F1 Pollen Sterility Research Articles

Domain Unknown Function DUF1668-Containing Genes in Multiple Lineages Are Responsible for F1 Pollen Sterility in Rice.

Postzygotic reproductive isolation maintains species integrity and uniformity and contributes to speciation by restricting the free gene flow between divergent species. In this study we identify causal genes of two Mendelian factors S22A and S22B on rice chromosome 2 inducing F1 pollen sterility in hybrids between Oryza sativa japonica-type cultivar Taichung 65 (T65) and a wild relative of rice species Oryza glumaepatula. The causal gene of S22B in T65 encodes a protein containing DUF1668 and gametophytically expressed in the anthers, designated S22B_j. The O. glumaepatula allele S22B-g, allelic to S22B_j, possesses three non-synonymous substitutions and a 2-bp deletion, leading to a frameshifted translation at the S22B C-terminal region. Transcription level of S22B-j and/or S22B_g did not solely determine the fertility of pollen grains by genotypes at S22B. Western blotting of S22B found that one major band with approximately 46 kDa appeared only at the mature stage and was reduced on semi-sterile heterozygotes at S22B, implying that the 46 kDa band may associated in hybrid sterility. In addition, causal genes of S22A in T65 were found to be S22A_j1 and S22A_j3 encoding DUF1668-containing protein. The allele of a wild rice species Oryza meridionalis Ng at S22B, designated S22B_m, is a loss-of-function allele probably due to large deletion of the gene lacking DUF1668 domain and evolved from the different lineage of O. glumaepatula. Phylogenetic analysis of DUF1668 suggested that many gene duplications occurred before the divergence of current crops in Poaceae, and loss-of-function mutations of DUF1668-containing genes represent the candidate causal genetic events contributing to hybrid incompatibilities. The duplicated DUF1668-domain gene may provide genetic potential to induce hybrid incompatibility by consequent mutations after divergence.

Polyploidy Enhances F1 Pollen Sterility Loci Interactions That Increase Meiosis Abnormalities and Pollen Sterility in Autotetraploid Rice.

Intersubspecific autotetraploid rice (Oryza sativa ssp. indica × japonica) hybrids have greater biological and yield potentials than diploid rice. However, the low fertility of intersubspecific autotetraploid hybrids, which is largely caused by high pollen abortion rates, limits their commercial utility. To decipher the cytological and molecular mechanisms underlying allelic interactions in autotetraploid rice, we developed an autotetraploid rice hybrid that was heterozygous (S(i)S(j)) at F1 pollen sterility loci (Sa, Sb, and Sc) using near-isogenic lines. Cytological studies showed that the autotetraploid had higher percentages (>30%) of abnormal chromosome behavior and aberrant meiocytes (>50%) during meiosis than did the diploid rice hybrid control. Analysis of gene expression profiles revealed 1,888 genes that were differentially expressed between the autotetraploid and diploid hybrid lines at the meiotic stage, among which 889 and 999 were up- and down-regulated, respectively. Of the 999 down-regulated genes, 940 were associated with the combined effect of polyploidy and pollen sterility loci interactions (IPE). Gene Ontology enrichment analysis identified a prominent functional gene class consisting of seven genes related to photosystem I (Gene Ontology 0009522). Moreover, 55 meiosis-related or meiosis stage-specific genes were associated with IPE in autotetraploid rice, including Os02g0497500, which encodes a DNA repair-recombination protein, and Os02g0490000, which encodes a component of the ubiquitin-proteasome pathway. These results suggest that polyploidy enhances epistatic interactions between alleles of pollen sterility loci, thereby altering the expression profiles of important meiosis-related or meiosis stage-specific genes and resulting in high pollen sterility.

Two linked genes on rice chromosome 2 for F1 pollen sterility in a hybrid between Oryza sativa and O. glumaepatula.

Hybrid incompatibility plays an important role in establishment of post-zygotic reproductive isolation. To unveil genetic basis of hybrid incompatibilities between diverged species of genus Oryza AA genome species, we conducted genetic dissection of hybrid sterility loci, S22(t), which had been identified in backcross progeny derived from Oryza sativa ssp. japonica (recurrent parent) and South American wild rice O. glumaepatula near the end of the short arm of chromosome 2. The S22(t) region was found to be composed of two loci, designated S22A and S22B, that independently induce F1 pollen sterility. Pollen grains containing either of the sterile alleles (S22A-glums or S22B-glums) were sterile if produced on a heterozygous plant. No transmission of the S22A-glums allele via pollen was observed, whereas a low frequency of transmission of S22B-glums was observed. Cytological analysis showed that the sterile pollen grains caused by S22A could reach the bicellular or tricellular stage, and the nearly-sterile pollen grains caused by S22B could reach the tricellular stage. Our genetic analysis showed repulsion linkage effect is possible to induce strong reproductive barrier by high pollen sterility based on recombination value and transmission ratio of hybrid sterility gene to the progeny was influenced by frequency of competitors on fertilization.

HYBRID INCOMPATIBILITY IS ACQUIRED FASTER IN ANNUAL THAN IN PERENNIAL SPECIES OF SUNFLOWER AND TARWEED

Hybrid sterility is an important species barrier, especially in plants where hybrids can often form between divergent taxa. Here we explore how life history affects the acquisition of hybrid sterility in two groups in the sunflower family. We analyzed genetic distance and F1 pollen sterility for interspecific crosses in annual and perennial groups. We find that reproductive isolation is acquired in a steady manner and that annual species acquire hybrid sterility barriers faster than perennial species. Potential causes of the observed sterility pattern are discussed.

Double‐Neutral Genes, Sa‐n and Sb‐n, for Pollen Fertility in Rice to Overcome Indica × Japonica Hybrid Sterility

A major obstacle in the development of superior intersubspecific hybrids between subspecies (indica and japonica) of Asian cultivated rice (Oryza sativa L.) is partial sterility of the F1 generation. Neutral genes, Sa‐n, Sb‐n, and Sc‐n, for pollen fertility have the potential to overcome F1 pollen sterility. The present study was planned to find double‐neutral genes for pollen fertility and to evaluate their effect on pollen fertility of intersubspecific hybrids. Five genotypes were crossed with eight isogenic lines to mine neutral genes for pollen fertility. Pollen fertility of hybrids was observed in F1 and F2, and segregation ratios were recorded in F2 to confirm the neutral genes. The results showed that DN18 and DN75 had double‐neutral genes (Sa‐n and Sb‐n) whereas DN22 had three neutral genes Sa‐n, Sb‐n, and Sc‐n at Sa, Sb, and Sc pollen sterility loci, respectively. Furthermore, we found a single neutral gene (Sc‐n) for pollen fertility at the Sc locus and neutral gene for embryo sac fertility at the S5 locus in DN13. DN18 and DN75 were crossed with numerous typical japonica and indica cultivars. Results showed that double‐neutral genes could overcome most of the intersubspecific hybrid pollen sterility. These lines could be used for the development of high yielding indica × japonica hybrids and neutral genes could be introgressed into elite cultivars of Asian cultivated rice for fertility restoration.

Identification of neutral alleles at pollen sterility gene loci of cultivated rice (Oryza sativa L.) from wild rice (O. rufipogon Griff.)

Oryza rufipogon Griff. is the ancestor of Asian cultivated rice (O. sativa L.) and possesses valuable genes for rice breeding. Pollen abortion is one of the major causes of indica–japonica hybrid sterility in rice and it happens due to allelic interaction at the pollen sterility gene loci. A total of six loci (Sa, Sb, Sc, Sd, Se, and Sf) have been found to be associated with F1 pollen sterility between indica and japonica rice, and five of them (all except Sf) have been mapped. Neutral alleles (Sn) at each locus have the potential to overcome the pollen sterility associated with the respective locus. Therefore, exploitation and utilization of neutral alleles have significant importance in overcoming indica–japonica hybrid sterility. In this study, an accession (IRW28) of O. rufipogon, native to China, was selected as paternal to cross with typical japonica (Taichung 65) and indica (Guanglu’ai 4) tester lines, and two F2 populations were developed. The simple sequence repeat markers tightly linked to five pollen sterility loci were applied for genotyping the F2 populations. Chi-squared tests were applied to examine the normal segregation/distortion at each locus. The expected and observed pollen sterility for each locus were estimated. As a result, the genotypes at five pollen sterility gene loci for IRW28 were identified as: Sai−1/Sai−1, Sbn/Sbn, Sci−2/Sci−2, Sdn/Sdn and Sen/Sen. Our results suggest that IRW28 (O. rufipogon) has the neutral alleles for pollen fertility at the Sb, Sd and Se loci, and these alleles have a good affinity with indica and japonica rice. These neutral alleles provide valuable gene resources to overcome the inter-subspecific hybrid pollen sterility in rice.

Allelic interaction of F1 pollen sterility loci and abnormal chromosome behaviour caused pollen sterility in intersubspecific autotetraploid rice hybrids

The intersubspecific hybrids of autotetraploid rice has many features that increase rice yield, but lower seed set is a major hindrance in its utilization. Pollen sterility is one of the most important factors which cause intersubspecific hybrid sterility. The hybrids with greater variation in seed set were used to study how the F1 pollen sterile loci (S-a, S-b, and S-c) interact with each other and how abnormal chromosome behaviour and allelic interaction of F1 sterility loci affect pollen fertility and seed set of intersubspecific autotetraploid rice hybrids. The results showed that interaction between pollen sterility loci have significant effects on the pollen fertility of autotetraploid hybrids, and pollen fertility further decreased with an increase in the allelic interaction of F1 pollen sterility loci. Abnormal ultra-structure and microtubule distribution patterns during pollen mother cell (PMC) meiosis were found in the hybrids with low pollen fertility in interphase and leptotene, suggesting that the effect-time of pollen sterility loci interaction was very early. There were highly significant differences in the number of quadrivalents and bivalents, and in chromosome configuration among all the hybrids, and quadrivalents decreased with an increase in the seed set of autotetraploid hybrids. Many different kinds of chromosomal abnormalities, such as chromosome straggling, chromosome lagging, asynchrony of chromosome disjunction, and tri-fission were found during the various developmental stages of PMC meiosis. All these abnormalities were significantly higher in sterile hybrids than in fertile hybrids, suggesting that pollen sterility gene interactions tend to increase the chromosomal abnormalities which cause the partial abortion of male gametes and leads to the decline in the seed set of the autotetraploid rice hybrids.

Identifying neutral allele Sb at pollen-sterility loci in cultivated rice with Oryza rufipogon origin

Pollen sterility is commonly found in the intra-specific hybrids of indica and japonica rice, which is one of the main constrains for the utilization of heterosis between indica and japonica. Six loci controlling the pollen sterility of F1 between indica and japonica have been identified from previous studies. Neutral alleles at each locus are potential to overcome the F1 pollen sterility associated with the locus. Therefore, exploitation and utilization of neutral alleles are of significant importance. The present research was based on fine mapping of the F1 pollen-sterility gene Sb and the abundant genetic diversity of Oryza rufipogon Griff. indigenous to Gaozhou, Guangdong Province (referred to as Gaozhou wild rice). Crosses were made using Taichung65 (with the genotype of S b j S b j and referred to as E1) and its near-isogenic line of F1 pollen sterility gene Sb (with the genotype of S b j S b j , E2) as female parents, and 12 different accessions of Gaozhou wild rice as male parents. F1 pollen fertility was examined to identify the materials having the neutral alleles at the F1 pollen-sterility locus. Segregation of 4 molecular markers tightly linked with the Sb locus was analyzed in the F2 populations derived from the F1s carrying the neutral gene. The pollen fertility related to the 3 genotypes of the molecular markers was also checked by statistical test to determine whether it was consistent with the hypothesis. The results showed that the pollen fertility of two F1s from one accession of Gaozhou wild rice (GZW099) with E1 and E2 was (89.22±1.07)% and (85.65±1.05)%, respectively. Both of them were fertile and showed no significant difference by t-test. Segregation of the 3 genotypes of the 4 molecular markers followed the expected Mendelian ratio (1:2:1) in the F2 populations. There was no significant difference for the averaged pollen fertility of the plants related to the 3 genotypes, suggesting that no interaction exists between the alleles at the Sb locus in GZW099 and Taichung65 or E2. Evidentially, GZW099 carried the neutral gene (named S b n S b n ) at the Sb locus, which provides valuable theoretical basis and resources for further studying and overcoming the sterility of indica-japonica hybrids.

Identification of two loci causing F1 pollen sterility in inter- and intraspecific crosses of rice

F1 sterility, one of the most common post-zygotic reproductive barriers, is frequently observed in both interspecific and intraspecific crosses of rice. Elucidating the genetic and cytological mechanisms of F1 pollen sterility is important to exploit genetic resources and to understand the evolutionary dynamics of post-zygotic reproductive isolation in rice. Here, we report two F1 pollen sterility loci, designated S36 and S25, found in an interspecific cross between O. sativa ssp. japonica (Taichung 65) and O. nivara (IRGC105444), and an intraspecific cross between O. sativa ssp. japonica (Asominori) and ssp. indica (IR24). Genetic analyses revealed that both loci are located on distal end of the short arm of chromosome 12 and that allelic interaction at the heterozygous locus caused the sterility of male gametes carrying the japonica alleles in both cases. Comparison of map positions of S36 and S25 suggested that these two loci might be the same locus. Cytological investigation revealed that abnormality of sterile pollen grains caused by S36 occurred mainly at the late bicellular stage after initiation of starch accumulation. This study would provide the better understanding on the genetic nature and the cytological mechanism of F1 pollen sterility in rice.

Identification and fine mapping of S-d, a new locus conferring the partial pollen sterility of intersubspecific F1 hybrids in rice (Oryza sativa L.)

The partial pollen abortion of hybrids between the indica and japonica subspecies of Asian cultivated rice is one of the major barriers in utilizing intersubspecific heterosis in hybrid rice breeding. Although a single hybrid pollen sterility locus may have little impact on spikelet fertility, the cumulative effect of several loci usually leads to a serious decrease in spikelet fertility. Isolating of the genes conferring hybrid pollen sterility is necessary to understand this phenomenon and to overcome the resulting genetic barrier. In this study, a new locus for F1 pollen sterility, S-d, was identified on the short arm of chromosome 1 by analyzing the genetic effect of substituted segments of the near-isogenic line E11-5 derived from the japonica variety Taichung 65 (recurrent parent) and the indica variety Dee-geo-woo-gen (donor parent). The S-d locus was first mapped to a 0.8 cM interval between SSR markers PSM46 and PSM80 using a F2 population of 125 individuals. The flanking markers were then used to identify recombinants from a population of 2,160 plants derived from heterozygotes of the primary F2 population. Simultaneously, additional markers were developed from genomic sequence divergence in this region. Analysis of the recombinants in the region resulted in the successful mapping of the S-d locus to a 67-kb fragment, containing 17 predicted genes. Positional cloning of this gene will contribute to our understanding of the molecular basis for partial pollen sterility of intersubspecific F1 hybrids in rice.

Cytological mechanism of pollen abortion resulting from allelic interaction of F1 pollen sterility locus in rice (Oryza sativa L.)

Pollen abortion is one of the major reasons causing the inter-subspecific F(1) hybrid sterility in rice and is due to allelic interaction of F(1) pollen sterility genes. The microsporogenesis and microgametogenesis of Taichung 65 and its three F(1) hybrids were comparatively studied by using techniques of differential interference contrast microscopy, semi-thin section light microscopy, epifluorescence microscopy and TEM. The results showed that there were differences among the cytological mechanisms of pollen abortion due to allelic interaction at the three F(1) pollen sterility loci. The allelic interaction at S-a locus resulted in microspores unable to extend the protoplasm membrane with the enlargement of the microspore at the middle microspore stage and finally producing empty abortive pollen. The allelic interaction at S-b locus caused asynchronous development of microspores at the middle microspore stage producing stainable abortive pollen. The allelic interaction at S-c locus mainly led to the non-dissolution of the generative cell wall and finally caused the hybrid F(1) mainly producing stainable abortive pollen. Genotypic identification indicated that the abortive pollen were those with S ( j ) allele.

Fine mapping of locus S-b for F1 pollen sterility in rice (Oryza sativa L.)

Hybrid sterility is the main barrier in utilizing the heterosis of subspecies in rice. A knowledge of the underlying molecular mechanism of the hybrid sterility will be useful for overcoming the barrier. In this research, the F1 pollen sterility locus, S-b, was mapped between SSR markers PSM8 and PSM202. To fine map the locus, one F2 mapping population of 3910 plants was developed using the near-isogenic lines of the locus. Ninety-seven recombinants between two markers were selected. Moreover, a series of markers, including two SSR markers, two InDel markers and four CAPS markers, were developed on the region. Linkage analysis showed that marker W4 was co-segregated with locus S-b, while makers A8 and A14 were located on the two sides of the locus with a distance of 0.026 and 0.038 cM, respectively. The markers were then integrated with the sequences of the clones of the region. Results showed that all the polymorphic markers were anchored on the three end-to-end jointed clones AC093089, AC079021 and AC 134931. According to the physical information of the markers, locus S-b was finally delimited to a region of 27 kb between A8 and A14. Seven ORFs were identified on the region based on the annotation results of RiceGAAS system. These results laid the foundation for further cloning the gene.

Molecular mapping of S-c, an F1 pollen sterility gene in cultivated rice

Hybrids between indica and japonica rice varieties usually show partial sterility, and are a major limiting factor in the utilization of heterosis at subspecific level. When studying male-gamete (pollen) abortion, a possibly important cause for sterility, six loci (S-a, S-b, S-c, S-d, S-e and S-f) for F1 pollen sterility were identified. Here we report genetic and linkage analysis of S-c locus using molecular markers in a cross between Taichung 65, a japonica variety carrying allele S-cj, and its isogenic line TISL5, carrying alleleS-cj. Our results show that pollen sterility occurring in the hybrids is controlled by one locus. We used 208 RFLP markers, as well as 500 RAPD primers, to survey the polymorphism between Taichung 65 and TISL5. Six RFLP markers located on a small region of chromosome 3, detected different RFLP patterns. Co-segregation analysis of fertility and RFLP patterns with 123 F2 plants confirmed that the markers RG227, RG391, R1420 were completely linked with the S-c locus. The genetic distances between the markers C730, RG166 and RG369 and the S-c locus were 0.5 cM, 3.4 cM, and 3.4 cM respectively. Distorted F2 ratios were also observed for these 4 RFLP markers in the cross. This result suggests that the `one locus sporo-gametophytic' model could explain F1 hybrid pollen sterility in cultivated rice. RG227, the completely linked marker, has been converted to STS marker for marker-assisted selection.

Molecular mapping of genes for F1 pollen sterility in rice

Molecular mapping of genes for F1 pollen sterility in rice

Origin of the genic diversity of cultivated rice (Oryza spp.): study of the polymorphism scored at 40 isozyme loci.

An electrophoretic survey of up to forty presumed isozyme loci was carried out in Asian and African cultivated rice (O. sativa and O. glaberrima) and in weedy and wild forms of African O. breviligulata on a total of 1, 948 strains. Hidden variability was checked by a test of heat stability at thirty isozyme loci in the same species.The mean gene diversity index (heterozygosity) was high (0.23) in O. Sativa, medium (0.14) in wild O. breviligulata and low (0.06 and 0.03) in weedy O. breviligulata and O. glaberrima.In contrast, a maximum of seven alleles at a single locus could be distinguished in wild O. breviligulata while only three at the most were found in O. sativa and two in O. glaberrima and the weedy forms of O. breviligulata.Calculations of genetic distances showed that the cultivated, wild and weedy African species formed a genetic group distinct from O. sativa. Multivariate analysis of the data on an individual strain basis confirmed this fact and showed in turn that most varieties of O, sativa tended to cluster in two groups which correspond to the so-called Indica and Japonica subspecies. There was however a continuous array of intermediates between the two groups.Multivariate analysis also showed that the endemic African strains with the most genetic affinity to O. sativa were certain strains of the weedy form of O. breviligulata.Analysis of F1 pollen sterility among 115 strains of O, sativa permitted the extraction of one small Indica and one small Japonica group of strains characterized by a high pollen sterility relationship but most strains were intermediate.Each group had little gene diversity, with more than 80% of the loci fixed for one allele. Their isozyme patterns were remarkably complementary in that most of the various gametic associations of alleles found in the numerous intermediate strains could be explained by hybridization between varieties belonging to one and the other group. Consequently, these were assumed to represent the ancestral isozyme patterns of the Indica and Japonica subsp.Similar genetic distances, which point to a divergence time of one to a few millions years ago were found between O. glaberrima and the ancestral types of Indica and Japonica in the three combinations.Assuming the neutral theory of isozyme polymorphism, the data confirmed that O. glaberrima was domesticated from O. breviligulata independently of O, sativa. They also suggest that, at the origin of O. sativa, the Indica and the Japonica types were also domesticated independently. The large diversity of O. sativa would result: (i) primarily from introgressions that occurred between the ancestral cultivated types, together with the selection imposed by man, and (ii) secondarily, following the dispersion of cultivars, from the introgression of genes of wild rice in various areas. Some of the weedy forms of O. breviligulata could have originated from natural hybrids between the two cultivated species, O. sativa and O. glaberrima.

REPRODUCTIVE BARRIERS DISTRIBUTED IN CULTIVATED RICE SPECIES AND THEIR WILD RELATIVES

F1 plants from 2, 728 crosses in total, made between strains of Oryza perennis Moench, O. sativa L., O. breviligulata Chev. et Roehr., and O. glaberrima Steud., were observed in a survey of hybrid sterility and other developmental abortions which constitute reproductive barriers. All the F1 plants observed showed no significant disturbance in chromosome pairing. Reproductive barriers were found as follows:1) A low crossability due to deterioration of young F1 zygotes was found between the African form of perennis (subsp. barthii) and other taxa.2) Vegetative weakness of F1 plants was frequently found among strains of glaberrima and breviligulata as well as in their hybrids with barthii and other taxa. It was sporadical among perennis and sativa strains.3) F1 pollen sterility was found in various cross-combinations. Strains of glaberrima and breviligulata were inter-fertile with a few exceptions, and generally showed high F1 sterility with most perennis and sativa strains. Strains of perennis could be largely divided into four inter-sterile groups, Asian, African, American and Oceanian, suggesting that isolation by the ocean might have played an important role in phylogenetic differentiation. But some African (barthii) strains showed partial fertilities with others.4) Embryosac fertility was shown by the percentage of anatomically normal embryosacs, which was comparable to the rate of successful back-crossing. No F1 hybrids were completely embryosac-sterile. They were divided into two groups, one in which pollen and embryosac fertilities were correlated and the other in which pollen fertility was near zero but embryosac fertility was apparently higher. Crosses between perennis-sativa and breviligulata-glaberrima series and some other distant crosses belonged to the latter group, in which embryosac tetrads tended to deteriorate.