Circular RNA hsa_circ_0011298 enhances Taxol resistance of non-small cell lung cancer by regulating miR-486-3p/CRABP2 axis.

BACKGROUND: Circular RNAs (circRNAs) serve as critical regulators in the chemoresistance of human cancers, including non-small cell lung cancer (NSCLC). We aimed to explore the role of hsa_circ_0011298 (circ_0011298) and its mechanism in Taxol resistance of NSCLC. METHODS: Circ_0011298, microRNA-486-3p (miR-486-3p), and CRABP2 mRNA expression were determined using qRT-PCR. EdU and MTT assays were used to detect cell proliferation. Cell cycle distribution and cell apoptosis were detected by flow cytometry. Cell migratory and invasive abilities were detected using transwell assay. Cellular glycolysis was determined by specific kits. Protein levels were examined by western blot. Dual-luciferase reporter and RIP assays were performed to confirm the relationship between miR-486-3p and circ_0011298 or CRABP2. Xenograft mice model was established to confirm the function of circ_0011298 in vivo. RESULTS: Circ_0011298 was overexpressed in Taxol-resistant NSCLC cells and tissues. Circ_0011298 knockdown enhanced Taxol sensitivity by decreasing cell proliferation, migration, invasion, and glycolysis and inducing apoptosis and cell cycle arrest in Taxol-resistant NSCLC cells. Circ_0011298 was a sponge of miR-486-3p, and the impact of circ_0011298 silencing on Taxol resistance was rescued by miR-486-3p inhibition. Moreover, miR-486-3p directly targeted CRABP2, and miR-486-3p inhibited Taxol resistance by targeting CRABP2. Furthermore, circ_0011298 regulated CRABP2 expression through targeting miR-486-3p. Importantly, circ_0011298 interference elevated Taxol sensitivity of NSCLC in vivo. CONCLUSION: Circ_0011298 elevated Taxol resistance of NSCLC by sponging miR-486-3p and upregulating CRABP2, providing a possible circRNA-targeted therapy for NSCLC.

Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Identification of the Viral Determinant of Hypovirulence and Host Range in Sclerotiniaceae of a Genomovirus Reconstructed from the Plant Metagenome.

Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified in the newly formed family Genomoviridae. Here, we determined the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fructicola, two economically important members of the family Sclerotiniaceae, and Fusarium oxysporum. SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea, and M. fructicola. SlaGemV-1 also replicates in Spodoptera frugiperda insect cells but not in Caenorhabditis elegans or plants. By expressing viral genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. IMPORTANCE Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant-pathogenic fungi and fall armyworm (S. frugiperda Sf9) insect cells but not a model nematode, C. elegans, or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species in the family Sclerotiniaceae, including Sclerotinia sclerotiorum, Botrytis cinerea, and Monilinia fructicola. The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenomes can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.

Assembling seed dormancy genes into a system identified their effects on seedbank longevity in weedy rice.

Seed dormancy (SD) and longevity (SL) may share developmental and genetic mechanisms, as both traits are developed in the same maternal environment and evolved to coordinate the timing of germination and the life span of seedbanks. To test the hypothesis, allelic variants at the SD1-2, 7-1, 7-2, and 12 loci from weedy and cultivated rice (Oryza sativa) were assembled into the same genetic background, and 16 homozygous lines selected as a tetragenic system. These lines were evaluated for SD measured by germination at 7, 21, 35, and 150 days of after-ripening (DAR), and for SL measured by the seed decay rate and survivability in the soil of a rice field for 7 months. Pyramiding the alleles from weedy rice lengthened the dormancy duration, and seeds survived in the soil remained dormant at the excavation. Germination levels at 7 to 150 DAR were correlated positively with the seed decay rate (r = 0.41-0.53) and negatively with the survivability (r = -0.45 to -0.28) in the tetragenic system. All four loci contributed to genotypic variation for each of the SD and SL measurements through main and/or epistatic (two- to four-order interactions) effects. SD7-1 (identical to the pericarp color gene Rc) played a major role in regulating seedbank longevity when interacted with the other SD gene(s). This research provided evidence that natural genes controlling SD are involved in regulation of soil seedbank longevity. Thus, accumulation of SD genes in a population could result in persistence of wild plants and weeds in conventional tillage systems.

Metatranscriptomic Analysis and In Silico Approach Identified Mycoviruses in the Arbuscular Mycorrhizal Fungus Rhizophagus spp.

Arbuscular mycorrhizal fungi (AMF), including Rhizophagus spp., can play important roles in nutrient cycling of the rhizosphere. However, the effect of virus infection on AMF's role in nutrient cycling cannot be determined without first knowing the diversity of the mycoviruses in AMF. Therefore, in this study, we sequenced the R. irregularis isolate-09 due to its previously demonstrated high efficiency in increasing the N/P uptake of the plant. We identified one novel mitovirus contig of 3685 bp, further confirmed by reverse transcription-PCR. Also, publicly available Rhizophagus spp. RNA-Seq data were analyzed to recover five partial virus sequences from family Narnaviridae, among which four were from R. diaphanum MUCL-43196 and one was from R. irregularis strain-C2 that was similar to members of the Mitovirus genus. These contigs coded genomes larger than the regular mitoviruses infecting pathogenic fungi and can be translated by either a mitochondrial translation code or a cytoplasmic translation code, which was also reported in previously found mitoviruses infecting mycorrhizae. The five newly identified virus sequences are comprised of functionally conserved RdRp motifs and formed two separate subclades with mitoviruses infecting Gigasporamargarita and Rhizophagusclarus, further supporting virus-host co-evolution theory. This study expands our understanding of virus diversity. Even though AMF is notably hard to investigate due to its biotrophic nature, this study demonstrates the utility of whole root metatranscriptome.

Two Contrasting Patterns and Underlying Genes for Coadaptation of Seed Dormancy and Flowering Time in Rice.

Association between seed dormancy (SD) and flowering time (FT) may generate a synergy in plant adaptation. This research aimed to identify patterns and underlying genes of the association in rice (Oryza sativa). Four F2 and two BC1F1 populations from crosses of weedy/cultivated rice, and two families of progeny lines from backcrosses were evaluated for variations in time to flowering and germination ability. The two measurements were correlated negatively in the F2 and BC1F1 populations, but positively in advanced generations of the progeny lines. The negative correlations were resulted from linkage disequilibria between SD and FT loci at 7-40 cM apart. The positive correlations arose from co-located SD and FT loci undetectable in the BC1F1 population. Two independent sets of co-localized loci were isolated as single Mendelian factors, and haplotypes that promote flowering and reduce germination derived from weedy and cultivated rice, respectively. The presence of negative and positive correlations indicates that the rice complex has maintained two contrasting patterns of SD-FT coadaptation, with the positive being "recessive" to the negative pattern. Modeling with isogenic lines suggests that a negative pattern could generate a greater synergy (difference between haplotype variants) than the positive one for seedbank persistence, or enhanced plant adaptation to seasonal changes in temperature or moisture. However, the early-flowering dormant genotype of a positive pattern could also have a selective advantage over its counterpart for weeds to avoid harvesting. The isolated haplotypes could be used to manipulate cultivars simultaneously for germination ability and growth duration.

The synergistic effects of Apatinib combined with cytotoxic chemotherapeutic agents on gastric cancer cells and in a fluorescence imaging gastric cancer xenograft model.

INTRODUCTION: Methylsulfonic apatinib (hereinafter referred to as Apatinib) is a small-molecule angiogenesis inhibitor highly and selectively targeted to vascular endothelial growth factor receptor-2. At present, a series of basic and clinical studies have confirmed that Apatinib mono-therapy can inhibit the growth of different carcinomas. Our experiment aimed to determine whether there is a synergistic effect between the combination of the traditional cytotoxic chemotherapy drugs paclitaxel (TAX), oxaliplatin (L-OHP), 5-fluorouracil (5-FU), and Apatinib. MATERIALS AND METHODS: We evaluated the combined effect using cytological experiments and a fluorescence imaging xenograft model. In vitro, the inhibition of cell proliferation increased notably when Apatinib was combined with TAX, L-OHP, and 5-FU. Then, for the mechanistic research, we selected the optimal dose of drugs that also had a synergistic effect. Apatinib combined with the aforementioned drugs, especially the combination of Apatinib and 5-FU, decreased the invasion and migration ability of the cells and increased the apoptosis ratio; expression of the anti-apoptotic protein Bcl-2 significantly decreased, and expression of the pro-apoptotic protein Bax increased. In vivo, when Apatinib was combined with TAX, L-OHP, and 5-FU, the volume of the xenograft model was significantly inhibited, the strength of the green fluorescence was weakened and the microvessel density decreased. RESULTS: The combination of Apatinib with TAX and 5-FU was synergistic (coefficient of drug interaction <1); the combination effect of Apatinib and L-OHP was only additive, with a shorter associated survival time. CONCLUSION: The combination of Apatinib and classical chemotherapy drugs may be an optimal choice for gastric cancer treatment.

Triploid Production from Interspecific Crosses of Two Diploid Perennial Helianthus with Diploid Cultivated Sunflower (Helianthus annuus L.).

Wild Helianthus species are a valuable genetic resource for the improvement of cultivated sunflower. We report the discovery and characterization of a unique high frequency production of triploids when cultivated sunflower was pollinated by specific accessions of diploid Helianthus nuttallii T. & G. and H. maximiliani Schr. Genomic in situ hybridization (GISH) analyses indicated that the triploid F1s had two genomes from the wild pollen sources and one from the cultivated line. Mitotic chromosome analyses indicated that the frequency of triploid progenies from the crosses of cultivated lines × H. nuttallii accession 102 (N102) was significantly higher than those of unexpected polyploid progenies from the crosses of wild perennial species × N102, and no unexpected polyploids were obtained from the reverse crosses. Pollen stainability analysis suggested the existence of a low percentage of unreduced (2n) male gametes in some accessions, especially N102 and H. maximiliani accession 1113 (M1113), which were generated at the telophase II and tetrad stages of meiosis. The triploid F1s could be the results of preferred fertilization of the low frequency of 2n male gametes with the female gametes of the cultivated sunflower, due to the dosage factors related to recognition and rejection of foreign pollen during fertilization. The triploids have been used to produce amphiploids and aneuploids. Future studies of the male gametes' fate from pollination through fertilization will further uncover the mechanism of this whole genome transmission. Studies of the genetic control of this trait will facilitate research on sunflower polyploidy speciation and evolution, and the utilization of this trait in sunflower breeding.

Map-Based Cloning of Seed Dormancy1-2 Identified a Gibberellin Synthesis Gene Regulating the Development of Endosperm-Imposed Dormancy in Rice.

Natural variation in seed dormancy is controlled by multiple genes mapped as quantitative trait loci in major crop or model plants. This research aimed to clone and characterize the Seed Dormancy1-2 (qSD1-2) locus associated with endosperm-imposed dormancy and plant height in rice (Oryza sativa). qSD1-2 was delimited to a 20-kb region, which contains OsGA20ox2 and had an additive effect on germination. Naturally occurring or induced loss-of-function mutations of the gibberellin (GA) synthesis gene enhanced seed dormancy and also reduced plant height. Expression of this gene in seeds (including endospermic cells) during early development increased GA accumulation to promote tissue morphogenesis and maturation programs. The mutant allele prevalent in semidwarf cultivars reduced the seed GA content by up to 2-fold at the early stage, which decelerated tissue morphogenesis including endosperm cell differentiation, delayed abscisic acid accumulation by a shift in the temporal distribution pattern, and postponed dehydration, physiological maturity, and germinability development. As the endosperm of developing seeds dominates the moisture equilibrium and desiccation status of the embryo in cereal crops, qSD1-2 is proposed to control primary dormancy by a GA-regulated dehydration mechanism. Allelic distribution of OsGA20ox2, the rice Green Revolution gene, was associated with the indica and japonica subspeciation. However, this research provided no evidence that the primitive indica- and common japonica-specific alleles at the presumably domestication-related locus functionally differentiate in plant height and seed dormancy. Thus, the evolutionary mechanism of this agriculturally important gene remains open for discussion.

Genotyping of endosperms to determine seed dormancy genes regulating germination through embryonic, endospermic, or maternal tissues in rice.

Seed dormancy is imposed by one or more of the embryo, endosperm, and maternal tissues that belong to two generations and represent two ploidy levels. Many quantitative trait loci (QTL) have been identified for seed dormancy as measured by gross effects on reduced germination rate or delayed germination in crop or model plants. This research developed an endosperm genotype-based genetic approach to determine specific tissues through which a mapped QTL regulates germination using rice as a model. This approach involves testing germination velocity for partially after-ripened seeds harvested from single plants heterozygous for a tested QTL and genotyping endosperms from individual germinated and nongerminated seeds with a codominant DNA marker located on the QTL peak region. Information collected about the QTL includes genotypic frequencies in germinated and/or nongerminated subpopulations; allelic frequency distributions during a germination period; endosperm or embryo genotypic differences in germination velocity; and genotypic frequencies for gametes involved in the double fertilization to form the sampled seeds. Using this approach, the seed dormancy loci SD12, SD1-2, and SD7-1 were determined to regulate germination through the embryo, endosperm, and maternal tissues, respectively; SD12 and SD1-2 acted additively on germination velocity in the offspring tissues; and SD12 also was associated with the preferential fertilization of male gametes in rice. This new genetic approach can be used to characterize mapped genes/QTL for tissue-specific functions in endospermic seeds and for marker-assisted selection of QTL alleles before or immediately after germination in crop breeding.

Quantitative trait locus and haplotype analyses of wild and crop-mimic traits in U.S. weedy rice.

Conspecific weeds retained characteristics from wild ancestors and also developed crop mimicries for adaptation and competitiveness. This research was conducted to identify quantitative trait loci (QTL) associated with the wild and crop-mimic traits and to determine haplotype variants for QTL-rich regions in U.S. weedy rice. An F2 population from the cross between a cultivated (EM93-1) and a U.S. weedy (US1) rice line was evaluated for six wild and eight crop-mimic traits in a greenhouse to identify the QTL. A core collection of 27 U.S. weedy red rice lines and 14 AA-genome wild rice lines were determined for the haplotype variants. A total of 49 QTL were identified, with 45 collocated as clusters on 14 genomic segments. The number of haplotypes across the 14 segments was lower in the weedy (6.1 ± 2.4) than in the wild (7.5 ± 1.8) rice sample. Both samples shared ~50% haplotypes (wild-like). The EM93-1-like haplotypes accounted for a greater proportion (30 ± 26%) of the haplotypes in the weedy than in the wild (7 ± 10%) rice. Based on haplotype patterns for the 14 QTL cluster regions, 26 of the 28 red rice lines were clustered into two groups corresponding to the black-hull awned and straw-hull awnless morphological types, respectively. The QTL analysis demonstrated that conspecific weed-crop differentiation involved many genomic segments with multiple loci regulating natural variation for adaptation and competitiveness. The haplotype analysis revealed that U.S. weedy rice retained large blocks of linkage disequilibrium for the multiple loci from the wild relatives and also incorporated haplotypes from cultivars.

Genetic and physiological characterization of two clusters of quantitative trait Loci associated with seed dormancy and plant height in rice.

Seed dormancy and plant height have been well-studied in plant genetics, but their relatedness and shared regulatory mechanisms in natural variants remain unclear. The introgression of chromosomal segments from weedy into cultivated rice (Oryza sativa) prompted the detection of two clusters (qSD1-2/qPH1 and qSD7-2/qPH7) of quantitative trait loci both associated with seed dormancy and plant height. Together, these two clusters accounted for >96% of the variances for plant height and ~71% of the variances for germination rate in an isogenic background across two environments. On the initial introgression segments, qSD1-2/qPH1 was dissected genetically from OsVp1 for vivipary and qSD7-2/qPH7 separated from Sdr4 for seed dormancy. The narrowed qSD1-2/qPH1 region encompasses the semidwarf1 (sd1) locus for gibberellin (GA) biosynthesis. The qSD1-2/qPH1 allele from the cultivar reduced germination and stem elongation and the mutant effects were recovered by exogenous GA, suggesting that sd1 is a candidate gene of the cluster. In contrast, the effect-reducing allele at qSD7-2/qPH7 was derived from the weedy line; this allele was GA-insensitive and blocked GA responses of qSD1-2/qPH1, including the transcription of a GA-inducible α-amylase gene in imbibed endosperm, suggesting that qSD7-2/qPH7 may work downstream from qSD1-2/qPH1 in GA signaling. Thus, this research established the seed dormancy-plant height association that is likely mediated by GA biosynthesis and signaling pathways in natural populations. The detected association contributed to weed mimicry for the plant stature in the agro-ecosystem dominated by semidwarf cultivars and revealed the potential benefit of semidwarf genes in resistance to preharvest sprouting.

Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones.

Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (~100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC-fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)-derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources.

Diversifying sunflower germplasm by integration and mapping of a novel male fertility restoration gene.

The combination of a single cytoplasmic male-sterile (CMS) PET-1 and the corresponding fertility restoration (Rf) gene Rf1 is used for commercial hybrid sunflower (Helianthus annuus L., 2n = 34) seed production worldwide. A new CMS line 514A was recently developed with H. tuberosus cytoplasm. However, 33 maintainers and restorers for CMS PET-1 and 20 additional tester lines failed to restore the fertility of CMS 514A. Here, we report the discovery, characterization, and molecular mapping of a novel Rf gene for CMS 514A derived from an amphiploid (Amp H. angustifolius/P 21, 2n = 68). Progeny analysis of the male-fertile (MF) plants (2n = 35) suggested that this gene, designated Rf6, was located on a single alien chromosome. Genomic in situ hybridization (GISH) indicated that Rf6 was on a chromosome with a small segment translocation on the long arm in the MF progenies (2n = 34). Rf6 was mapped to linkage group (LG) 3 of the sunflower SSR map. Eight markers were identified to be linked to this gene, covering a distance of 10.8 cM. Two markers, ORS13 and ORS1114, were only 1.6 cM away from the gene. Severe segregation distortions were observed for both the fertility trait and the linked marker loci, suggesting the possibility of a low frequency of recombination or gamete selection in this region. This study discovered a new CMS/Rf gene system derived from wild species and provided significant insight into the genetic basis of this system. This will diversify the germplasm for sunflower breeding and facilitate understanding of the interaction between the cytoplasm and nuclear genes.

Association between seed dormancy and pericarp color is controlled by a pleiotropic gene that regulates abscisic acid and flavonoid synthesis in weedy red rice.

Seed dormancy has been associated with red grain color in cereal crops for a century. The association was linked to qSD7-1/qPC7, a cluster of quantitative trait loci for seed dormancy/pericarp color in weedy red rice. This research delimited qSD7-1/qPC7 to the Os07g11020 or Rc locus encoding a basic helix-loop-helix family transcription factor by intragenic recombinants and provided unambiguous evidence that the association arises from pleiotropy. The pleiotropic gene expressed in early developing seeds promoted expression of key genes for biosynthesis of abscisic acid (ABA), resulting in an increase in accumulation of the dormancy-inducing hormone; activated a conserved network of eight genes for flavonoid biosynthesis to produce the pigments in the lower epidermal cells of the pericarp tissue; and enhanced seed weight. Thus, the pleiotropic locus most likely controls the dormancy and pigment traits by regulating ABA and flavonoid biosynthetic pathways, respectively. The dormancy effect could be eliminated by a heat treatment, but could not be completely overcome by gibberellic acid or physical removal of the seed maternal tissues. The dormancy-enhancing alleles differentiated into two groups basically associated with tropical and temperate ecotypes of weedy rice. Of the pleiotropic effects, seed dormancy could contribute most to the weed adaptation. Pleiotropy prevents the use of the dormancy gene to improve resistance of white pericarp cultivars against pre-harvest sprouting through conventional breeding approaches.

The qSD12 underlying gene promotes abscisic acid accumulation in early developing seeds to induce primary dormancy in rice.

Seeds acquire primary dormancy during their development and the phytohormone abscisic acid (ABA) is known to play a role in inducing the dormancy. qSD12 is a major seed dormancy quantitative trait locus (QTL) identified from weedy rice. This research was conducted to identify qSD12 candidate genes, isolate the candidates from weedy rice, and determine the relation of the dormancy gene to ABA. A fine mapping experiment, followed by marker-assisted progeny testing for selected recombinants, narrowed down qSD12 to a genomic region of <75 kb, where there are nine predicted genes including a cluster of six transposon/retrotransposon protein genes and three putative (a PIL5, a hypothetic protein, and a bHLH transcription factor) genes based on the annotated Nipponbare genome sequence. The PIL5 and bHLH genes are more likely to be the QTL candidate genes. A bacterial artificial chromosome (BAC) library equivalent to 8-9 times of the haploid genome size was constructed for the weedy rice. One of the two BAC contigs developed from the library covers the PIL5 to bHLH interval. A pair of lines different only in the QTL-containing region of <200 kb was developed as isogenic lines for the qSD12 dormancy and non-dormancy alleles. The dormant line accumulated much higher ABA in 10-day developing seeds than the non-dormant line. In the QTL-containing region there is no predicted gene that has been assigned to ABA biosynthetic or metabolic pathways. Thus, it is concluded that the qSD12 underlying gene promotes ABA accumulation in early developing seeds to induce primary seed dormancy.

Inheritance and molecular mapping of a downy mildew resistance gene, Pl (13) in cultivated sunflower (Helianthus annuus L.).

The inheritance of resistance to sunflower downy mildew (SDM) derived from HA-R5 conferring resistance to nine races of the pathogen has been determined and the new source has been designated as Pl ( 13 ) . The F(2) individuals and F(3) families of the cross HA-R5 (resistant) x HA 821 (susceptible) were screened against the four predominant SDM races 300, 700, 730, and 770 in separate tests which indicated dominant control by a single locus or a cluster of tightly linked genes. Bulked segregant analysis (BSA) was carried out on 116 F(2) individuals with 500 SSR primer pairs that resulted in the identification of 10 SSR markers of linkage groups 1 (9 markers) and 10 (1 marker) of the genetic map (Tang et al. in Theor Appl Genet 105:1124-1136, 2002) that distinguished the bulks. Of these, the SSR marker ORS 1008 of linkage group 10 was tightly linked (0.9 cM) to the Pl (13) gene. Genotyping the F(2) population and linkage analysis with 20 polymorphic primer pairs located on linkage group 10 failed to show linkage of the markers with downy mildew resistance and the ORS 1008 marker. Nevertheless, validation of polymorphic SSR markers of linkage group 1 along with six RFLP-based STS markers of linkage group 12 of the RFLP map of Jan et al. (Theor Appl Genet 96:15-22, 1998) corresponding to linkage group 1 of the SSR map, mapped seven SSR markers (ORS 965-1, ORS 965-2, ORS 959, ORS 371, ORS 716, and ORS 605) including ORS 1008 and one STS marker (STS10D6) to linkage group 1 covering a genetic distance of 65.0 cM. The Pl (13) gene, as a different source with its location on linkage group 1, was flanked by ORS 1008 on one side at a distance of 0.9 cM and ORS 965-1 on another side at a distance of 5.8 cM. These closely linked markers to the Pl (13) gene provide a valuable basis for marker-assisted selection in sunflower breeding programs.

Physical localization and genetic mapping of the fertility restoration gene Rfo in canola (Brassica napus L.).

The Ogu cytoplasm for male sterility and its fertility restorer gene Rfo in canola (Brassica napus L.) were originally introgressed from radish (Raphanus sativus L.) and have been widely used for canola hybrid production and breeding. The objective of this study was to determine the physical location of the Rfo locus in the canola genome using fluorescence in situ hybridization and genetic mapping. For physical localization of the Rfo gene, two bacterial artificial chromosome (BAC) clones, G62 and B420, which were closely linked to the Rfo gene, were used as probes to hybridize with the somatic metaphase chromosomes of a canola hybrid variety, PHI-46 (46H02), containing the Rfo fragment. The results showed that both clones were physically located at the end of one large metacentric chromosome. By simultaneous use of two BAC clones and 45S rDNA repeated sequences as the probes, we demonstrated that the large metacentric chromosome probed with the two BAC clones did not carry 45S rDNA repeated sequences. The chromosome was 3.65 +/- 0.74 microm in average length (20 cells) and ranked second in size among the chromosomes without 45S rDNAs. The centromere index of the chromosome (20 cells) was calculated as 43.74 +/- 4.19. A comparison with previously reported putative karyotypes of B. napus (AACC) and its diploid ancestors Brassica rapa L. (AA) and Brassica oleracea L. (CC) suggests that the chromosome carrying the Rfo fragment might belong to one of three large metacentric chromosomes of the C genome. Genetic mapping has confirmed the localization of the Rfo fragment to the distal region of linkage group N19, which corresponds to the C genome in B. napus. This study has provided the evidence of the location of the Rfo gene on canola chromosomes and established a basic framework for further physical mapping and manipulation of the gene.

Introgression and molecular tagging of Rf (4), a new male fertility restoration gene from wild sunflower Helianthus maximiliani L.

Cytoplasmic male sterility (CMS) and its fertility restoration (Rf) genes are critical tools for hybrid seed production to utilize heterosis. In sunflower, CMS PET1 and the associated Rf gene Rf (1) is the only source extensively used in commercial hybrid production. The objective of this research was to develop new sources of CMS and fertility restorers to broaden the genetic diversity of hybrid seed production. We identified a new type of CMS, named as CMS GIG2, from an interspecific cross between Helianthus giganteus accession1934 and H. annuus cv. HA 89. Based on reactions to a set of standard Rf testers, CMS GIG2 is different from all previously reported CMS types, including the CMS GIG1 from another H. giganteus accession. We also identified an Rf gene for CMS GIG2 from wild species H. maximiliani accession 1631. The CMS GIG2 and its restoration gene were introduced into HA 89 background through recurrent backcross and single plant selection techniques. Genetic analysis revealed that the CMS GIG2-Rf system is controlled by a completely dominant gene, named as Rf (4), and the gene additive and dominance effects were estimated as 39.9 and 42.2%, respectively, in the HA 89 background. The gene Rf (4) was mapped onto linkage group 3 with simple sequence repeat (SSR) markers and RFLP-derived STS-marker, and is about 0.9 cM away from the SSR marker ORS1114 based on a segregation population of 933 individuals. The CMS GIG2-Rf (4) system tagged by molecular markers provides an alternative genetic source for hybrid breeding in the sunflower crop.

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.