Separated Transcriptomes of Male Gametophyte and Tapetum in Rice: Validity of a Laser Microdissection (LM) Microarray

In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum

Ultra-High-Resolution Computed Tomography of the Lung: Image Quality of a Prototype Scanner

Purpose: The image noise and image quality of a prototype ultra-high-resolution computed tomography (U-HRCT) scanner was evaluated and compared with those of conventional high-resolution CT (C-HRCT) scanners. Materials and Methods: This study was approved by the institutional review board. A U-HRCT scanner prototype with 0.25 mm × 4 rows and operating at 120 mAs was used. The C-HRCT images were obtained using a 0.5 mm × 16 or 0.5 mm × 64 detector-row CT scanner operating at 150 mAs. Images from both scanners were reconstructed at 0.1-mm intervals; the slice thickness was 0.25 mm for the U-HRCT scanner and 0.5 mm for the C-HRCT scanners. For both scanners, the display field of view was 80 mm. The image noise of each scanner was evaluated using a phantom. U-HRCT and C-HRCT images of 53 images selected from 37 lung nodules were then observed and graded using a 5-point score by 10 board-certified thoracic radiologists. The images were presented to the observers randomly and in a blinded manner. Results: The image noise for U-HRCT (100.87 ± 0.51 Hounsfield units [HU]) was greater than that for C-HRCT (40.41 ± 0.52 HU; P <.0001). The image quality of U-HRCT was graded as superior to that of C-HRCT (P <.0001) for all of the following parameters that were examined: margins of subsolid and solid nodules, edges of solid components and pulmonary ves sels in subsolid nodules, air bronchograms, pleural indentations, margins of pulmonary vessels, edges of bronchi, and interlobar fissures. Conclusion: Despite a larger image noise, the prototype U-HRCT scanner had a significantly better image quality than the C-HRCT scanners

Whole Mitochondrial Genome Sequencing and Re-Examination of a Cytoplasmic Male Sterility-Associated Gene in Boro-Taichung-Type Cytoplasmic Male Sterile Rice

<div><p>Nuclear genome substitutions between subspecies can lead to cytoplasmic male sterility (CMS) through incompatibility between nuclear and mitochondrial genomes. Boro-Taichung (BT)-type CMS rice was obtained by substituting the nuclear genome of <i>Oryza sativa</i> subsp. <i>indica</i> cultivar Chinsurah Boro II with that of <i>Oryza sativa</i> subsp. <i>japonica</i> cultivar Taichung 65. In BT-type CMS rice, the mitochondrial gene <i>orf79</i> is associated with male sterility. A complete sequence of the Boro-type mitochondrial genome responsible for BT-type CMS has not been determined to date. Here, we used pyrosequencing to construct the Boro-type mitochondrial genome. The contiguous sequences were assembled into five circular DNA molecules, four of which could be connected into a single circle. The two resulting subgenomic circles were unable to form a reliable master circle, as recombination between them was scarcely detected. We also found an unequal abundance of DNA molecules for the two loci of <i>atp6</i>. These results indicate the presence of multi-partite DNA molecules in the Boro-type mitochondrial genome. Expression patterns were investigated for Boro-type mitochondria-specific <i>orf</i>s, which were not found in the mitochondria from the standard <i>japonica</i> cultivar Nipponbare. Restorer of fertility 1 (RF1)-dependent RNA processing has been observed in <i>orf79</i>-containing RNA but was not detected in other Boro-type mitochondria-specific <i>orf</i>s, supporting the conclusion that <i>orf79</i> is a unique CMS-associated gene in Boro-type mitochondria.</p></div

Genetic analysis of male sterility obtained from a rice cultivar Lebed backcrossed with Taichung 65

Abstract Background Male sterility is a useful agronomic trait for breeding of self-pollinating crops and is often observed in the progenies of hybrids of distantly related species, for example, Oryza sativa L. subsp. indica and O. sativa L. subsp. japonica. To explore new male sterile lines in rice, we performed successive backcrosses using a japonica cultivar Taichung 65 (T65) as a recurrent pollen parent and various indica cultivars as the initial female parents. Findings We observed male sterile plants in the backcross progeny from an indica cultivar, Lebed. Both fertile and sterile plants were present in the BC4F1 generation. The sterile plants segregated for fertile and sterile plants when backcrossed with T65 in BC5F1, BC6F1 and BC7F1 with a ratio of 1:1. Conversely, all the backcross progenies from the fertile BC4F1 were consistently fertile. Anthers of the male sterile line were stunted and did not shed pollen; cross-sectional observations revealed defects in sporophytic cells. The male sterility appears to be caused by heterozygous alleles derived from T65 and Lebed. A male sterility gene was mapped between two INDEL markers on the long arm of chromosome 10, which corresponded to a 407 kb region in the Nipponbare genome. Conclusions Since the heterozygous Lebed allele acts as dominant sporophytic pollen killer, it would be useful for recurrent selection breeding of japonica rice

Boro-type mitochondria sequences are assembled into five circular chromosomes.

<p>(a) Compositions of contigs in five assembled mitochondrial chromosomes (MC). The arrow box indicates a contig and its direction. Arrows represent primers used for confirmation of contig connections. The same contigs are filled with the same colors. (b) Each of the five circular chromosomes was validated by PCR analysis using the primer sets indicated. (c) The inverse order of the contigs in MC-3 was detected by PCR analysis. (d) Integration of MC-5 into MC-2 was validated by PCR analysis.</p

Recombination between subgenome-1 and subgenome-2 via the 450-bp identical sequence is possible but rare.

<p>(a) Schematic model of recombination between subgenome-1 and subgenome-2. Arrows indicate primers used for detection of recombination. <i>Eco</i>RI sites are indicated as E. (b) Recombinant fragments were detected by PCR. (c) Southern blot analysis detected the recombination molecule between subgenome-1 and subgenome-2; however, the frequency of recombination was lower than that of the non-recombinant subgenome-1 molecule.</p

Expression patterns of <i>orf</i>s specific to Boro-type mitochondria were not altered in the presence or absence of the restorer of fertility gene, <i>Rf1</i>.

<p>Northern blot analysis was performed using a BT-type CMS rice (BTA), a fertility restorer with <i>Rf1</i> (BTR), a transgenic BTA complemented with <i>Rf1</i> (Compl), and a maintainer line (T65).</p