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Combining QTL mapping and RNA-Seq reveals candidate genes controlling flag leaf width in foxtail millet.

BACKGROUND: The flag leaf, a crucial component of plant architecture, significantly influences final grain yield in crops, including foxtail millet (Setaria italica L.). Optimizing flag leaf size is considered an effective strategy for enhancing grain yield potential under higher planting densities. However, the genetic mechanism underlying flag leaf size, particularly flag leaf width (FLW), remains largely unknown under varying planting densities in foxtail millet. RESULTS: An FLW phenotype variation analysis was conducted across multiple planting densities using a recombinant inbred line (RIL) population derived from Heizhigu (narrow leaf) and Changnong 35 (wide leaf). Based on a high-density genetic map with 3795 Bin markers, 11 flag leaf width (FLW) QTLs were identified on chromosomes 3, 5, and 6, explaining 2.35%-36.06%. Among these, qFLW5-2 was a major QTL, detected consistently across 3 environments and explaining a large proportion of FLW variation. The QTL was further validated with 9 InDel markers with its candidate region across different planting densities. Moreover, RNA-seq revealed 2,293 and 2,338 differentially expressed genes (DEGs) between biparents at heading stage and grain filling stage, respectively. There were 11 and 9 DEGs within the location range of qFLW5-2 among 2 comparison groups (HZG-H_vs_CN35-H and HZG-G_vs_CN35-G). Combining QTL mapping and RNA-seq, we speculated that Seita.5g134600 (encoding an auxin responsive protein Aux/IAA) and Seita.5G123900 (encoding a cytochrome P450 family protein) as key candidate genes for qFLW5-2. Furthermore, variation analysis confirmed that the lines or germplasm with Seita.5G1346005UTR277+ allele, both within the RIL population and natural populations, exhibited significantly wider leaves than those with Seita.5G1346005UTR277- allele. These findings advance our understanding of the genetic and molecular regulatory mechanisms governing flag leaf growth. CONCLUSIONS: This study elucidates genetic and molecular mechanism regulating flag leaf growth and development in foxtail millet. The results provide a theoretical foundation for improving plant architecture and facilitating molecular marker-assisted breeding in this crop.

Quantitative Trait Loci

Rates of electron transfer and of non-cyclic photophosphorylation for chloroplasts isolated from maize populations selected for differences in juvenile productivity and in leaf widths.

In vitro studies with chloroplasts isolated from maize populations differing in juvenile productivity by the sixth leaf stage of growth demonstrated differences in rates of ferricyanide reduction and of non-cyclic photophosphorylation. Similar results were obtained for two independent sets of replicate selections for high and for low productivity. Chloroplasts from low productive selections had about 115 percent of the reduction rate and 129 percent of the phosphorylation rate found for the high productive selections. Differences in ferricyanide reduction held for a fourfold range of experimentally induced rates. The contrasting rates of electron flow did not result from differential extraction environments following cell disruption nor from the failure to protect chloroplasts during extraction. Light-dependent phosphate uptake gave evidence, though inconclusive, of being so modified. No evidence was found for reciprocal effects between low-high and high-low hybrids. No difference in activities was found between chloroplasts isolated from two populations selected for narrow and for wide leaf widths. These two selections differed markedly in leaf areas. While in vitro measures of chloroplast efficiency reflected differences which were under genetic control, the results did not follow those expected from photosynthetic studies. The differences in rates appeared to reflect modification of processes other than those in the photosynthetic pathway.

Biological Transport

Exploring genomic regions and genes modulating plant height and flag leaf morphology in rice.

Plant height and flag leaf morphology critically affect plant yield because they determine above-ground plant biomass and photosynthate production. However, few genetic basis analyses and gene mining studies on plant height, flag leaf length, and flag leaf width have been performed, and there is little available information about the evolution and utilization of the underlying natural alleles. This study conducted a genome-wide association study (GWAS) using 689 rice accessions collected from diverse regions across the globe. The GWAS identified 73, 159, and 158 significant loci associated with plant height, flag leaf length, and flag leaf width, respectively. SD1HAP1 and NAL1A were also identified as superior alleles that could be used to improve plant architecture by reducing plant height and increasing flag leaf width, respectively. LEAF1 and its elite allele LEAF1G, which simultaneously modulated plant height and flag leaf morphology, were isolated, and the LEAF1 knockout lines showed reduced flag leaf length and plant height, whereas LEAF1G-complementary lines in the LEAF1A background had the opposite phenotypes. The results also showed that LEAF1G and SD1HAP1 evolved directly from wild rice and were mainly found in the Xian subgroup, whereas NAL1A might have originated from de novo mutation during domestication and was mainly found in the Geng subgroup. A joint haplotype analysis revealed that pyramiding SD1HAP1, NAL1A, and LEAF1G in Type I accessions optimized plant architecture, reduced plant height, and enlarged the flag leaves. In addition, genomic regions and genes that had been convergently selected for these traits were identified by combining a population genetics analysis with a GWAS. These findings provide valuable genetic targets for molecular breeding that will improve plant height and flag leaf morphology in rice.

Oryza

Natural variation in the cytokinin oxidase gene ZmCKX6 influences leaf morphology and yield-related traits in maize.

Leaf width (LW) is a critical determinant of maize architecture and yield. To uncover its genetic basis, we performed a genome-wide association study (GWAS) on 348 maize inbred lines and identified ZmCKX6, encoding cytokinin oxidase/dehydrogenase, as a key gene associated with LW. Natural variation in the ZmCKX6 promoter significantly influenced its expression levels, leading to differences in LW across various haplotypes. Functional validation using CRISPR/Cas9 revealed that ZmCKX6 knockout results in pleiotropic effects, including narrower leaves, reduced plant height, and decreased grain yield components. These phenotypes were accompanied by elevated levels of active cytokinins but reduced levels of auxin, gibberellins, and salicylic acid. Transcriptome analysis revealed a significant downregulation of photosynthesis-related genes, corresponding to reduced photosynthetic rates in knockout lines. Evolutionary analysis demonstrated that the allele associated with narrower leaves were preferentially selected during maize domestication and breeding. This study highlights the role of ZmCKX6 in modulating cytokinin homeostasis and its subsequent impact on multiple agronomic traits in maize, providing insights into the complex genetic control of plant architecture and yield. The identified natural variations could be valuable for marker-assisted selection aimed at optimizing plant architecture and improving yield.

Zea mays

Cytokinin content and tissue distribution in plants transformed by a reconstructed isopentenyl transferase gene.

The cytokinin gene, isopentenyl transferase (ipt), was placed under the control of a heat-inducible promoter from the Drosophila melanogaster hsp70 gene and introduced into Nicotiana plumbaginifolia by cocultivation with Agrobacterium tumefaciens. Transformants were analyzed for organ-specific expression, cytokinin levels and effects on plant development before and after the heat induction. The ipt gene transcripts were detected in leaves and stems but not roots of transgenic plants following a 2 hour, 45 degrees C treatment. Maximum mRNA levels observed occurred 2 hours after heat treatment and 46 hours later were detected only in leaves. Zeatin and zeatinriboside concentrations 2 hours after heat shock ranged from over 900 to 2000 pmol/g, representing a greater than 140- to 200-fold increase over uninduced levels. After 46 hours, approximately 50% of the cytokinins are still present in the leaves as opposed to much reduced levels in the stems. Transgenic plants were greener, shorter, had an underdeveloped root system, reduced leaf width, and increased growth of axillary buds. After a single heat treatment, plants exhibited a darker green pigment and continued growth of lateral buds. Transient accumulations of endogenous cytokinins following thermal induction did not appear to alter the plant's preprogrammed pattern of differentiation.

Adenosine

[Inheritance of certain quantitative and qualitative traits in a system of diallele barley crosses].

Unequal inheritance of characters, i.e. flower glume colour, degree of lateral spikelets development, hairy rechis of the ear, were observed in diallel crosses among the varieties Nutans 244, Trumpf, DZ-02389, Hiproly and Brachitic. Recessive inheritance was marked for short rachilla hairs. The dominant effect of genes for the leaf length and overdominance for the plant height were ascertained. The partial dominance for the number of internodes and leaf width was found. The character of the upper internode length did not correspond to additive-dominant model.

Alleles

Stomatal patterning in Tradescantia: an evaluation of the cell lineage theory.

The cell lineage theory, which explains stomatal patterning in monocot leaves as a consequence of orderly divisions, was studied in Tradescantia. Data were collected to test the theory at three levels of organization: the individual stoma; stomata distributed in one dimension, in linear fashion along cell files; and stomata apportioned in two dimensions, across the length and breadth of the leaf. In an attempt to watch the patterning process through regeneration, stomata in all visible stages of development were laser ablated. The results showed that the formation of stomatal initials was highly regular, and measurements of stomatal frequency and spacing showed that pattern was determined near the basal meristem when the stomatal initials arose. Following the origin of initials, the pattern was not readjusted by division of epidermal cells. Stomatal initials were not committed when first present and a small percentage of them arrested. The arrested cells, unlike stomata, were consistently positioned in cell files midway between a developed pair of stomata. At the one-dimensional level of pattern, stomata in longitudinal files were separated by a variable number of epidermal cells and the frequency of these separations was not random. The sequential spacing of stomata also was not random, and stomata separated by single epidermal cells were grouped into more short and long series than expected by chance. The stomatal pattern across the width of the leaf resulted from cell files free of stomata which alternated with cell files containing stomata, but not with a recurring periodicity. Files lacking stomata were found only over longitudinal vascular bundles. Laser ablations of developing stomata did not disrupt the pattern in nearby cells or result in stomatal regeneration. We conclude that the cell lineage theory explains pattern as an individual stomatal initial arises from its immediate precursor and satisfactorily accounts for the minimum spacing of stomata in a cell file, i.e., stoma-epidermal cell-stoma. However, the theory does not explain the collective stomatal pattern along the cell files, at the one-dimensional level of patterning. Nor does the theory account for the for the two-dimensional distribution of stomata in which regions devoid of stomata alternate with regions enriched with stomata, but not in a highly regular nor haphazard manner. We suggest that the grouping of epidermal cells and stomata separated by single epidermal cells in cell files may result from cell lineages at a specific position in the cell cycle as they traverse the zone where stomatal initials form.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Division

Evaluation of multileaf collimator design for a photon beam.

Various aspects of multileaf collimator (MLC) design are examined relative to clinical requirements. The characteristics studied included: (a) irregular field edge definition or "effective" penumbra, (b) optimum field coverage for the multileaf portion of the field, and (c) leaf velocity. A film dosimetry technique was developed to measure the rapid 2-dimensional change in dose at an edge defined by a multileaf collimator with the segments staggered. The method applies a correction factor which allows for the changing ratio of scattered to primary photons at the field edge so that the energy dependence of the film is corrected. Stepped lead alloy blocks were irradiated with 6 MV photons to obtain films simulating a double-focused multileaf collimator, and the results were compared to films of fields shaped with standard divergent blocks. The effect of the shape of the leaf face (the end of the leaf) on penumbra was also studied. Proper shaping of the leaf ends may eliminate the need to exactly match beam divergence so that the mechanical of the collimator system is simplified. Leaves having several different end shapes and moving horizontally to intercept a vertical beam were compared to the divergent design where a straight face moves along an arc. The measurements showed that the "effective" penumbra (measured as the distance from the 80 to 20% isodose lines) for the multileaf collimator is a function of the angle between the direction of leaf motion and the edge defined by the leaves. In addition, all leaf end shapes showed some increase in penumbra compared to standard divergent blocking and also had increasing penumbra width as they moved over or back from the field center line. A total of 459 treatment fields and six disease sites were examined to determine the percentage of fields potentially shaped by multileaf segments of specified length. This study showed 93% of the fields had lengths of 30 cm or less and 99% had widths of 25 cm or less. A study conducted to determine the required leaf velocity to shape various target volume configurations during complete rotation (at 1 RPM) showed that a leaf speed of at least 1.5 cm/sec at isocenter is needed for dynamic conformal treatment.

Equipment Design

[The first record of human natural infection of Echinochasmus liliputanus].

Human natural infection with Echinochasmus liliputanus was found for the first time from the inhabitants of Hexian county of Anhui Province in Spring, 1991. Sixty worms collected from 3 infected persons were studied morphologically. The adult worms are leaf-shaped, 1519.4-2056.3 x 466.4-564.0 microns in size. The width of the collar is 235.8-297.3 microns. A row of 24 collar spines with size of 22.5-35.6 x 8.8-10.6 microns is arranged on the collar symmetrically with dorsal and ventral interruption. The oral sucker is terminal, 107.6-148.6 x 102.5-148.6 microns. The length of the prepharynx is 25.6-66.7 microns, the pharynx, 97.3-127.8 microns and the oesophagus 117.9-205.0 microns. The caeca 764.3-1,248.8 x 21.0-39.0 microns extends to the distal end of the body. The acetabulum is anterior to the middle level of the body, 205.0-240.9 x 205.0-235.8 microns in size. Two testes, situated in the posterior one third of the body, is slightly oval, in tandem; the anterior one is 133.3-199.9 x 199.9-256.4 microns in size, and the posterior one, 178.9-251.3 x 164.0-246.0 microns. The Cirrus pouch is kidney-shaped, 211.5-248.5 x 112.8-194.8 microns in size, located between the bifurcation of the intestine and the acetabulum, containing the seminal vesicle and cirrus. The ovary is oval in shape, 71.8-92.3 x 76.8-97.4 microns in size, situated in the middle of the body. The vitellaria are distributed on each side from the acetabulum to subterminal, consisting of many follicles. The uterus is short, convoluted between the anterior testis and the acetabulum, containing 0-6 eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent

Background correction by wavelength modulation for pulsed-laser-excited atomic fluorescence spectrometry.

Instrumentation was constructed to modulate the dye laser wavelength for background correction in laser-excited atomic fluorescence spectrometry (LEAFS). To achieve wavelength modulation a piezoelectric pusher was used to drive the wavelength tuning mirror in a laboratory-constructed grazing incidence dye laser. The laser pulses were synchronized with the piezoelectric pusher movement so that alternate laser pulses measured the atomic fluorescence signal at the analytical atomic spectral line (on-line) and the background signal at a wavelength displaced to one side of the atomic line (off-line). The background-corrected signal was obtained by subtracting the off-line "background" from the on-line "signal plus background". The spectral line width (fwhm) of the dye laser was 0.003 nm, while the wavelength modulation interval was controllable over the range from 0 to 0.2 nm with a spectral resolution limited only by the spectral line width of the laser. This type of background correction could, in principle, be applied to other types of tunable lasers such as pulsed Ti: sapphire lasers. The performance of background correction by wavelength modulation (WM) was demonstrated by measurement of sodium resonance fluorescence in an air-acetylene flame and by thallium nonresonance fluorescence in a graphite furnace. The experimental data indicated that the wavelength modulation corrected, effectively and quantitatively, for flame background, blackbody emission from a graphite furnace, and scatter of laser radiation off aluminum chloride (1 mg/mL as AI) matrix particles in both the furnace and the flame. Analytical results were in good agreement with certified values for the determination of sodium in standard reference materials by the use of modulated LEAFS.

Lasers

Electron microscopy of the novel barley yellow streak mosaic virus.

Unique particles of barley yellow streak mosaic virus (BYSMV) were detected in diseased barley, wheat, and several species of grass. They appeared to be about 64 nm in width and from 127 nm to an astonishing 4000 nm in length. Individual particles were circular in transverse section. The outermost layer of each particle seemed to be a membrane-like envelope. The internal structure of many particles was bead-like. Some particles had centers that were translucent. The BYSMV particles were distributed throughout the leaf, sheath, root, and awn organs of barley. Virus particles were present in all cell types of the epidermis, mesophyll, phloem, and xylem. However, mesophyll cells contained the greatest number of particles. Most BYSMV particles occurred in large clusters of quasi-parallel arrays. Both individual and groups of particles were located within the cavities of ER elements. Ribosomes were attached to some outer surfaces of the ER bounding membrane. BYSMV particles are unique because they do not resemble any in presently classified groups or families of plant viruses: they are, however, similar to those of some unclassified viruses that infect insects.

Hordeum

Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts.

We have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one-dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell-free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters. We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse-width time-of-flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15-55 micron (r2 greater than 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia. These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.

Cell Separation

Microstructures of the osseous spiral laminae in the bat cochlea: a scanning electron microscopic study.

The architecture and surface structures of the primary and secondary osseous spiral laminae in the cochlea of the bat, an animal able to hear high frequency sounds, were examined by scanning electron microscopy to understand the micromechanical adaptations of the bony supportive elements in the inner ear to the specific hearing function. The bat used was Myotis frater kaguyae. The myotis bat cochlea was seen to consist of a hook and a spiral portion with one and three-quarter turns and was characterized by: 1) a distinct ridge-like projection running spirally along the middle line on the vestibular leaf of the primary osseous spiral lamina; 2) a wide secondary osseous spiral lamina; and 3) a narrow spiral fissure between the primary and secondary osseous spiral laminae. The ridge on the primary osseous spiral lamina was 150 microns high in the hook and basal turn, then lowered toward apex, and flattened before the apical end. The surface structures appeared to provide a firm anchorage of the auditory teeth. The secondary osseous spiral lamina, which anchors the fibers of the basilar membrane, was sharply projected and measured 150 microns in width in the hook, and then narrowed gradually toward apex to disappear in the helicotrema. The spiral fissure for the basilar membrane was about 40 microns in width in the hook and about 120 microns in the apical turn. The findings suggest the presence of a narrow and rigid basilar membrane with a high amount of fibers and a strong bony support to the auditory teeth for scaffolding the tectorial membrane during intense vibration in response to high frequency sounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Dose distributions of x-ray fields as shaped with multileaf collimators.

Multileaf collimators (MLC) with various blade widths were simulated using standard cerrobend blocks, and three-dimensional dose computations were carried out to study the resultant radiation field edges. Film measurements made with 6 and 18 MV x-ray beams were compared with calculations that employed a three-dimensional Fourier convolution. A spatial accuracy of better than 3 mm was found in the 50% isodose line of the penumbral region with a calculation voxel size of 5 mm x 5 mm x 5 mm. The computer simulation was used to study the deviation of the calculated 50% isodose line from the desired geometric field edge using various MLC blade positions. The study suggests that multileaf collimation to the outside of the desired field edge will lead to overdose outside the field, whereas multileaf collimation to the inside of the desired field edge will lead to underdose inside the field. When the direction of travel of the leaves with respect to the field edge is near 45 degrees, the 50% isodose of a multileaf-collimated beam will fall close to the desired edge with no underdose when the leaf corners are allowed to insert into the desired field edge by 1.2 mm for 6 MV x-rays and 1.4 mm for 18 MV x-rays using a 1 cm wide leaf. These blade offsets account for the scattering of photons and electrons in the medium within the penumbral region.

Computer Simulation