A novel discovery of a long terminal repeat retrotransposon-induced hybrid weakness in rice.

Hybrid weakness is a post-zygotic hybridization barrier frequently observed in plants, including rice. In this study, we describe the genomic variation among three temperate japonica rice (Oryza sativa ssp. japonica) varieties 'Aranghyangchalbyeo' ('CH7'), 'Sanghaehyangheolua' ('CH8') and 'Shinseonchalbyeo' ('CH9'), carrying different hybrid weakness genes. The reciprocal progeny obtained from crossing any two varieties displayed characteristic hybrid weakness traits. We mapped and cloned a new locus, Hwc3 (hybrid weakness 3), on chromosome 4. Sequence analysis identified that a long terminal repeat (LTR) retrotransposon was inserted into the promoter region of the Hwc3 gene in 'CH7'. A 4-kb DNA fragment from 'CH7' containing the Hwc3 gene with the inserted LTR retrotransposon was able to induce hybrid weakness in hybrids with 'CH8' plants carrying the Hwc1 gene by genetic complementation. We investigated the differential gene expression profile of F1 plants exhibiting hybrid weakness and detected that the genes associated with energy metabolism were significantly down-regulated compared with the parents. Based on our results, we propose that LTR retrotransposons could be a potential cause of hybrid weakness in intrasubspecific hybrids in japonica rice. Understanding the molecular mechanisms underlying intrasubspecific hybrid weakness is important for increasing our knowledge on reproductive isolation and could have significant implications for rice improvement and hybrid breeding.

Distribution of Hwc2-1, a causal gene of a hybrid weakness, in the World Rice Core collection and the Japanese Rice mini Core collection: its implications for varietal differentiation and artificial selection.

A pair of complementary genes, Hwc1-1 at HWC1 locus and Hwc2-1 at HWC2 locus, cause a weakness phenomenon in rice. For this study, we performed haplotype analysis around the HWC2 locus in two core collections comprising 119 accessions. We also examined reactions to phenol and Xanthomonas oryzae pv. oryzae (Xoo) Japanese race I. To elucidate the genetic relations among all accessions, we analyzed their banding patterns of 40 Indel markers covering the rice genome. The classification by Indel markers was almost consistent with that using 4,357 SNPs. The testcross with Hwc1-1 carrier indicated that 37 accessions carried Hwc2-1 allele, whereas 82 carried hwc2-2 allele. Strong association between HWC2 and Ph genes was observed. Based on 14 DNA markers around HWC2 locus and Ph genotype, the 119 accessions were divided into 50 haplotypes. To examine the HWC2 candidate chromosomal region specifically, the 'haplotype group' characterized by the six DNA markers closely linked with HWC2 were analyzed. Hwc2-1 carriers had the same haplotype group. Some hwc2-2 haplotype groups were associated with resistance against the Xoo race. The relation between varietal differentiation and haplotypes around the HWC2 locus was discussed, along with its breeding significance.

Hybrid weakness and continuous flowering caused by compound expression of FTLs in Chrysanthemum morifolium × Leucanthemum paludosum intergeneric hybridization.

Hybridization is an important evolutionary mechanism ubiquitous to plants. Previous studies have shown that hybrid polyploidization of cultivated chrysanthemum, 'Zhongshanzigui', and Leucanthemum paludosum exhibit spring-flowering traits. This study explores the function of the LpFTLs gene via the phenotype of A. thaliana after heterologous transformation of the LpFTLs gene, and analyzes the mechanism ofthe continuous flowering phenotype and heterosis of hybrid offspring. The results suggest that the flowering phenotype of hybrid offspring in spring may be related to the expression of the LpFTLs gene. Ectopic expression of Leucanthemum paludosumLpFTLs in Arabidopsis thaliana resulted in earlier flowering, indicating that the LpFTLs gene also affects the flowering time in L. paludosum. Compound expression of FTLs in C. morifolium × L. paludosum intergeneric hybridization directly leads to serious heterosis in the hybrid offspring. Moreover, continuous flowering appears to be accompanied by hybrid weakness under the balance of vegetative and reproductive growth. Therefore, in future studies on chrysanthemum breeding, a suitable balance point must be established to ensure the target flowering time under normal growth.

A Novel Combination of Genes Causing Temperature-Sensitive Hybrid Weakness in Rice.

Reproductive isolation is an obstacle for plant breeding when a distant cross is demanded. It can be divided into two main types based on different growth stages: prezygotic isolation and postzygotic isolation. The hybrid weakness, which is a type of postzygotic isolation, can become a problem in crop breeding. In order to overcome reproductive isolation, it is necessary to elucidate its mechanism. In this study, genetic analysis for low temperature-dependent hybrid weakness was conducted in a rice F2 population derived from Taichung 65 (T65, Japonica) and Lijiangxintuanheigu (LTH, Japonica). The weak and severe weak plants in F2 showed shorter culm length, late heading, reduced panicle number, decreased grain numbers per panicle, and impaired root development in the field. Our result also showed that hybrid weakness was affected by temperature. It was observed that 24°C enhanced hybrid weakness, whereas 34°C showed recovery from hybrid weakness. In terms of the morphology of embryos, no difference was observed. Therefore, hybrid weakness affects postembryonic development and is independent of embryogenesis. The genotypes of 126 F2 plants were determined through genotyping-by-sequencing and a linkage map consisting of 862 single nucleotide polymorphism markers was obtained. Two major quantitative trait loci (QTLs) were detected on chromosomes 1 [hybrid weakness j 1 (hwj1)] and 11 [hybrid weakness j 2 (hwj2)]. Further genotyping indicated that the hybrid weakness was due to an incompatible interaction between the T65 allele of hwj1 and the LTH allele of hwj2. A large F2 populations consisting of 5,722 plants were used for fine mapping of hwj1 and hwj2. The two loci, hwj1 and hwj2, were mapped in regions of 65-kb on chromosome 1 and 145-kb on chromosome 11, respectively. For hwj1, the 65-kb region contained 11 predicted genes, while in the hwj2 region, 22 predicted genes were identified, two of which are disease resistance-related genes. The identified genes along these regions serve as preliminary information on the molecular networks associated with hybrid weakness in rice.

Transcriptome Analyses Indicate Significant Association of Increased Non-Additive and Allele-Specific Gene Expression with Hybrid Weakness in Rice (Oryza sativa L.).

The heterosis in hybrid rice is highly affected by the environment and hybrid weakness occurs frequently depending on the genotypes of the hybrid and its parents. Hybrid weakness was also observed in our field experiments on nine rice hybrids produced by 3 × 3 incomplete diallel crosses. Among the nine hybrids, five displayed mid-parent heterosis (MPH) for grain yield per plant, while four showed mid-parent hybrid weakness (MPHW). A sequencing analysis of transcriptomes in panicles at the seed-filling stage revealed a significant association between enhanced non-additive gene expression (NAE) and allele-specific gene expression (ASE) with hybrid weakness. High proportions of ASE genes, with most being of mono-allele expression, were detected in the four MPHW hybrids, ranging from 22.65% to 45.97%; whereas only 4.80% to 5.69% of ASE genes were found in the five MPH hybrids. Moreover, an independence test indicated that the enhancements of NAE and ASE in the MPHW hybrids were significantly correlated. Based on the results of our study, we speculated that an unfavorable environment might cause hybrid weakness by enhancing ASE and NAE at the transcriptome level.

A Novel Embryo Phenotype Associated With Interspecific Hybrid Weakness in Rice Is Controlled by the MADS-Domain Transcription Factor OsMADS8.

A novel hybrid weakness gene, DTE9, associated with a dark tip embryo (DTE) trait, was observed in CR6078, an introgression line derived from a cross between the Oryza sativa spp. japonica "Hwayeong" (HY) and the wild relative Oryza rufipogon. CR6078 seeds exhibit protruding embryos and flowers have altered inner floral organs. DTE9 was also associated with several hybrid weakness symptoms including decreased grain weight. Map-based cloning and transgenic approaches revealed that DTE9 is an allele of OsMADS8, a MADS-domain transcription factor. Genetic analysis indicated that two recessive complementary genes were responsible for the expression of the DTE trait. No sequence differences were observed between the two parental lines in the OsMADS8 coding region; however, numerous single nucleotide polymorphisms were detected in the promoter and intronic regions. We generated overexpression (OX) and RNA interference (RNAi) transgenic lines of OsMADS8 in HY and CR6078, respectively. The OsMADS8-OX lines showed the dark tip embryo phenotype, whereas OsMADS8-RNAi recovered the normal embryo phenotype. Changes in gene expression, including of ABCDE floral homeotic genes, were observed in the OsMADS8-OX and OsMADS8-RNAi lines. Overexpression of OsMADS8 led to decreased expression of OsEMF2b and ABA signaling-related genes including OsVP1/ABI3. HY seeds showed higher ABA content than CR6078 seeds, consistent with OsMADS8/DTE9 regulating the expression of genes related ABA catabolism in CR6078. Our results suggest that OsMADS8 is critical for floral organ determination and seed germination and that these effects are the result of regulation of the expression of OsEMF2b and its role in ABA signaling and catabolism.

HWA1- and HWA2-Mediated Hybrid Weakness in Rice Involves Cell Death, Reactive Oxygen Species Accumulation, and Disease Resistance-Related Gene Upregulation.

Hybrid weakness is a type of reproductive isolation in which F1 hybrids of normal parents exhibit weaker growth characteristics than their parents. F1 hybrid of the Oryza sativa Indian cultivars 'P.T.B.7' and 'A.D.T.14' exhibits hybrid weakness that is associated with the HWA1 and HWA2 loci. Accordingly, the aim of the present study was to analyze the hybrid weakness phenotype of the 'P.T.B.7' × 'A.D.T.14' hybrids. The height and tiller number of the F1 hybrid were lower than those of either parent, and F1 hybrid also exhibited leaf yellowing that was not observed in either parent. In addition, the present study demonstrates that SPAD values, an index correlated with chlorophyll content, are effective for evaluating the progression of hybrid weakness that is associated with the HWA1 and HWA2 loci because it accurately reflects degree of leaf yellowing. Both cell death and H2O2, a reactive oxygen species, were detected in the yellowing leaves of the F1 hybrid. Furthermore, disease resistance-related genes were upregulated in the yellowing leaves of the F1 hybrids, whereas photosynthesis-related genes tended to be downregulated. These results suggest that the hybrid weakness associated with the HWA1 and HWA2 loci involves hypersensitive response-like mechanisms.