The Experimental Evidence For The Discovery Of Indica-type Cytoplasmic In Oryza Sativa L. Ssp. Japonica And Application Estimation

Hybridization of indica and japonica rice and utilization of an ideal plant type have enabled development of high-yielding japonica rice in northeast China. The genetic and physiological mechanisms research of heterosis of indica-japonica hybrid also more thorough. However, little is known about the genetic contribution of cytoplasmic inheritance in indica-japonica hybrid. In this study, cytoplasmic inheritance and nuclear-cytoplasm interaction also contribute to japonica super-rice breeding through bioinformatics and biological techniques combined with analytical methods of classical genetics, and found the experimental evidence for the discovery of inter-subspecific nuclear-cytoplasmic interaction by construction of isonuclear alloplasmic lines in Oryza sativa L. ssp. Japonica.The main research results were as follows:1. The subspecies differentiation fragment of cytoplasmic genome and nuclear genome were highly homologous by alignment of the sequence between indica 93-11 and japonnica Nipponbare. A view is presented based on alignment; false-positive is inevitable if PCR studies of cytoplasmic sequence are operated by using total DNA as a template.2. Using indica-japonica specific markers, chloroplast DNA and mitochondrial DNA as a template, indica-japonica differentiation of cytoplasm is detected on 44 main cultivated rice varieties in the northeast region. The results showed that seven varieties are indica-genotypes in the eight cytoplasm gene locimentioned above, such as Shennong 265, Shennong 9741, Liaogeng 454, Liaogeng326, Liaoyan241, Ying8433, Liaoyan283 et al, high-yielding or super high-yielding varieties. Plasmon is hardly to link and to recombine with the nuclear genome, thus these varieties possess indica-cytoplasm.3. With the rapid development of sequencing technology, the cytoplasm genetic background and SNP loci polymorphism can parse depth by Specific-locus amplified fragment sequencing, The results found that japonica-indica cytoplasm material Shennong 265, Shennong 9741, Shennong9816, Liaogeng454 and Liaogeng326 were clustered with indica rice. Haplotype analysis showed that Twenty-one chloroplast and 43 mitochondrial haplotypes were found in the 70 accessions. We found that the five target japonica varieties shared the same or genetically similar haplotypes as the indica varieties in both the chloroplast and mitochondrial networks. The neighbor-joining tree and haplotype analysis confirmed that the five target varieties had an indica cytoplasmic genetic background.4. To further reveal the types of functional genes and whether new mutations occurred in the chloroplast and mitochondrial genomes, we selected 14 important functional genes from the chloroplast and mitochondrial genomcs, respectively, for sequence comparison, The results of sequencing and alignment showed that the five target varieties possessed the same genotype combinations as the two typical indica varieties 93-11 for all 14 cytoplasmic functional genes segments, whereas the four non-target japonica varieties possessed the same genotypes as the four typical japonica controls。the results implied that the japonica-type nucleus interacting with the indica-type cytoplasm might have contributed to the higher yield potential of the five target varieties rather than novel mutations.5. To further reveal whether the indica-cytoplasm gene contribute to the yield-increasing in japonica, the genetic analysis of yield traits were analyzed by using genetic analysis based on diallel cross groups. The parents are three indica-cytoplasm japonica Shennong 265, Shennong 9741, Liaojing 454 and four japonica-cytoplasm japonica Akihikari, Feng Jin, Yanfeng 47, Liaojing 5. The results as follows, additive variance, dominance variance, narrow sense heritability and generalized genetic rates on tiller number, panicle number per plant, panicles and grain weight of F1 hybrids present significant at 0.01 level, and the same as in maternal heritability and maternal inheritance ratio, which indicate that the four main agronomic traits are influenced by combined effect between nucleus and cytoplasmic genetic effects.Analyzing of seven parental variance components shows that there is no maternal effects on parental plant height and panicle length, however, the dominant variance is significant at 0.01 level. Maternal effect of tiller number, panicle number, effective panicles per plant in Liaojing 5 and Fengjin are significant at 0.01 level, and variance components is negative. Although effect of indica-cytoplasm from Shennong 265 and Shenong 9741 on yield components is fail to present significant level, mostly effects of that are positive. Meanwile,the indica-cytoplasm of Shennong 265 and Shennong 9741 are significant positive effect on grain weight, but it is oppsite in japonica-cytoplasm of Akihikari on that.6. In this study, we substituted the nuclear genomes of these four target japonica varieties with japonica-type nuclear genomes from Toyonishiki and Liaogeng5 to construct seven nuclear substitution lines, The results showed that three indica-type cytoplasms showed a significant effect on tiller number per m2, number of panicle per plant and grain number per panicle under the nuclear genome backgrounds of Toyonishiki and Liaogeng5.