Hybrid Sterility in Rice (Oryza sativa L.) Involves the Tetratricopeptide Repeat Domain Containing Protein.

Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7(cp) and S7(i) were aborted in S7(ai)/S7(cp) and S7(ai)/S7(i), respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7(n) S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7(ai) in heterozygote S7(ai)/S7(cp); sterility occurred in the transformants Cpslo17-S7(ai) Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement.

Pollen semi-sterility1 encodes a kinesin-1-like protein important for male meiosis, anther dehiscence, and fertility in rice.

In flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1-like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1-green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice.

Genetic analysis of root elongation induced by phosphorus deficiency in rice (Oryza sativa L.): fine QTL mapping and multivariate analysis of related traits.

Root elongation induced by phosphorus deficiency has been reported as one of the adaptive mechanisms in plants. Genetic differences were found in rice for the root elongation under phosphorus deficiency (REP), for which a distinct quantitative trait locus (QTL) was detected on the long arm of chromosome 6. Subsequently, the effect and position of the QTL, designated as qREP-6, were confirmed using chromosome segment substitution lines (CSSLs), in which the background of a japonica cultivar, 'Nipponbare' with non-REP, was partially substituted by chromosomal segments from an indica cultivar, 'Kasalath' with remarkable REP. Out of 54 CSSLs, two lines (CSSL28 and CSSL29) that retain a common 'Kasalath'-derived segment on the long arm of chromosome 6 showed a significantly high REP. The high REP lines also showed high adaptabilities such as enhanced tillering ability and shoot phosphorus content. Accordingly, conditional dependencies between the related traits were assessed using a graphical Gaussian model (GGM). Direct interactions between REP and root length, and between root length and tiller number were detected under P deficiency in CSSLs. Furthermore, qREP-6 for REP and qTNP-6 for tiller number under P deficiency were fine-mapped with an F(2) population of a cross between Nipponbare and CSSL29. A region containing qREP-6 accounted for more than half of the phenotypic variance, the most plausible interval of which contained 37 candidate genes. The result provides a foundation for cloning of the qREP-6 gene which will be applicable to study P deficiency-dependent response and to improve rice's adaptability to P deficiency stress.

Fine mapping of pss1, a pollen semi-sterile gene in rice (Oryza sativa L.).

During routine seed increase procedures in rice, semi-sterile plants are common; however, such semi-sterility mutants in rice varieties have been only rarely analyzed genetically. W207-2 is a semi-sterile selection from the japonica rice variety Nipponbare. In this report, we found the female gamete of W207-2 was normal, and its semi-sterility was unaffected by growth duration but was conditioned by a recessive nuclear gene whose action leads to pollen semi-sterility and anther indehiscence, and the gene was named as pss1 (pollen semi-sterile). Using an F(2) population derived from the two parents W207-2 and Dular and a pooled DNA strategy, pss1 was mapped to an interval on chromosome 8 defined by the two SSR loci RM6356 and RS41. The position of pss1 was confirmed in another F(2) population derived from the cross W207-2 x Nipponbare. Over 2,000 homozygous pss1 segregants from the large W207-2 x Dular F(2) population were used to fine map pss1 to a 0.04 cM segment flanked by a CAPs marker L2 and a dCAPs L3 marker. Sequences for both markers are present on a single PAC clone, and the physical distance between them is about 28 kb. Analysis of the PAC sequence predicts the presence of five open reading frames, they are as follows: putative ribonuclease PH, putative avr9 elicitor response protein, kinesin1-like protein, putative protein RNP-D precursor and putative 40S ribosomal protein S13. This result would be helpful in cloning the pss1 gene.

Fine mapping of S32(t), a new gene causing hybrid embryo sac sterility in a Chinese landrace rice (Oryza sativa L.).

Ketan Nangka, the donor of wide compatibility genes, showed sterility when crossed to Tuanguzao, a landrace rice from Yunnan province, China. Genetic and cytological analyses revealed that the semi-sterility was primarily caused by partial abortion of the embryo sac. Genome-wide analysis of the linkage map constructed from the backcross population of Tuanguzao/Ketan Nangka//Ketan Nangka identified two independent loci responsible for the hybrid sterility located on chromosomes 2 and 5, which explained 18.6 and 20.1% of phenotypic variance, respectively. The gene on chromosome 5 mapped to the previously reported sterility gene S31(t), while the gene on chromosome 2, a new hybrid sterility gene, was tentatively designated as S32(t). The BC1F2 was developed for further confirmation and fine mapping of S32(t). The gene S32(t) was precisely mapped to the same region as that detected in the BC1F1 but its position was narrowed down to an interval of about 1.9 cM between markers RM236 and RM12475. By assaying the recombinant events in the BC1F2, S32(t) was further narrowed down to a 64 kb region on the same PAC clone. Sequence analysis of this fragment revealed seven predicted open reading frames, four of which encoded known proteins and three encoded putative proteins. Further analyses showed that wide-compatibility variety Dular had neutral alleles at loci S31(t) and S32(t) that can overcome the sterilities caused by these two genes. These results are useful for map-based cloning of S32(t) and for marker-assisted transferring of the neutral allele in hybrid rice breeding.

QTL-based analysis of leaf senescence in an indica/japonica hybrid in rice (Oryza sativa L.).

In order to identify quantitative trait loci (QTLs) for leaf senescence and related traits in rice (Oryza sativa L.), we developed two backcross populations, indica/japonica// japonica and indica/japonica//indica, using IR36 as the indica parent and Nekken-2 as the japonica parent. The QTLs were mapped using a set of simple sequence-repeat markers (SSRs) in the BC(1)F(1) population. Senescence was characterized in these plants by measuring the leaf chlorophyll content 25 days after flowering (DAF), the reduction in chlorophyll content (the difference between the chlorophyll content at flowering and at 25 DAF), and the number of late-discoloring leaves per panicle at 25 DAF in five plants from each BC(1)F(2) line. These plants were moved into a temperature-controlled growth cabinet at the time of flowering and allowed to mature under identical conditions. Eleven QTLs were detected in the two populations. The major of QTLs for senescence were found on the short arm of chromosome 6 and on the long arm of chromosome 9. Of these, one QTL on chromosome 6 and two on chromosome 9 were verified by confirming the effects of the genotypes on the phenotypes of the BC(1)F(3) lines. The japonica parent was found to contribute to late senescence at all but one QTL. Based on a comparison of the effects of heterozygotes and homozygotes on the phenotypic values of each QTL genotype, we concluded that the differential senescence observed in the indica-japonica hybrid was not due to over-dominance; rather, it was the result of partial-dominance genes that were donated from either of the parents.

[Inheritance of bc1 gene in intersubspecific hybrids of rice (Oryza sativa L.)].

Distorted segregation of the brittle culm-1 gene (bc1) on rice chromosome 3 was found with greatly increased or decreased frequency of bc1 bc1 genotype in inter-subspecific hybrids, although the gene normally transmitted to its offspring following the Mendelian Law in intra-subspecific hybrids. In a combination of Kamairazu//Ketan Nangka/Kamairazu,an increased frequency of bc1 bc1 in F1, normal segregation in F2, and increased and decreased frequency in a few F3 and F4 lines were observed. In a cross of IR36/Kamairazu, decreased frequency in F2, both normal and decreased segregations in F3 and F4, and a few lines of increased ratio in F4 were found. In F2 of Ketan Nangka/IR36//Kamairazu, increased and decreased and normal segregations were all observed. There was no significant correlation between the frequency of bc1 bc1 and pollen fertility. It implied that distorted segregation of bc1 was caused by selective fertilization of male gametes, which were governed by gametophyte genes of ga2, ga3 and ga14 on chromosome 3.

Phosphorus deficiency-induced root elongation and its QTL in rice (Oryza sativa L.).

A significant level of root elongation was induced in rice (Oryza sativa) grown under phosphorus-deficient conditions. The root elongation clearly varied among a total of 62 varieties screened under two different phosphorus levels. Two contrasting varieties, 'Gimbozu', with a low elongating response and 'Kasalath', with a high elongating response, were chosen and crossed to produce a hybrid population for QTL analyses. QTLs for the phosphorus deficiency-induced root elongation were detected by two linkage maps, i.e., one with 82 F3 families constructed by 97 simple sequence repeat (SSR) and sequence-tag site markers and another with 97 F8 lines by 790 amplified fragment length polymorphism and SSR markers. A single QTL for the elongation response was detected on chromosome 6, with a LOD score of 4.5 in both maps and explained about 20% of total phenotypic variance. In addition, this QTL itself, or a region tightly linked with it, partly explained an ability to reduce accumulation of excess iron in the shoots. The identified QTL will be useful to improve rice varieties against a complex nutritional disorder caused by phosphorus deficiency and iron toxicity.