM6863 onchromosome 8, showed polymorphisms between the two bulks and between the two parents.Based on the linkage map of chromosomes 5, 7 and 8, which were constructed using 182random individuals from W207-2/Dular F₂ population, only one major QTL (pss1)controlling the pollen semi-sterility was identified with a LOD score of 48.3 and a PVE(phenotypic variation explained) of 70.5% in the region between markers RS41 andRM6356 on chromosome 8.6. The pss1 locus that located in the region between markers RS41 and RM6356 onchromosome 8 was confirmed by using another 291 individuals in F₂ population ofW207-2/Nipponbare. To fine-map pss1, 2100 recessive homozygous semi-sterile plantswere selected from a large W207-2/Dular F₂ population to identify the recombinationbetween the pss1 locus and the tightly linked molecular markers. Finally, pss1was mappedon the region between one CAPs L2 and one dCAPs L3 marker with the genetic distancesof 0.02 cM, respectively. Both L2 and L3 markers were located on a same PAC cloneP0470F10 with physical size of about 28 kb. Five open reading frames were predicted by asequence analysis of this fragment. They were as follows: putative ribonuclease PH,putative avr9 elicitor response protein, kinesin1-like RNA-binding protein, putativeRNA-binding protein RNP-D precursor and putative 40S ribosomal protein S13.