Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility (TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F2 populations derived from crosses between IR68301S, an indica TGMS rice line, and IR14632 (tropical japonica), Supanburi 91062 (indica) and IR67966-188-2-2-1 (tropical japonica), respectively. Approximately 1:3 ratio between sterile and normal pollen of F2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat (SSR) and insertion-deletion (InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively. InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S. Keywords: hybrid rice, thermo-sensitive genic male sterility, insertion/deletion, simple sequence repeat, marker-assisted selectio

Transcriptome Analysis Reveals Genes Involved in Responses of Eucalyptus to Gall Wasp Infestation

Leptocybe invasa is a gall wasp causing significant damage to Eucalyptus species. This study used RNA sequencing (RNA-seq) to identify differentially expressed genes (DEGs) associated with early L. invasa infestation in eucalyptus parents and their F1-progenies. A total of 14,648 significant DEGs were identified from U22-tolerant and C153-susceptible parents, and extremely tolerant and susceptible pools of their F1-progenies. A total of 324/632 and 182/205 DEGs specific for the tolerant group and the susceptible group were up-regulated, respectively. Expression analysis by qRT-PCR of the selected DEGs was comparable with the results of RNA-seq. Expression analysis of the selected genes using the top five progenies from each pool was consistent with that in the parents. Three genes (RCA, SUI1, GCN5) were up-regulated after infestation in all tested tolerant plants, suggesting their important roles in the tolerant phenotype. Using expression and STRING analysis, our results suggest that early response at three days after gall wasp infestation increased protein and terpenoid synthesis, and increased transportation of these molecules. In addition, wounding also increased photosynthesis and glycolysis. These processes involved the interaction of several plant hormones, such as JA, Auxin, and ABA. The information obtained from this study could be useful for future Eucalyptus breeding programs

Identification of Rice Accessions Having Cold Tolerance at the Seedling Stage and Development of Novel Genotypic Assays for Predicting Cold Tolerance

Rice is susceptible to cold stress at the seedling stage, which can delay growth and decrease yield. We evaluated 187 rice accessions for cold tolerance at the seedling stage and developed genotypic assays for three markers. All japonica (20/20) and 20/140 indica accessions were highly cold tolerant. Two SNP markers specific for COLD1 and LOC_Os10g34840 were practical to use by normal agarose gel. The SNP marker specific for COLD1 was highly specific for predicting cold tolerance. However, the sensitivity of this marker was low as several cold-tolerant indica accessions lacked the cold-tolerant allele. The LOC_Os10g34840 marker was slightly more sensitive than the COLD1 marker for predicting highly cold-tolerant accessions. An insertion/deletion variant in the NAC6 gene was identified as a novel cold tolerance marker. The NAC6 marker predicted more highly cold-tolerant accessions compared with the other two markers. The SNP marker specific for LOC_Os10g34840 and the NAC6 marker were present in several tested subgroups, suggesting their wide effects and distribution. The three markers combined predicted the most highly cold-tolerant accessions, indicating that the marker combination is superior for applications such as marker-assisted breeding. The cold-tolerant accessions and the genotypic marker assays will be useful for future rice breeding

Total ceramide analysis.

<p>6A) Total ceramide analysis in leafs of RNAi rice lines and WT rice plants. 6B) Total ceramide analysis in panicles of tms2 mutant and WT rice plants. Ceramide levels were determined from the leaves of <i>LOC_Os07g26940</i> RNAi (3ES-41 and 7ES-3) or <i>ORMDL</i> RNAi (29KS38 and 29KS-40) transgenic rice plants, and compared with equivalent tissue from WT. Results were expressed as absolute levels of ceramides (nmol/g dw). Similar analysis was performed on panicles of either WT or tms2 mutant rice, and data is presented as absolute levels of ceramides (nmol/g dw). Absolute levels (A, B) represent the mean of 6 technical replicates for each independent line. Bar indicates the standard error (n = 6). * indicates statistical significance at p-value of 0.05 as determined by t-test. 3ES-41 and 7ES-3 or 29KS38 and 29KS-40 are two independent transgenic lines from the same construct.</p

Expression analysis and pollen staining of RNAi transgenic rice plants.

<p>A) qRT-PCR analysis of <i>ORMDL LOC_Os07g26940</i> and other <i>ORMDL</i> gene expression in leaves of <i>LOC_Os07g26940</i> RNAi (3ES-41 and 7ES-3). B) qRT-PCR analysis of <i>ORMDL LOC_Os07g26940</i> and other <i>ORMDL</i> gene expression in leaves of <i>ORMDL</i> RNAi (29KS38 and 29KS-40) transgenic rice plants. 3ES-41 and 7ES-3 or 29KS38 and 29KS-40 are two independent transgenic lines from the same construct. The expression level of <i>EF-1α</i> was used as internal control. All data are representative from at least two biological repeats, each based on three technical replicates; similar results were obtained in the repeated experiments. Bars indicate the standard error (n = 3). which was calculated form technical replicates. Each biological sample was a mixture of 3 plants. C) Pollen staining of wild type, <i>LOC_Os07g26940</i> RNAi, and <i>ORMDL</i> RNAi transgenic rice plants. Similar results were obtained from the two tested transgenic lines form each construct. The pictures are a representative from each construct.</p

Sphingolipid long-chain base composition ng LCB mg⁻¹ dry weight in wild type and <i>tms2</i> mutant rice plants in leaf tissue(A) and in panicles(B).

<p>Sphingolipid long-chain base composition ng LCB mg⁻¹ dry weight in wild type and <i>tms2</i> mutant rice plants in leaf tissue(A) and in panicles(B).</p

Rice <i>ORMDL</i> Controls Sphingolipid Homeostasis Affecting Fertility Resulting from Abnormal Pollen Development

<div><p>The orosomucoids (ORM) are ER-resisdent polypeptides encoded by <i>ORM</i> and <i>ORMDL</i> (ORM-like) genes. In humans, ORMDL3 was reported as genetic risk factor associated to asthma. In yeast, ORM proteins act as negative regulators of sphingolipid synthesis. Sphingolipids are important molecules regulating several processes including stress responses and apoptosis. However, the function of <i>ORM</i>/<i>ORMDL</i> genes in plants has not yet been reported. Previously, we found that temperature sensitive genetic male sterility (TGMS) rice lines controlled by <i>tms2</i> contain a deletion of about 70 kb in chromosome 7. We identified four genes expressed in panicles, including an <i>ORMDL</i> ortholog, as candidates for <i>tms2</i>. In this report, we quantified expression of the only two candidate genes normally expressed in anthers of wild type plants grown in controlled growth rooms for fertile and sterile conditions. We found that only the <i>ORMDL</i> gene (<i>LOC_Os07g26940</i>) showed differential expression under these conditions. To better understand the function of rice <i>ORMDL</i> genes, we generated RNAi transgenic rice plants suppressing either <i>LOC_Os07g26940</i>, or all three <i>ORMDL</i> genes present in rice. We found that the RNAi transgenic plants with low expression of either <i>LOC_Os07g26940</i> alone or all three <i>ORMDL</i> genes were sterile, having abnormal pollen morphology and staining. In addition, we found that both sphingolipid metabolism and expression of genes involved in sphingolipid synthesis were perturbed in the <i>tms2</i> mutant, analogous to the role of ORMs in yeast. Our results indicated that plant ORMDL proteins influence sphingolipid homeostasis, and deletion of this gene affected fertility resulting from abnormal pollen development.</p></div

Ceramide composition in leaves of <i>Japonica</i> WT and RNAi rice lines.

<p>Unit: nmol g dw⁻¹, N: a minimum of 3± SE.</p><p>Ceramide composition in leaves of <i>Japonica</i> WT and RNAi rice lines.</p

Expression analysis by quantitative RT-PCR.

<p>(A) qRT-PCR analysis of <i>LOC_Os07g26930</i> and all three splicing forms of <i>ORMDL LOC_Os07g26940</i> in panicles and anthers of wild type rice plants grown in control growth room at 26°C and 32°C. The expression level of <i>Actin1</i> (<i>Os05g36290</i>) was used as internal control. (B) qRT-PCR analysis of all three splicing forms of <i>LOC_Os07g26940</i> in wild type rice tissues, including stems, leaves, roots, small panicles (1–2 cm), medium panicles (3–14 cm), large panicles (15–25 cm) and anthers. The expression level of <i>EF-1α</i> (<i>Os03g08020</i>) was used as internal control. All data are representative from at least two biological repeats, each based on three technical replicates; similar results were obtained in the repeated experiments. Bars indicate the standard error (n = 3), which was calculated from technical replicates. Each biological sample was a mixture of 3 plants.</p

Ceramide composition in panicles of <i>Indica</i> WT and <i>tms</i>2 rice lines.

<p>Unit: nmol g dw⁻¹, N: a minimum of 3± SE.</p><p>Ceramide composition in panicles of <i>Indica</i> WT and <i>tms</i>2 rice lines.</p