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Article

Identification and Gene Cloning of a Brittle Culm Mutant (bc22) in Rice

by
Xiying Cao
1,2,†,
Tao Zhou
1,†,
Yue Sun
2,
Yuhan Zhang
3,
Huan Xu
2,
Wei Liu
1,
Yu Zou
1,
Qingquan Chen
2,
Hui Ma
1,
Dongfang Gu
1,4,* and
Jinlong Ni
1,4,*
1
Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230001, China
2
College of Agronomy, Anhui Agricultural University, Hefei 230036, China
3
School of Life Sciences, Zhejiang University, Hangzhou 310058, China
4
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2024, 14(2), 235; https://doi.org/10.3390/agriculture14020235
Submission received: 12 December 2023 / Revised: 26 January 2024 / Accepted: 29 January 2024 / Published: 31 January 2024
(This article belongs to the Section Genotype Evaluation and Breeding)
Browse Figures
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Abstract

:
The mechanical strength of rice culm, an essential factor for lodging resistance and yield maintenance, is influenced by the composition and structure of the cell wall. In this study, we characterized a rice brittle culm mutant 22 (bc22), derived from LR005 through ethyl methanesulfonate (EMS) mutagenesis. The bc22 culm exhibited increased fragility and reduced mechanical strength compared to LR005. The mutant displayed pleiotropic effects, including a shorter plant height and panicle length, a smaller grain size, and the absence of the glume hairs. Scanning electron microscopy revealed a decrease in cell density and a looser structure in the bc22 culms. Biochemical analysis demonstrated a significant increase in hemicellulose content and a marked reduction in lignin content in the culm of bc22. Genetic analysis indicated that the brittle culm trait was governed by a single recessive gene. After employing bulked segregant analysis (BSA), whole-genome resequencing, and MutMap methods, LOC_Os02g25230 was identified as the candidate gene responsible for bc22. In bc22, a point mutation from proline (Pro) to leucine (Leu) in its coding region led to the pleiotropic phenotype. A complementation test further confirmed that the missense mutation causing the proline to leucine amino acid substitution in LOC_Os02g25230 was causative of the observed bc22 phenotype. Additionally, gene expression analysis showed that BC22 had higher expression levels in the culms, leaves, and spikelets compared to the roots. Taken together, our findings indicate that BC22 is a pleiotropic gene, and the influence of BC22 on brittleness may be associated with cell wall biosynthesis in rice culm.

1. Introduction

Brittle mutants have been extensively studied in various plant species, including rice [1,2], Arabidopsis thaliana [3,4], and barley [5]. The culms of these mutants typically exhibit decreased mechanical strength and increased brittleness. In the case of rice, more than 20 brittle culm mutants have been characterized and their lodging resistance was found to be weakened due to a reduction in mechanical strength in the stem or leaves [6]. Conversely, ruminants also have greater chewing efficiency and digestibility on brittle culm rice due to their lower cellulose and higher lignin contents [7,8].
The brittleness of rice culms is closely related to various factors, among which the main factor is the physicochemical properties of their cell walls [9,10,11]. The plant cell wall is mainly composed of cellulose, hemicellulose, and lignin, which constitute the reinforcing rods of the wall and contribute to the strength and firmness of the cell wall in plants [12,13,14]. Alteration in the cell wall composition and structure affects the mechanical strength of rice straws and leaves [15]. Several genes associated with brittle mutations have been characterized in rice, and these genes are involved in various metabolic processes, including cellulose synthesis, secondary cell wall construction, lignin synthesis, and plant hormone synthesis pathways. For instance, OsCesA4, OsCesA7, and OsCesA9 encode cellulose synthases, and their mutants often exhibit reduced cellulose content and increased plant brittleness [1,16,17,18]. Mutants like bc7 and bc11 result from the alleles of OsCesA4, the brittleness of which is caused by a decrease in cellulose content due to OsCesA4’s loss of function [18,19]. Another brittle culm mutant, S1-24, has lower cellulose and higher hemicellulose contents on account of the mutation of OsCesA7 [17]. The brittle trait observed in bc6, osfc16, and oscesA9 mutants is also associated with gene mutations that affect cellulose accumulation in the secondary cell wall [16,20,21]. Other genes, such as BC1, BC3, BC10, BC12, and BC15, are associated with a reduction in cellulose content, involving the biosynthesis of secondary cell walls and influencing rice brittleness [22,23,24,25,26]. Hemicellulose, composed of complex polysaccharides, interacts with cellulose to enhance cell wall stiffness and toughness [27]. For example, cslf6 mutants affect the deposition of mixed-linkage glucan in rice tissues, the mechanical strength of the cell wall, and disease resistance [28]. Different from cellulose, lignin polymers, by being incorporated into the cell wall, increase the cell wall’s stiffness and defense responses against pathogens [29]. One crucial enzyme in lignin synthesis, cinnamyl alcohol dehydrogenase (CAD), accelerates the creation of lignin monomers and plays a role in controlling the mechanical strength of rice. Fc1 mutants display decreased cell wall thickness, lower lignin content, and increased brittleness [30]. The lignin content and cell layer number of the bc17 mutant were found to be lower than those of the wild type, showing mechanical characteristics with higher brittleness [15]. This study further shows that BC17 is involved in the synthesis of lignin precursors through the polymerization of caffeyl alcohol by regulating laccase expression [15]. TAC4 and SG2, alleles of BC17, have also been reported to regulate tiller angle and grain size in rice [31,32].
Plant cell walls are complex dynamic network structures composed of cellulose, hemicellulose, pectin, and lignin. The current understanding of the mechanisms by which plant cell wall components and substances accumulate is limited. There are numerous genes involved in cell wall synthesis that have not been thoroughly investigated. In this study, we identified a brittle culm mutant from a rice EMS mutant library, named bc22, which is characterized by its brittleness, dwarf stature, small grains, dispersed architecture, and hairlessness. This mutant was used to identify and clone a key gene that regulates brittle rice culms.

2. Materials and Methods

2.1. Plant Materials

LR005, the wild type (WT) indica rice bred by the Rice Research Institute of Anhui Academy of Agricultural Sciences, was used in this study. The brittle culm 22 (bc22) mutant was screened from a mutagenesis pool constructed by treating LR005 with ethyl methanesulfonate (EMS). All plants used in this study were cultivated in an experimental field at Anhui Academy of Agricultural Sciences, Hefei, China, using standard fertilizer and water management strategies.

2.2. Investigation of Agronomic Traits

During the maturity stage, 10 plants each of LR005 and the bc22 mutant were randomly selected from the field to assess their agronomic traits, including plant height, 1000-grain weight, grain length, and grain width. The data were subjected to recording and statistical analysis using SPSS 18.0, and graphs were drawn with GraphPad Prism 8.0.1.

2.3. Determination of Mechanical Properties

In the heading stage, 30 fresh rice plants each from LR005 and bc22 were randomly selected to test the mechanical properties. The second internodes of the culm base were placed on an equidistant scaffold with a fulcrum distance of 6 cm. A plant culm mechanical strength tester (http://www.tpyn.net/productshow_96.html; accessed on 5 October 2022; model YYD-1) with an accuracy of 0.001 N was used to apply vertical pressure slowly until the culm broke. The indicator reading on the dynamometer at the point of breakage represented the culm breaking resistance. The data were recorded and analyzed statistically using SPSS 18.0.

2.4. Scanning Electron Microscopy (SEM)

To understand the specific histological changes responsible for the decreased mechanical strength in the mutant at the tissue level, we prepared cross-sectional sections of culms from LR005 and the bc22 mutant and observed them using scanning electron microscopy. Approximately 2–4 mm rice stem tissues from the same parts of LR005 and bc22 mutant culms in the rice heading stage were fixed in PBS buffer (pH = 7.4) containing 2.5% glutaraldehyde and then stored at 4 °C. After rinsing with 0.1 M PBS, the samples were fixed with 1% osmic acid for 1 h. Subsequently, the samples were dehydrated with a gradient of ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 15 min each. The dehydrated samples were then treated with isoamyl acetate for 15 min, fully dried using a critical-point dryer (Quorum, K850, Laughton, UK), conductively treated with ion sputter (Hitachi, MC1000, Tokyo, Japan), and, finally, scanned with the use of a scanning electron microscope (Hitachi, SU-8100, Tokyo, Japan), which was also used to take photos.

2.5. Determination of the Content of Cell Wall Components

The main substances accumulated during vegetative and reproductive rice growth are cellulose, hemicellulose, and lignin. To detect the effect of the changes in the dominating substances of the bc22 mutant on its brittleness, internodes of 20 rice plants each from LR005 and the bc22 mutant in the heading stage were collected to determine the cell wall component contents. All samples were dried in an oven at 80 °C to a constant weight. Cellulose content was measured following the Updegraff method described by Li et al. [33]. In brief, firstly, lignin, hemicellulose, and xylosans were extracted using an acetic acid/nitric acid reagent derived from the dried samples. Secondly, 67% H2SO4 was added to dissolve the remaining cellulose. Finally, the supernatant mixed with anthrone reagents was used to determine the cellulose content at 620 nm by comparing the absorbances of the samples with a standard curve. Hemicellulose and lignin contents were measured according to the method of Xiong et al. [34]. The reducing sugar contents were determined based on 3,5-dinitrosalicylic acid (DNS) colorimetry, 90% of which was hemicellulose content. Lignin content was determined via the redox titration of potassium permanganate and sodium thiosulfate.

2.6. Genemapping Using the MutMap Method

MutMap is a gene localization method based on next-generation sequencing and combined with association analysis algorithms, which can be said to be a resequencing-based Bulked Segregant Analysis (BSA) [35]. The stable mutant bc22 obtained by means of EMS mutagenesis was crossed with the wild-type parent LR005 to obtain the F2 generation localization population, and 30 plants with extreme mutant phenotypes were selected from the F2 population to construct the mutant mixing pools, and then, LR005 and the mutant mixing pools were re-sequenced using the MGI-DNBSeq T7 PE100 sequencing platform, obtaining a total of 42.68 Gb of raw data. We used FastQc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/; accessed on 15 January 2022) and Trimmomatic (http://www.usadellab.org/cms/index.php?page= Trimmomatic; accessed on 15 January 2022) for quality control and the cleaning of raw data to obtain clean data, bwa (https://bio-bwa.sourceforge.net/; accessed on 15 January 2022) to align the clean data to the Nipponare genome (http://rice.uga.edu/; accessed on 20 January 2023), and then bcftools (https://github.com/samtools/bcftools; accessed on 25 January 2023) to identify 1183332 SNPs and 215350 InDel. To minimize false positives generated by sequencing or alignment errors, sites with a sequencing depth lower than 8 or an SNP index less than 0.3 were screened out, and a total of 3927 SNP and InDel sites were obtained. The rate at which each SNP or InDel appeared in the mutant mixing pool was calculated (SNP-index = mutant-depth/depth). Theoretically, the SNP index is ≈0.5 for trait-independent loci, while some loci adjacent to candidate genes will have an SNP index greater than 0.5, or even equal to 1, due to the gene chain effect. By using the sliding window method with 2 Mb as the window and 100 kb as the step to plot the SNP index, a highly significant peak is obtained, which is the region linked to the candidate gene.

2.7. Complementary Genetic Test

To confirm the candidate genes, the 5.8 kb genomic region of BC22 (including the 1.5 kb promoter sequence, the entire 3.8 kb open reading frame, and the 0.5 kb downstream region) was amplified from LR005 genomic DNA and inserted into the pRHE vector. The construct was verified by means of sequencing and then transformed into the callus of the bc22 mutant using an Agrobacterium-mediated transformation approach. The amplified primers and verification primers were designed using Primer Premier 5 software (Supplementary Table S1), and the sequencing results were compared using SnapGene software version 4.2.7.

2.8. Gene Expression Analysis

Total RNA was isolated from the roots, seedlings, seedling culms, seedling leaves, culms, leaves, and spikes of LR005 and the bc22 mutant using the TRIzol reagent. First-strand cDNA was synthesized from 1 µg of total RNA using the Hiscript® III 1st Strand cDNA Synthesis kit (Vazyme, R312, Nanjing, China). qRT-PCR was performed by using gene-specific primers and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711, Nanjing, China) on an ABI7500 system. The rice actin gene was used as the internal control, and relative gene expression levels were calculated using the 2−ΔΔCt method [36]. The data presented for gene expression were derived from three biological replicates and three technical repeats for each tissue.

2.9. Statistical Analyses

SPSS 18.0 was used for all statistical analyses. All data are presented as the mean ± standard deviation of multiple biological replicates (n ≥ 3). All graphs were created using GraphPad Prism 9.0. Significant differences were determined using Student’s t-test and are indicated by * (p < 0.05) and ** (p < 0.01).

3. Results

3.1. Characterization of LR005 and Brittle Culm 22

We successfully obtained a genetically stable brittle culm mutant bc22 from a mutant library constructed by treating LR005 with EMS. When subjected to external bending force, the culm of the bc22 mutant exhibited increased fragility and was more prone to breakage compared to that of LR005. The fracture of LR005 remained connected by fibers, whereas the bc22 mutant showed complete breakage (Figure 1a). Compared with the LR005 wild type, the bc22 brittle culm mutant was significantly dwarfed, accompanied by a tiller-spreading phenotype, and its panicle length was remarkably decreased (Figure 1b,c, Table 1). Furthermore, the bc22 grains were smaller, and the grain length, grain width, and 1000-grain weight values of the bc22 mutant were reduced by 16.6%, 7.8%, and 34.5%, respectively, compared to those of the LR005 wild type (Figure 1d,e, Table 1). Additionally, the electron microscopy image of the cross-section of the leaves revealed that the number of trichomes on the vascular bundles of the bc22 mutant was significantly reduced compared to parent LR005 (Figure S1). The trichomes on the surface of glumes were rare or even absent in the bc22 mutant (Figure 1f), and the bc22 grains were smoother than those of LR005. The above observations indicate that the bc22 mutant showed pleiotropic phenotypes with brittle culms, dwarfed plants, tiller-spreading, and small grains and that they were less hairy.

3.2. Determination of Culm Mechanical Strength

To further characterize the mechanical properties of the bc22 mutant, the tensile strength and bending resistance of their stems were measured. The results regarding culm bending resistance indicated that the culm base fracture resistance of bc22 was 14.370 N, while LR005 exhibited a higher resistance of 23.85 N (Figure 2). This significant difference suggests that the bc22 mutations cause a dramatic reduction in the mechanical strength of stems.

3.3. Histological Characterisation of the Mutation Effect

The electron microscopy of the cross-sectional section of culms revealed that the number of cell layers in the stem epidermis of the bc22 mutant was lower and the thickness of cell wall was significantly thinner than that of LR005 (Figure 3). These findings indicated that the bc22 mutant’s reduction in mechanical strength was probably caused by histological changes.

3.4. Measurements of Cell Wall Component Contents

Among the three components measured, the bc22 mutant showed significantly higher hemicellulose content and significantly lower lignin content than LR005 (Figure 4). These findings indicated that the brittleness of the bc22 mutant is related to the changes in the hemicellulose and lignin contents of the cell wall.

3.5. Genetic Analysis

An F2 population including 921 individuals was derived from the bc22 mutant backcrossed to LR005. We found that 210 plants showed pleiotropic defects (including brittle culms, dwarfed plants, tiller-spreading, small grains, and less hairy glumes) identical to the bc22 mutant, while the other 711 individuals resembled those of LR005, and the segregation ratio of the normal to pleiotropic defects in the F2 population was close to 3:1 (χ2 = 2.45 < χ20.05 = 3.84, p > 0.05). These results suggest that the pleiotropic defect trait of bc22 is controlled by a single recessive gene.

3.6. Gene Mapping and Candidate Gene Prediction

Based on the analysis results, the candidate interval was identified on chromosome 2, spanning from 13 to 15 Mb, containing three SNP loci with a SNP-index value of 1 (Figure 5a). Among these three SNPs, only SNP14683899 occurred within the LOC_Os02g25230 CDS region. The Rice Genome Annotation Project (http://rice.uga.edu/; accessed on 5 February 2023) revealed that LOC_Os02g25230 consists of a single exon with a full-length genome of 2433 base pairs encoding 810 amino acids. The identified point mutation corresponded to substitution of a cytosine (C) into thymine (T) at position 1919 in the exon of the candidate gene, thereby resulting in the substitution of proline (Pro) with leucine (Leu) at position 640 (Figure 5d).
Subsequently, we designed PCR primers (Supplementary Table S1) using SnapGene 6.0 software based on the sequence of candidate genes from the Rice Gene Annotation Database and amplified the LOC_Os02g25230 gene from both the LR005 wild type and the bc22 mutant. These products were sequenced using Sanger sequencing technology, and the alignment results confirmed the existence of this mutation (Figure 5b,c). Hence, LOC_Os02g25230 was preliminarily identified as a candidate gene for BC22.

3.7. Functional Verification of the Gene

A plasmid with 5.8 kb wild type genomic DNA fragments was constructed to confirm the candidate gene. Then, the plasmid was introduced via A. tumefaciens to transform the bc22 mutant rice, and seven positive seedlings of bc22 complementary (bc22-COM) plants were obtained via selection using hygromycin. The T1 transgenic plants had a similar plant height and tiller angle to the LR005 wild type (Figure 6a–c). When the rice culms were bent due to being subjected to external force, the breaking points of the transgenic plants were visually indistinguishable from those of the LR005 wild type (Figure 6d,f). Likewise, the bc22-COM plants were phenotypically identical to the LR005 plants in terms of grain size and trichome formation (Figure 6g,h). The complementation analysis provided further evidence supporting the functional role of the candidate gene LOC_Os02g25230. Hence, it was confirmed that the candidate gene LOC_Os02g25230 could restore the trait of the bc22 mutant, validating it as BC22.

3.8. Expression Patterns of BC22

To determine the expression patterns of BC22 in rice, qRT-PCR was conducted to evaluate the transcription levels of BC22 in LR005 tissues such as the roots, culms, leaves, and spikelets. The results showed that BC22 was expressed in all tissues and that it was the most abundant in the culms and leaves (Figure 7).

4. Discussions

Mutants that cause culm fragility have been commonly observed in rice. Some mutants, such as bcm581-1 [37], fc17 [38], and bc19 [39], show no significant differences compared to their wild types, except for the brittle phenotype. However, the bc22 mutant in this study displays brittle culms, along with several other prominent characteristics, including a semidwarf stature, short panicles, and small grains and being less hairy. The missense mutation in the LOC_Os02g25230 gene was confirmed by means of complementation analysis to be causative of the bc22 mutant phenotypes. In previous studies, LOC_Os02g25230 has been shown to be a pleiotropic gene and encoded a plant-specific RNaseH-like protein [15,31,32,40]. The tiller angles observed in the tac4 mutant, the small grains observed in the sg2 mutant, the fragile stems observed in the bc17 mutant, and the less hairy leaves observed in the lhl1 mutant are all attributed to the premature termination of candidate gene expression, which is caused by point mutations or several base deletions [15,31,32,40]. Gene mapping and sequencing revealed that BC22 is a new allele of TAC4, BC17, LHL1, and SG2, arising from the mutation of C to T at position 1919 in the exon. Nevertheless, the point mutation alters an amino acid from Pro to Leu and results in the above phenotype, suggesting that Pro is critical to its function. BC22 encodes an RNaseH-like domain-containing protein. Interestingly, this mutation site did not disturb the formation of RNaseH-like domain but results in the loss of BC22 function. It is a reasonable guess that BC22 may have a more complex molecular mechanism influencing multiple agronomic parameters.
The cell wall plays a crucial role in maintaining the morphogenesis of plant cells and the mechanical strength of plant tissues [12], and changes in the contents of cell wall components can impact a plant’s mechanical properties [9]. Cellulose, hemicellulose, and lignin are key components that form the cell wall framework and provide support to plant cells. Changes in the contents of cell wall composition, a reduction in cell wall thickness, and abnormal cell wall development all affect the mechanical strength of plants and tissue brittleness [41,42,43]. However, mechanisms leading to plant brittleness are diverse. For instance, in the rice bc1 mutant, brittleness is associated with lower cellulose content, higher lignin content, and thinner cell walls [22]. Similarly, several genes, such as BC7, BC11, BC15, and BC19, alter mechanical strength by disturbing cellulose deposition and synthesis [18,24,26,39]. In general, cellulose is responsible for the stability of the cell wall skeleton, and reductions in the stability of the cell wall skeleton cause brittle culm characteristics [24]. The bc22 mutant exhibited fragile culms with no differences compared to those of the LR005 in terms of cellulose content, but the hemicellulose and lignin contents significantly increased and decreased, respectively, which is consistent with the results reported by the authors of [15] for the bc17 mutant. Thinner cell walls were also observed in the bc22 mutant (Figure 3). Lignin, as the main material responsible for secondary cell wall thickening, interacts with cellulose and hemicellulose to maintain mechanical strength [44,45]. BC17 was found to promote the lignin synthesis pathway by regulating the expression of laccase [15]. Recently, the same gene locus, TAC4, was also shown to regulate plant architecture by indirectly affecting indole acetic acid content and distribution [32]. In rice, auxin response factors 6 and 17 (abbreviated as ARF6 and ARF17) directly independently and synergistically regulate the synthesis of secondary cell walls [46] and may greatly affect lignin biosynthesis. Another identical locus, SG2, positively regulated the brassinosteroid (BR) signaling pathway to control grain size [31]. The role of brassinosteroids in increasing lignin content and cell wall thickness has been demonstrated in woody plants such as populus tomentosa [47]. Lignin is closely related to the stiffness of the cell wall and plays a vital role in pathogen defense [29]. It has been reported that the inhibition of BR signal transduction promotes rice blast fungus M. oryzae infection [48]. We speculated that the BR signaling pathway may confer blast resistance by promoting lignin synthesis, but this hypothesis still needs further experimental validation. Hence, how LOC_Os02g25230 is involved in cell wall synthesis through the auxin and BR signaling pathways should be further investigated in future studies.
Brittle-stalked rice plants have changed cell wall components and are easy to crush, making them not only susceptible to microbial degradation but also more easily digestible and absorbable by ruminants [49,50]. Therefore, investigating the brittleness genes and mechanisms in rice with brittle culms can offer a practical and theoretical foundation for breeding grain-forage rice [51,52].

5. Conclusions

We have demonstrated that Brittle Culm 22, a new allele of TAC4, BC17, SG2, and LHL1, is a pleiotropic gene encoding an RNaseH-like potein. The loss of function of BC22 leads to an increase in brittleness and tiller angle and a reduction in grain, plant height, and the trichomes of leaves and glumes. We propose that the decrease in the lignin content and the thickness of cell wall was confirmed to be causative of the bc22 mutant in brittle culms. This study provides a novel allelic mutant for deeper explorations of the function of the BC22 gene.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14020235/s1, Table S1: Primers used in the study. Figure S1: SEM of the trichome phenotype of LR005 and bc22 mutant.

Author Contributions

J.N., D.G., X.C. and Q.C. designed the study. X.C., T.Z., Y.S., Y.Z. (Yuhan Zhang), H.X., W.L., Y.Z. (Yu Zou) and H.M. performed the field experiments, data collection, and analysis. X.C., D.G. and J.N. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study received support from the Anhui Province Key Research and Development Project (Grant No. 2023h11020007, 202104g01020013, and 202204c06020018) and the Anhui Academy of Agricultural Sciences Young Talent Programme (grant No. QNYC2022), Key Laboratory of Rice Genetic Breeding of Anhui Province Programme (SDKF-2022-04).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

Acknowledgments

The authors would like to express sincere thanks to all those involved in this work. Special thanks are given to the Rice Research Institute of Anhui Academy of Agricultural Sciences for providing the experimental platform and Anhui Agricultural University for giving us this learning opportunities.

Conflicts of Interest

The authors declare no competing interests among them.

References

  1. Tanaka, K.; Murata, K.; Yamazaki, M.; Onosato, K.; Miyao, A.; Hirochika, H. Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall. Plant Physiol. 2003, 133, 73–83. [Google Scholar] [CrossRef] [PubMed]
  2. Johnson, S.E.; Angeles, O.R.; Brar, D.S.; Buresh, R.J. Faster anaerobic decomposition of a brittle straw rice mutant: Implications for residue management. Soil Biol. Biochem. 2006, 38, 1880–1892. [Google Scholar] [CrossRef]
  3. Hu, Y.; Zhong, R.; Morrison, W.H., 3rd; Ye, Z.H. The Arabidopsis RHD3 gene is required for cell wall biosynthesis and actin organization. Planta 2003, 217, 912–921. [Google Scholar] [CrossRef] [PubMed]
  4. Mitsuda, N.; Iwase, A.; Yamamoto, H.; Yoshida, M.; Seki, M.; Shinozaki, K.; Ohme-Takagi, M. NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 2007, 19, 270–280. [Google Scholar] [CrossRef]
  5. Guo, B.J.; Huang, X.Y.; Qi, J.; Sun, H.W.; Lv, C.; Wang, F.F.; Zhu, J.; Xu, R.G. Brittle culm 3, encoding a cellulose synthase subunit 5, is required for cell wall biosynthesis in barley (Hordeum vulgare L.). Front. Plant Sci. 2022, 13, 989406. [Google Scholar] [CrossRef]
  6. Tong, C.; Tong, J.P.; Sun, C. Reseach progress on functions of brittle culm genes in rice. Mol. Plant Breed. 2013, 11, 286–292. [Google Scholar]
  7. Wang, H.F.; Liu, J.X.; Qian, Q.; Wu, Y.M. Feeding brittle culm1 whole crop rice replacing wheat by-products to growing-finishing pigs. Acta Agric. Scand. Sect. A 2006, 56, 155–160. [Google Scholar]
  8. Van Soest, P.J.; Robertson, J.B.; Lewis, B.A. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 1991, 74, 3583–3597. [Google Scholar] [CrossRef]
  9. Zhang, Y.Y.; Wang, Y.H.; Wang, C.M.; Rautengarten, C.; Duan, E.C.; Zhu, J.P.; Zhu, X.P.; Lei, J.; Peng, C.; Wang, Y.L.; et al. BRITTLE PLANT1 is required for normal cell wall composition and mechanical strength in rice. J. Integr. Plant Biol. 2021, 63, 865–877. [Google Scholar] [CrossRef]
  10. Zhang, R.; Hu, H.Z.; Wang, Y.M.; Hu, Z.; Ren, S.F.; Li, J.Y.; He, B.Y.; Wang, Y.T.; Xia, T.; Chen, P.; et al. A novel rice fragile culm 24 mutant encodes a UDP-glucose epimerase that affects cell wall properties and photosynthesis. J. Exp. Bot. 2020, 71, 2956–2969. [Google Scholar] [CrossRef]
  11. Zhang, B.C.; Zhou, Y.H. Rice brittleness mutants: A way to open the ‘black box’ of monocot cell wall biosynthesis. J. Integr. Plant Biol. 2011, 53, 136–142. [Google Scholar] [CrossRef]
  12. Burton, R.A.; Gidley, M.J.; Fincher, G.B. Heterogeneity in the chemistry, structure and function of plant cell walls. Nat. Chem. Biol. 2010, 6, 724–732. [Google Scholar] [CrossRef]
  13. Hofte, H.; Voxeur, A. Plant cell walls. Curr. Biol. 2017, 27, R865–R870. [Google Scholar] [CrossRef] [PubMed]
  14. Miyagi, A.; Mori, K.; Ishikawa, T.; Ohkubo, S.; Adachi, S.; Yamaguchi, M.; Ookawa, T.; Kotake, T.; Kawai-Yamada, M. Metabolomic analysis of rice brittle culm mutants reveals each mutant-specific metabolic pattern in each organ. Metabolomics 2022, 18, 95. [Google Scholar] [CrossRef] [PubMed]
  15. Li, G.Z.; Zeng, X.F.; Li, Y.; Li, J.R.; Huang, X.Z.; Zhao, D.G. BRITTLE CULM17, a Novel Allele of TAC4, Affects the Mechanical Properties of Rice Plants. Int. J. Mol. Sci. 2022, 23, 5305. [Google Scholar] [CrossRef]
  16. Wang, D.F.; Yuan, S.J.; Yin, L.; Zhao, J.F.; Guo, B.T.; Lan, J.H.; Li, X.Y. A missense mutation in the transmembrane domain of CESA9 affects cell wall biosynthesis and plant growth in rice. Plant Sci. 2012, 196, 117–124. [Google Scholar] [CrossRef]
  17. Wang, D.F.; Qin, Y.L.; Fang, J.J.; Yuan, S.J.; Peng, L.X.; Zhao, J.F.; Li, X.Y. A Missense Mutation in the Zinc Finger Domain of OsCESA7 Deleteriously Affects Cellulose Biosynthesis and Plant Growth in Rice. PLoS ONE 2016, 11, e0153993. [Google Scholar] [CrossRef] [PubMed]
  18. Zhang, B.C.; Deng, L.W.; Qian, Q.; Xiong, G.Y.; Zeng, D.L.; Li, R.; Guo, L.B.; Li, J.Y.; Zhou, Y.H. A missense mutation in the transmembrane domain of CESA4 affects protein abundance in the plasma membrane and results in abnormal cell wall biosynthesis in rice. Plant Mol. Biol. 2009, 71, 509–524. [Google Scholar] [CrossRef]
  19. Yan, C.J.; Yan, S.; Zeng, X.H.; Zhang, Z.Q.; Gu, M.H. Fine mapping and isolation of Bc7(t), allelic to OsCesA4. J. Genet. Genom. 2007, 34, 1019–1027. [Google Scholar] [CrossRef]
  20. Kotake, T.; Aohara, T.; Hirano, K.; Sato, A.; Kaneko, Y.; Tsumuraya, Y.; Takatsuji, H.; Kawasaki, S. Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls. J. Exp. Bot. 2011, 62, 2053–2062. [Google Scholar] [CrossRef]
  21. Yu, H.; Hu, M.; Hu, Z.; Liu, F.; Yu, H.Z.; Yang, Q.M.; Gao, H.R.; Xu, C.B.; Wang, M.L.; Zhang, G.F.; et al. Insights into pectin dominated enhancements for elimination of toxic Cd and dye coupled with ethanol production in desirable lignocelluloses. Carbohydr. Polym. 2022, 286, 119298. [Google Scholar] [CrossRef]
  22. Li, Y.H.; Qian, Q.; Zhou, Y.H.; Yan, M.X.; Sun, L.; Zhang, M.; Fu, Z.M.; Wang, Y.H.; Han, B.; Pang, X.M.; et al. BRITTLE CULM1, which encodes a COBRA-like protein, affects the mechanical properties of rice plants. Plant Cell 2003, 15, 2020–2031. [Google Scholar] [CrossRef]
  23. Hirano, K.; Kotake, T.; Kamihara, K.; Tsuna, K.; Aohara, T.; Kaneko, Y.; Takatsuji, H.; Tsumuraya, Y.; Kawasaki, S. Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis. Planta 2010, 232, 95–108. [Google Scholar] [CrossRef]
  24. Zhang, M.; Zhang, B.C.; Qian, Q.; Yu, Y.C.; Li, R.; Zhang, J.W.; Liu, X.L.; Zeng, D.L.; Li, J.Y.; Zhou, Y.H. Brittle Culm 12, a dual-targeting kinesin-4 protein, controls cell-cycle progression and wall properties in rice. Plant J. 2010, 63, 312–328. [Google Scholar] [CrossRef]
  25. Zhou, Y.H.; Li, S.B.; Qian, Q.; Zeng, D.L.; Zhang, M.; Guo, L.B.; Liu, X.L.; Zhang, B.C.; Deng, L.W.; Liu, X.F.; et al. BC10, a DUF266-containing and Golgi-located type II membrane protein, is required for cell-wall biosynthesis in rice (Oryza sativa L.). Plant J. 2009, 57, 446–462. [Google Scholar] [CrossRef] [PubMed]
  26. Wu, B.; Zhang, B.C.; Dai, Y.; Zhang, L.; Shang-Guan, K.K.; Peng, Y.G.; Zhou, Y.H.; Zhu, Z. Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice. Plant Physiol. 2012, 159, 1440–1452. [Google Scholar] [CrossRef] [PubMed]
  27. Scheller, H.V.; Ulvskov, P. Hemicelluloses. Annu. Rev. Plant Biol. 2010, 61, 263–289. [Google Scholar] [CrossRef] [PubMed]
  28. Vega-Sanchez, M.E.; Verhertbruggen, Y.; Christensen, U.; Chen, X.; Sharma, V.; Varanasi, P.; Jobling, S.A.; Talbot, M.; White, R.G.; Joo, M.; et al. Loss of Cellulose synthase-like F6 function affects mixed-linkage glucan deposition, cell wall mechanical properties, and defense responses in vegetative tissues of rice. Plant Physiol. 2012, 159, 56–69. [Google Scholar] [CrossRef] [PubMed]
  29. Keegstra, K. Plant cell walls. Plant Physiol. 2010, 154, 483–486. [Google Scholar] [CrossRef]
  30. Li, X.J.; Yang, Y.; Yao, J.L.; Chen, G.X.; Li, X.H.; Zhang, Q.F.; Wu, C.Y. FLEXIBLE CULM 1 encoding a cinnamyl-alcohol dehydrogenase controls culm mechanical strength in rice. Plant Mol. Biol. 2009, 69, 685–697. [Google Scholar] [CrossRef]
  31. Huang, Y.S.; Dong, H.; Mou, C.L.; Wang, P.; Hao, Q.X.; Zhang, M.; Wu, H.M.; Zhang, F.L.; Ma, T.F.; Miao, R.; et al. Ribonuclease H-like gene SMALL GRAIN2 regulates grain size in rice through brassinosteroid signaling pathway. J. Integr. Plant Biol. 2022, 64, 1883–1900. [Google Scholar] [CrossRef]
  32. Li, H.; Sun, H.Y.; Jiang, J.H.; Sun, X.Y.; Tan, L.B.; Sun, C.Q. TAC4 controls tiller angle by regulating the endogenous auxin content and distribution in rice. Plant Biotechnol. J. 2021, 19, 64–73. [Google Scholar] [CrossRef]
  33. Bashline, L.; Li, S.; Zhu, X.; Gu, Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis. Proc. Natl. Acad. Sci. USA 2015, 112, 12870–12875. [Google Scholar] [CrossRef]
  34. Xiong, S.M.; Zuo, X.F.; Zhu, Y.Y. Determination of cellulose, hemicellulose and lignin in rice hull. Cereal Feed Ind. 2005, 8, 40–41. [Google Scholar]
  35. Abe, A.; Kosugi, S.; Yoshida, K.; Natsume, S.; Takagi, H.; Kanzaki, H.; Matsumura, H.; Yoshida, K.; Mitsuoka, C.; Tamiru, M.; et al. Genome sequencing reveals agronomically important loci in rice using MutMap. Nat. Biotechnol. 2012, 30, 174–178. [Google Scholar] [CrossRef]
  36. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29, e45. [Google Scholar] [CrossRef] [PubMed]
  37. Shen, G.Z.; Wang, X.Q.; Wang, J.; Wan, X.S.; Li, L.; Zhang, J.L. The morphology observation, mechanics intensity test and genetic analysis of brittle culm mutant bcm581-1 in rice. Shi Yan Sheng Wu Xue Bao 2002, 35, 307–312. [Google Scholar] [PubMed]
  38. Li, F.C.; Liu, S.T.; Xu, H.; Xu, Q. A novel FC17/CESA4 mutation causes increased biomass saccharification and lodging resistance by remodeling cell wall in rice. Biotechnol. Biofuels 2018, 11, 298. [Google Scholar] [CrossRef] [PubMed]
  39. Ma, X.Z.; Li, C.M.; Huang, R.; Zhang, K.; Wang, Q.; Fu, C.Y.; Liu, W.G.; Sun, C.G.; Wang, P.R.; Wang, F.; et al. Rice Brittle Culm19 Encoding Cellulose Synthase Subunit CESA4 Causes Dominant Brittle Phenotype But has No Distinct Influence on Growth and Grain Yield. Rice 2021, 14, 95. [Google Scholar] [CrossRef] [PubMed]
  40. Chen, H.Y.; Zhu, Z.; Wang, X.W.; Li, Y.Y.; Hu, D.L.; Zhang, X.F.; Jia, L.Q.; Cui, Z.B.; Sang, X.C. Less hairy leaf 1, an RNaseH-like protein, regulates trichome formation in rice through auxin. J. Integr. Agric. 2023, 22, 31–40. [Google Scholar] [CrossRef]
  41. Zhang, B.C.; Liu, X.L.; Qian, Q.; Liu, L.F.; Dong, G.J.; Xiong, G.Y.; Zeng, D.L.; Zhou, Y.H. Golgi nucleotide sugar transporter modulates cell wall biosynthesis and plant growth in rice. Proc. Natl. Acad. Sci. USA 2011, 108, 5110–5115. [Google Scholar] [CrossRef]
  42. Ruan, N.; Dang, Z.J.; Wang, M.H.; Cao, L.Y.; Wang, Y.; Liu, S.T.; Tang, Y.J.; Huang, Y.W.; Zhang, Q.; Xu, Q.; et al. FRAGILE CULM 18 encodes a UDP-glucuronic acid decarboxylase required for xylan biosynthesis and plant growth in rice. J. Exp. Bot. 2022, 73, 2320–2335. [Google Scholar] [CrossRef] [PubMed]
  43. Aohara, T.; Kotake, T.; Kaneko, Y.; Takatsuji, H.; Tsumuraya, Y.; Kawasaki, S. Rice BRITTLE CULM 5 (BRITTLE NODE) is involved in secondary cell wall formation in the sclerenchyma tissue of nodes. Plant Cell Physiol. 2009, 50, 1886–1897. [Google Scholar] [CrossRef] [PubMed]
  44. Zhong, R.; Ye, Z.H. Regulation of cell wall biosynthesis. Curr. Opin. Plant Biol. 2007, 10, 564–572. [Google Scholar] [CrossRef] [PubMed]
  45. Zhong, R.; Burk, D.H.; Ye, Z.H. Fibers. A model for studying cell differentiation, cell elongation, and cell wall biosynthesis. Plant Physiol. 2001, 126, 477–479. [Google Scholar]
  46. Huang, G.; Hu, H.; van de Meene, A.; Zhang, J.; Dong, L.; Zheng, S.; Zhang, F.; Betts, N.S.; Liang, W.; Bennett, M.J.; et al. AUXIN RESPONSE FACTORS 6 and 17 control the flag leaf angle in rice by regulating secondary cell wall biosynthesis of lamina joints. Plant Cell 2021, 33, 3120–3133. [Google Scholar] [CrossRef]
  47. Li, N.; Liu, B.M.; Chen, X.M. Influences of brassinolide synthesis gene DWF1 and DET2 on xylem formation in Populus tomentosa. Guangdong Agric. Sci. 2016, 43, 51–56. [Google Scholar]
  48. Meng, F.; Zheng, X.; Wang, J.; Qiu, T.; Yang, Q.; Fang, K.; Bhadauria, V.; Peng, Y.L.; Zhao, W. The GRAS protein OsDLA involves in brassinosteroid signalling and positively regulates blast resistance by forming a module with GSK2 and OsWRKY53 in rice. Plant Biotechnol. J. 2024, 22, 363–378. [Google Scholar] [CrossRef]
  49. Li, H. Principles and Techniques of Plant Physiological and Biochemical Experiments; Higher Education Press: Beijing, China, 2000; pp. 134–137. [Google Scholar]
  50. Shen, H.S.; Chen, J.C.; Zeng, D.L.; Tu, J.F.; Tang, B.S.; Teng, S. Dynamic analysis on composition of cell wall for low–fiber mutation rice. Sci. Agric. Sin. 2004, 37, 943–947. [Google Scholar]
  51. Multani, D.S.; Jiao, S.; Jung, M.T.; Simcox, K.D. Stalk strength improvement in crop plants: A progress report. Ann. Plant Rev. Online 2021, 4, 357–395. [Google Scholar]
  52. Dang, Z.J.; Wang, Y.; Wang, M.H.; Cao, L.Y.; Ruan, N.; Huang, Y.W.; Li, F.C.; Xu, Q.; Chen, W.F. The Fragile culm19 (FC19) mutation largely improves plant lodging resistance, biomass saccharification, and cadmium resistance by remodeling cell walls in rice. J. Hazard. Mater. 2023, 458, 132020. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Morphological characteristics of LR005 and the bc22 mutant. (a) Plant culm breaking traits. (b) Plant architecture in the heading stage. (c) Panicle phenotype in the maturity stage. (d) Grain length. (e) Grain width. (f) Chorionic characters on the surface of the glume. Scale bars: 2 cm (a); 10 cm (b,c); 5 mm (d–f).
Figure 1. Morphological characteristics of LR005 and the bc22 mutant. (a) Plant culm breaking traits. (b) Plant architecture in the heading stage. (c) Panicle phenotype in the maturity stage. (d) Grain length. (e) Grain width. (f) Chorionic characters on the surface of the glume. Scale bars: 2 cm (a); 10 cm (b,c); 5 mm (d–f).
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Figure 2. Culm base bending resistance of LR005 and the mutant bc22. ** indicates a significant difference at p < 0.01 (determined using Student’s t-test).
Figure 2. Culm base bending resistance of LR005 and the mutant bc22. ** indicates a significant difference at p < 0.01 (determined using Student’s t-test).
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Figure 3. Brittle analysis of culms of LR005 and the bc22 mutant. (a) Culm-breaking phenotype of LR005 in the maturity stage; bar = 2 cm. (b,c) Scanning electron microscopy (SEM) image of the cross-section of the LR005 culm; bar = 20 μm. (d) Culm-breaking phenotype of the bc22 mutant in the maturity stage; bar = 2 cm. (e,f) SEM image of the cross-section of the bc22 culm; bar = 20 μm. The red boxes in (b,e) are enlarged to show (c,f), respectively.
Figure 3. Brittle analysis of culms of LR005 and the bc22 mutant. (a) Culm-breaking phenotype of LR005 in the maturity stage; bar = 2 cm. (b,c) Scanning electron microscopy (SEM) image of the cross-section of the LR005 culm; bar = 20 μm. (d) Culm-breaking phenotype of the bc22 mutant in the maturity stage; bar = 2 cm. (e,f) SEM image of the cross-section of the bc22 culm; bar = 20 μm. The red boxes in (b,e) are enlarged to show (c,f), respectively.
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Figure 4. Analysis of the main components of the cell walls of LR005 and bc22 mutant culms. Cellulose contents (a), hemicellulose contents (b), and lignin contents (c) in culm internodes of LR005 and the bc22 mutant. No significant difference is indicated by ns at p ˃ 0.05 level. ** indicates a significant difference at p < 0.01 (determined using Student’s t-test).
Figure 4. Analysis of the main components of the cell walls of LR005 and bc22 mutant culms. Cellulose contents (a), hemicellulose contents (b), and lignin contents (c) in culm internodes of LR005 and the bc22 mutant. No significant difference is indicated by ns at p ˃ 0.05 level. ** indicates a significant difference at p < 0.01 (determined using Student’s t-test).
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Figure 5. Gene mapping and candidate prediction of bc22. (a) SNP index of the candidate gene on rice chromosome 2 (statistical confidence intervals; green = 95%; yellow = 99%). The peak marked by a black circle and arrow means the candidate region of a major genetic locus for the brittle culm trait. Orange line: 99% confidence interval of simulated SNP-index; green line: 95% confidence interval of simulated SNP-index; red line: mean SNP-index. (b) Sanger sequencing peak map of the LR005 wild type. The red box represents a codon encoding Pro. (c) The Sanger sequencing peak map of the bc22 mutant. The red box represents a codon encoding Leu. (d) Representation of the structure and base mutation of the candidate gene LOC_Os02g25230.
Figure 5. Gene mapping and candidate prediction of bc22. (a) SNP index of the candidate gene on rice chromosome 2 (statistical confidence intervals; green = 95%; yellow = 99%). The peak marked by a black circle and arrow means the candidate region of a major genetic locus for the brittle culm trait. Orange line: 99% confidence interval of simulated SNP-index; green line: 95% confidence interval of simulated SNP-index; red line: mean SNP-index. (b) Sanger sequencing peak map of the LR005 wild type. The red box represents a codon encoding Pro. (c) The Sanger sequencing peak map of the bc22 mutant. The red box represents a codon encoding Leu. (d) Representation of the structure and base mutation of the candidate gene LOC_Os02g25230.
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Figure 6. Analysis of the traits of the LR005 wild type, the bc22 mutant, and a complementary line. (a–c) Plant morphologies of a LR005 plant (a), a bc22 mutant plant (b), and a plant from the complementary line BC22-COM (c). (d–f) Culm fracture phenotypes of the LR005 wild type (d), the bc22 mutant (e), and the complementary line BC22-COM (f). (g,h) Grain length (g) and grain width (h) of the LR005, bc22 mutant, and the complementary line BC22-COM. Scale bar: 20 cm (a–c), 2 cm (d,f) and 5 mm (g,h).
Figure 6. Analysis of the traits of the LR005 wild type, the bc22 mutant, and a complementary line. (a–c) Plant morphologies of a LR005 plant (a), a bc22 mutant plant (b), and a plant from the complementary line BC22-COM (c). (d–f) Culm fracture phenotypes of the LR005 wild type (d), the bc22 mutant (e), and the complementary line BC22-COM (f). (g,h) Grain length (g) and grain width (h) of the LR005, bc22 mutant, and the complementary line BC22-COM. Scale bar: 20 cm (a–c), 2 cm (d,f) and 5 mm (g,h).
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Figure 7. Analysis of the expression of BC22 in different LR005 tissues. The rice actin gene was used as the internal control. Values represent the mean ± SD (n ≥ 3). ** indicates a significant difference at p < 0.01 (determined using Student’s t-test).
Figure 7. Analysis of the expression of BC22 in different LR005 tissues. The rice actin gene was used as the internal control. Values represent the mean ± SD (n ≥ 3). ** indicates a significant difference at p < 0.01 (determined using Student’s t-test).
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Table 1. Morphologic characterization of LR005 and the mutant bc22.
Table 1. Morphologic characterization of LR005 and the mutant bc22.
TraitPlant Height/cmPanicle Length/cmGrain Length/cmGrain Width/cm1000-Grain Weight/g
LR00593.24 ± 0.9422.22 ± 0.488.56 ± 0.092.56 ± 0.0420.16 ± 0.09
bc2259.78 ± 0.59 **14.18 ± 0.30 **7.14 ± 0.08 **2.36 ± 0.05 *13.23 ± 0.22 **
The data in the table are listed as the mean ± standard deviation. * means significant difference at p < 0.05, ** means significant difference at p < 0.01 according to Student’s t-test.
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MDPI and ACS Style

Cao, X.; Zhou, T.; Sun, Y.; Zhang, Y.; Xu, H.; Liu, W.; Zou, Y.; Chen, Q.; Ma, H.; Gu, D.; et al. Identification and Gene Cloning of a Brittle Culm Mutant (bc22) in Rice. Agriculture 2024, 14, 235. https://doi.org/10.3390/agriculture14020235

AMA Style

Cao X, Zhou T, Sun Y, Zhang Y, Xu H, Liu W, Zou Y, Chen Q, Ma H, Gu D, et al. Identification and Gene Cloning of a Brittle Culm Mutant (bc22) in Rice. Agriculture. 2024; 14(2):235. https://doi.org/10.3390/agriculture14020235

Chicago/Turabian Style

Cao, Xiying, Tao Zhou, Yue Sun, Yuhan Zhang, Huan Xu, Wei Liu, Yu Zou, Qingquan Chen, Hui Ma, Dongfang Gu, and et al. 2024. "Identification and Gene Cloning of a Brittle Culm Mutant (bc22) in Rice" Agriculture 14, no. 2: 235. https://doi.org/10.3390/agriculture14020235

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