| Rice (Oryza sativa L.) is the most important food crop in China, and increasing the yield of rice is of great significance to ensure food security in our country. In order to further improve the yield of rice, more and more rice breeders recognized that the utilization of strong heterosis between different Oryza species and subspecies of the Asian cultivated rice, as well as the favorable genes within them, is the only way to breed new cultivars with higher and more stable yield, especially the utilization of favorable genes between Indica and Japonica. However, the reproductive isolations between the two subspecies not only limit the utilization of the strong hybrid vigor, but also hinders the genetic communication between them. Reproductive isolation between intersubspecific hybrid of Indica and Japonica is the result of the continuously accumulated genetic divergences between the two subspecies. Reproductive isolation is beneficial to the survival and development of natural species. However, it also limits the communication between genes in different species, which is the greatest obstacle in the use of favorable alleles in Indica and Japonica.Hybrid sterility and Hybrid breakdown are the two major forms of post-zygotic reproductive isolation between Indica and Japonica. A lot of previous studies have been carried out on hybrid sterility, while the genetic mechanism responsible for hybrid breakdown was still understood poorly. Rice is an important food crop, but also a model plant of Gramineae in biological research. Therefore, research on the hybrid breakdown between two subspecies is of great theoretical significance and practical value in both the fields of evolutionary biology and breeding.In this study, hybrid breakdown appeared in the chromosome segment substitution lines and another backcross inbred lines derived from Sasanishiki/Habataki. And these two populations were used to conduct genetic dissection and gene cloning of hybrid breakdown in Rice(Oryza Sativa L.). The main results were as follows:Part1 Genetic analysis of hybrid breakdown through characterizing CSSL and BIL population1. The phenomenon of hybrid breakdown was observed in the CSSL population across different environments, with some CSSL showing low spikelet fertility. Furthermore, the QTL conferring spikelet fertility was conducted, and the results of QTL mapping showed that a QTL, qSF-12, was identified and located around the marker RM6998 on chromosome 12 in Yangzhou and Hainan during different years, which proved to be a locus responsible for hybrid breakdown.2. The results of QTL mapping for spikelet fertility in BIL showed that, two QTL, qSF-8 and qSF-12, were identified and located around marker C1121 and C1069 on chromosome 8 and 12, respectively, in different locations. The QTL for spikelet fertility identified on chromosome 12 is the same QTL identified in CSSL.3. Furthermore, on the base of the spikelet fertilities and genotypes of the 85 BILs, we analyzed the relationship between two QTL, qSF-8 and qSF-12. The lines with the segments on qSF-8 and qSF-12 both from Sasanishiki or Habataki showed normal spikelet fertility, otherwise, the spikelet fertility depressed. This result clearly demonstrated a hybrid incompatible genetic model played an important role in controlling rice hybrid breakdown.Part 2 Fine mapping and Functional analysis of HB-12, a key gene responsible for hybrid breakdown in rice (Oryza sativa L.)A secondary F2 population derived from a cross of SL438/Sasanishiki was generated for fine mapping of the gene responsible for hybrid breakdown on qSF-12.The main results were as follows:1. The spikelet fertility (about 30%) of SL438 is much lower than that of Sasanishiki. Further measurement of the pollen fertility and the embryo sac fertility of SL438 showed that, the pollen fertility of SL438 is normal(about 90%), while its embryo sac fertility (about 25%) was significantly lower than those of the two parents suggesting that the partial embryo sac abortion is the main reason for the low spikelet fertility in SL438.2. An F2 population derived from a cross of SL438/Sasanishiki was employed for fine mapping qSF-12 (HB-12, hybrid breakdown. HB-12 was finally restricted to a 137-kb DNA fragment, containing 19 ORFs, of which,11 ORFs had full length cDNA supports. Based on the analysis of DNA sequence and RNA-seq, LOC_Os12g38850 is considered to be the putative candidate gene of HB-12, which was further confirmed by complementary test and RNAi experiment.3. The genome and CDS sequence length of LOC_Os12g38850 is 5125bp and 1698bp, respectively. And it encodes a DUF1336 protein of 566 amino acid residues. Sequencing results showed that a 3-bp difference (GAT) in the exonlO of LOC_Os12g38850 existing between Habataki and Sasanishiki. The 3-bp in Habataki was located exactly before the terminator codon, encoding an aspartic acid. This difference exists between Indica and Japonica commonly.4.GUS staining and RT-PCR analysis showed that HB-12 constitutively expressed in all the tested tissues or organs, especially in the stem, spikelet and young embryo sac. Transient expression of the HB-12-GFP fusion protein in rice protoplasts showed that the protein was subcellular located in cytoplasm and chloroplast.5.Real-time PCR quantitative analysis showed that the expression level of LOC_Os12g38850 in the functional megaspore formation period in the development of embryo sac is the highest. The reference genes expression analysis in the young panicle between Sasanishiki and SL438 showed that E3 is a critical point of the development of embryo sac. In the E1-2 period, the reference gene expression in Sasanishiki was significantly lower than that of the SL438.While in E3, the expression of reference gene in SL438 and Sasanishiki were considerable, and in the E4-6, the expression of reference gene in Sasanishiki was significantly higher than SL438. Further analysis showed that expression levels of pre-meiotic and meiotic stages related gene in Sasanishiki and SL438 had no difference. These results showed that HB-12 mainly play a role in the funtional megaspore formation period of the development of embryo sac. |
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