Assessing the Response of Small RNA Populations to Allopolyploidy Using Resynthesized Brassica napus Allotetraploids.

Allopolyploidy, combining interspecific hybridization with whole genome duplication, has had significant impact on plant evolution. Its evolutionary success is related to the rapid and profound genome reorganizations that allow neoallopolyploids to form and adapt. Nevertheless, how neoallopolyploid genomes adapt to regulate their expression remains poorly understood. The hypothesis of a major role for small noncoding RNAs (sRNAs) in mediating the transcriptional response of neoallopolyploid genomes has progressively emerged. Generally, 21-nt sRNAs mediate posttranscriptional gene silencing by mRNA cleavage, whereas 24-nt sRNAs repress transcription (transcriptional gene silencing) through epigenetic modifications. Here, we characterize the global response of sRNAs to allopolyploidy in Brassica, using three independently resynthesized Brassica napus allotetraploids originating from crosses between diploid Brassica oleracea and Brassica rapa accessions, surveyed at two different generations in comparison with their diploid progenitors. Our results suggest an immediate but transient response of specific sRNA populations to allopolyploidy. These sRNA populations mainly target noncoding components of the genome but also target the transcriptional regulation of genes involved in response to stresses and in metabolism; this suggests a broad role in adapting to allopolyploidy. We finally identify the early accumulation of both 21- and 24-nt sRNAs involved in regulating the same targets, supporting a posttranscriptional gene silencing to transcriptional gene silencing shift at the first stages of the neoallopolyploid formation. We propose that reorganization of sRNA production is an early response to allopolyploidy in order to control the transcriptional reactivation of various noncoding elements and stress-related genes, thus ensuring genome stability during the first steps of neoallopolyploid formation.

Independent introductions and admixtures have contributed to adaptation of European maize and its American counterparts.

Through the local selection of landraces, humans have guided the adaptation of crops to a vast range of climatic and ecological conditions. This is particularly true of maize, which was domesticated in a restricted area of Mexico but now displays one of the broadest cultivated ranges worldwide. Here, we sequenced 67 genomes with an average sequencing depth of 18x to document routes of introduction, admixture and selective history of European maize and its American counterparts. To avoid the confounding effects of recent breeding, we targeted germplasm (lines) directly derived from landraces. Among our lines, we discovered 22,294,769 SNPs and between 0.9% to 4.1% residual heterozygosity. Using a segmentation method, we identified 6,978 segments of unexpectedly high rate of heterozygosity. These segments point to genes potentially involved in inbreeding depression, and to a lesser extent to the presence of structural variants. Genetic structuring and inferences of historical splits revealed 5 genetic groups and two independent European introductions, with modest bottleneck signatures. Our results further revealed admixtures between distinct sources that have contributed to the establishment of 3 groups at intermediate latitudes in North America and Europe. We combined differentiation- and diversity-based statistics to identify both genes and gene networks displaying strong signals of selection. These include genes/gene networks involved in flowering time, drought and cold tolerance, plant defense and starch properties. Overall, our results provide novel insights into the evolutionary history of European maize and highlight a major role of admixture in environmental adaptation, paralleling recent findings in humans.

High throughput genotyping of structural variations in a complex plant genome using an original Affymetrix® axiom® array.

BACKGROUND: Insertions/deletions (InDels) and more specifically presence/absence variations (PAVs) are pervasive in several species and have strong functional and phenotypic effect by removing or drastically modifying genes. Genotyping of such variants on large panels remains poorly addressed, while necessary for approaches such as association mapping or genomic selection. RESULTS: We have developed, as a proof of concept, a new high-throughput and affordable approach to genotype InDels. We first identified 141,000 InDels by aligning reads from the B73 line against the genome of three temperate maize inbred lines (F2, PH207, and C103) and reciprocally. Next, we designed an Affymetrix® Axiom® array to target these InDels, with a combination of probes selected at breakpoint sites (13%) or within the InDel sequence, either at polymorphic (25%) or non-polymorphic sites (63%) sites. The final array design is composed of 662,772 probes and targets 105,927 InDels, including PAVs ranging from 35 bp to 129kbp. After Affymetrix® quality control, we successfully genotyped 86,648 polymorphic InDels (82% of all InDels interrogated by the array) on 445 maize DNA samples with 422,369 probes. Genotyping InDels using this approach produced a highly reliable dataset, with low genotyping error (~ 3%), high call rate (~ 98%), and high reproducibility (> 95%). This reliability can be further increased by combining genotyping of several probes calling the same InDels (< 0.1% error rate and > 99.9% of call rate for 5 probes). This "proof of concept" tool was used to estimate the kinship matrix between 362 maize lines with 57,824 polymorphic InDels. This InDels kinship matrix was highly correlated with kinship estimated using SNPs from Illumina 50 K SNP arrays. CONCLUSIONS: We efficiently genotyped thousands of small to large InDels on a sizeable number of individuals using a new Affymetrix® Axiom® array. This powerful approach opens the way to studying the contribution of InDels to trait variation and heterosis in maize. The approach is easily extendable to other species and should contribute to decipher the biological impact of InDels at a larger scale.

Sequence analysis of European maize inbred line F2 provides new insights into molecular and chromosomal characteristics of presence/absence variants.

BACKGROUND: Maize is well known for its exceptional structural diversity, including copy number variants (CNVs) and presence/absence variants (PAVs), and there is growing evidence for the role of structural variation in maize adaptation. While PAVs have been described in this important crop species, they have been only scarcely characterized at the sequence level and the extent of presence/absence variation and relative chromosomal landscape of inbred-specific regions remain to be elucidated. RESULTS: De novo genome sequencing of the French F2 maize inbred line revealed 10,044 novel genomic regions larger than 1 kb, making up 88 Mb of DNA, that are present in F2 but not in B73 (PAV). This set of maize PAV sequences allowed us to annotate PAV content and to analyze sequence breakpoints. Using PAV genotyping on a collection of 25 temperate lines, we also analyzed Linkage Disequilibrium in PAVs and flanking regions, and PAV frequencies within maize genetic groups. CONCLUSIONS: We highlight the possible role of MMEJ-type double strand break repair in maize PAV formation and discover 395 new genes with transcriptional support. Pattern of linkage disequilibrium within PAVs strikingly differs from this of flanking regions and is in accordance with the intuition that PAVs may recombine less than other genomic regions. We show that most PAVs are ancient, while some are found only in European Flint material, thus pinpointing structural features that may be at the origin of adaptive traits involved in the success of this material. Characterization of such PAVs will provide useful material for further association genetic studies in European and temperate maize.

Management and dissemination of MS proteomic data with PROTICdb: example of a quantitative comparison between methods of protein extraction.

High throughput MS-based proteomic experiments generate large volumes of complex data and necessitate bioinformatics tools to facilitate their handling. Needs include means to archive data, to disseminate them to the scientific communities, and to organize and annotate them to facilitate their interpretation. We present here an evolution of PROTICdb, a database software that now handles MS data, including quantification. PROTICdb has been developed to be as independent as possible from tools used to produce the data. Biological samples and proteomics data are described using ontology terms. A Taverna workflow is embedded, thus permitting to automatically retrieve information related to identified proteins by querying external databases. Stored data can be displayed graphically and a "Query Builder" allows users to make sophisticated queries without knowledge on the underlying database structure. All resources can be accessed programmatically using a Java client API or RESTful web services, allowing the integration of PROTICdb in any portal. An example of application is presented, where proteins extracted from a maize leaf sample by four different methods were compared using a label-free shotgun method. Data are available at http://moulon.inra.fr/protic/public. PROTICdb thus provides means for data storage, enrichment, and dissemination of proteomics data.

BioMercator V3: an upgrade of genetic map compilation and quantitative trait loci meta-analysis algorithms.

SUMMARY: Compilation of genetic maps combined to quantitative trait loci (QTL) meta-analysis has proven to be a powerful approach contributing to the identification of candidate genes underlying quantitative traits. BioMercator was the first software offering a complete set of algorithms and visualization tool covering all steps required to perform QTL meta-analysis. Despite several limitations, the software is still widely used. We developed a new version proposing additional up to date methods and improving graphical representation and exploration of large datasets. AVAILABILITY AND IMPLEMENTATION: BioMercator V3 is implemented in JAVA and freely available (http://moulon.inra.fr/biomercator) CONTACT: joets@moulon.inra.fr.

Allopolyploidy has a moderate impact on restructuring at three contrasting transposable element insertion sites in resynthesized Brassica napus allotetraploids.

The role played by whole-genome duplication (WGD) in evolution and adaptation is particularly well illustrated in allopolyploids, where WGD is concomitant with interspecific hybridization. This 'Genome Shock', usually accompanied by structural and functional modifications, has been associated with the activation of transposable elements (TEs). However, the impact of allopolyploidy on TEs has been studied in only a few polyploid species, and not in Brassica, which has been marked by recurrent polyploidy events. Here, we developed sequence-specific amplification polymorphism (SSAP) markers for three contrasting TEs, and compared profiles between resynthesized Brassica napus allotetraploids and their diploid Brassica progenitors. To evaluate restructuring at TE insertion sites, we scored changes in SSAP profiles and analysed a large set of differentially amplified SSAP bands. No massive structural changes associated with the three TEs surveyed were detected. However, several transposition events, specific to the youngest TE originating from the B. oleracea genome, were identified. Our study supports the hypothesis that TE responses to allopolyploidy are highly specific. The changes observed in SSAP profiles lead us to hypothesize that they may partly result from changes in DNA methylation, questioning the role of epigenetics during the formation of a new allopolyploid genome.

OptiMAS: a decision support tool for marker-assisted assembly of diverse alleles.

Current advances in plant genotyping lead to major progress in the knowledge of genetic architecture of traits of interest. It is increasingly important to develop decision support tools to help breeders and geneticists to conduct marker-assisted selection methods to assemble favorable alleles that are discovered. Algorithms have been implemented, within an interactive graphical interface, to 1) trace parental alleles throughout generations, 2) propose strategies to select the best plants based on estimated molecular scores, and 3) efficiently intermate them depending on the expected value of their progenies. With the possibility to consider a multi-allelic context, OptiMAS opens new prospects to assemble favorable alleles issued from diverse parents and further accelerate genetic gain.

Secretome of the free-living mycelium from the ectomycorrhizal basidiomycete Laccaria bicolor.

The ectomycorrhizal basidiomycete Laccaria bicolor has a dual lifestyle with a transitory soil saprotrophic phase and a longer mutualistic interaction with tree roots. Recent evidence suggests that secreted proteins play key roles in host plant colonisation and symbiosis development. However, a limited number of secreted proteins have been characterized, and the full spectrum of effectors involved in the mycobiont invasion and survival remains unknown. We analyzed the extracellular proteins secreted in growth medium by free-living mycelium of L. bicolor as a proxy for its saprotrophic phase. The proteomic analyses (two-dimensional electrophoresis and shotgun proteomics) were substantiated by whole-genome expression transcript profiling on ectomycorrhizal roots. Among the 224 proteins identified were carbohydrate-acting enzymes likely involved in the cell wall remodelling linked to hyphal growth as well as secreted proteases possibly digesting soil organic compounds and/or fending off competitors, pathogens, and predators. Evidence of gene expression was found in ectomycorrhizal roots for 210 of them. These findings provide the first global view of the secretome of a mutualistic symbiont and shed some light on the mechanisms controlling cell wall remodelling during the hyphal growth. They also revealed many novel putative secreted proteins of unknown function, including one mycorrhiza-induced small secreted protein.

Genetic Architecture of Powdery Mildew Resistance Revealed by a Genome-Wide Association Study of a Worldwide Collection of Flax (Linum usitatissimum L.).

Powdery mildew is one of the most important diseases of flax and is particularly prejudicial to its yield and oil or fiber quality. This disease, caused by the obligate biotrophic ascomycete Oïdium lini, is progressing in France. Genetic resistance of varieties is critical for the control of this disease, but very few resistance genes have been identified so far. It is therefore necessary to identify new resistance genes to powdery mildew suitable to the local context of pathogenicity. For this purpose, we studied a worldwide diversity panel composed of 311 flax genotypes both phenotyped for resistance to powdery mildew resistance over 2 years of field trials in France and resequenced. Sequence reads were mapped on the CDC Bethune reference genome revealing 1,693,910 high-quality SNPs, further used for both population structure analysis and genome-wide association studies (GWASs). A number of four major genetic groups were identified, separating oil flax accessions from America or Europe and those from Asia or Middle-East and fiber flax accessions originating from Eastern Europe and those from Western Europe. A number of eight QTLs were detected at the false discovery rate threshold of 5%, located on chromosomes 1, 2, 4, 13, and 14. Taking advantage of the moderate linkage disequilibrium present in the flax panel, and using the available genome annotation, we identified potential candidate genes. Our study shows the existence of new resistance alleles against powdery mildew in our diversity panel, of high interest for flax breeding program.

BraSto, a Stowaway MITE from Brassica: recently active copies preferentially accumulate in the gene space.

We characterized a Brassica miniature inverted repeat transposable element (MITE) from the Stowaway superfamily, designated BraSto (Bra ssica Sto waway). BraSto copy number was assessed using real-time quantitative PCR in the two diploid species B. rapa (genome A) and B. oleracea (genome C) and the corresponding allotetraploid species B. napus (genome AC). Phylogenetic relationships among a set of 131 BraSto copies were then analyzed. BraSto appears to have been only moderately amplified in the Brassica genome and was still active recently with marks of proliferation in both diploid Brassica species, which diverged 3.75 million years ago, but also in the allotetraploid species after reuniting of the two differentiated genomes. We characterized insertion sites for low-divergence BraSto copies among the gene space of the B. rapa genome using bioinformatics approaches. For BraSto copies localized nearby or within genes, we observed frequent associations of BraSto with putative promoters and regulatory regions of genes, but exclusion from coding regions. In addition, BraSto was significantly similar to several Brassica expressed sequence tags (ESTs), including stress-induced ESTs. We also demonstrated the enrichment of BraSto sequences in binding sites for transcription factors and other regulatory elements. Our results lead to the question of a role for BraSto in the regulation of gene expression: this putative role, if further confirmed experimentally, would help to obtain a new insight into the significance of MITEs in the functional plant genome.

EuGène-maize: a web site for maize gene prediction.

MOTIVATION: A large part of the maize B73 genome sequence is now available and emerging sequencing technologies will offer cheap and easy ways to sequence areas of interest from many other maize genotypes. One of the steps required to turn these sequences into valuable information is gene content prediction. To date, there is no publicly available gene predictor specifically trained for maize sequences. To this end, we have chosen to train the EuGène software that can combine several sources of evidence into a consolidated gene model prediction. AVAILABILITY: http://genome.jouy.inra.fr/eugene/cgi-bin/eugene_form.pl.

Transcriptome Analysis Reveals Putative Target Genes of APETALA3-3 During Early Floral Development in Nigella damascena L.

Even though petals are homoplastic structures, their identity consistently involves genes of the APETALA3 (AP3) lineage. However, the extent to which the networks downstream of AP3 are conserved in species with petals of different evolutionary origins is unknown. In Ranunculaceae, the specificity of the AP3-III lineage offers a great opportunity to identify the petal gene regulatory network in a comparative framework. Using a transcriptomic approach, we investigated putative target genes of the AP3-III ortholog NdAP3-3 in Nigella damascena at early developmental stages when petal identity is determined, and we compared our data with that from selected eudicot species. We generated a de novo reference transcriptome to carry out a differential gene expression analysis between the wild-type and mutant NdAP3-3 genotypes differing by the presence vs. absence of petals at early stages of floral development. Among the 1,620 genes that were significantly differentially expressed between the two genotypes, functional annotation suggested a large involvement of nuclear activities, including regulation of transcription, and enrichment in processes linked to cell proliferation. Comparing with Arabidopsis data, we found that highly conserved genes between the two species are enriched in homologs of direct targets of the AtAP3 protein. Integrating AP3-3 binding site data from another Ranunculaceae species, Aquilegia coerulea, allowed us to identify a set of 18 putative target genes that were conserved between the three species. Our results suggest that, despite the independent evolutionary origin of petals in core eudicots and Ranunculaceae, a small conserved set of genes determines petal identity and early development in these taxa.

Identification of Key Tissue-Specific, Biological Processes by Integrating Enhancer Information in Maize Gene Regulatory Networks.

Enhancers are key players in the spatio-temporal coordination of gene expression during numerous crucial processes, including tissue differentiation across development. Characterizing the transcription factors (TFs) and genes they connect, and the molecular functions underpinned is important to better characterize developmental processes. In plants, the recent molecular characterization of enhancers revealed their capacity to activate the expression of several target genes. Nevertheless, identifying these target genes at a genome-wide level is challenging, particularly for large-genome species, where enhancers and target genes can be hundreds of kilobases away. Therefore, the contribution of enhancers to plant regulatory networks remains poorly understood. Here, we investigate the enhancer-driven regulatory network of two maize tissues at different stages: leaves at seedling stage (V2-IST) and husks (bracts) at flowering. Using systems biology, we integrate genomic, epigenomic, and transcriptomic data to model the regulatory relationships between TFs and their potential target genes, and identify regulatory modules specific to husk and V2-IST. We show that leaves at the V2-IST stage are characterized by the response to hormones and macromolecules biogenesis and assembly, which are regulated by the BBR/BPC and AP2/ERF TF families, respectively. In contrast, husks are characterized by cell wall modification and response to abiotic stresses, which are, respectively, orchestrated by the C2C2/DOF and AP2/EREB families. Analysis of the corresponding enhancer sequences reveals that two different transposable element families (TIR transposon Mutator and MITE Pif/Harbinger) have shaped part of the regulatory network in each tissue, and that MITEs have provided potential new TF binding sites involved in husk tissue-specificity.

Comparative proteomics of leaf, stem, and root tissues of synthetic Brassica napus.

Comparative proteomics was applied to three vegetative organs of Brassica napus, the leaf, stem, and root using 2-DE. Among the >1600 analyzed spots, 43% were found to be common to all three organs, suggesting the existence of a "basal" or ubiquitous proteome composed of housekeeping proteins. The green organs, leaf, and stem, were closely related (approximately 80% common spots) while the root displayed more organ-specific polypeptides (approximately 10%). Reference maps were established using MS, allowing the identification of 93, 385, and 266 proteins in leaf, stem, and root proteomes, respectively. Bioinformatic analyses were also performed; in silico functional categorization and cellular localization allow obtaining a precise picture of the cell molecular network within vegetative organs. These proteome maps can be explored using the PROTICdb software at the following address: http://bioinformatique.moulon.inra.fr/proticdb/web_view/.

The CACTA transposon Bot1 played a major role in Brassica genome divergence and gene proliferation.

We isolated and characterized a Brassica C genome-specific CACTA element, which was designated Bot1 (Brassica oleracea transposon 1). After analysing phylogenetic relationships, copy numbers and sequence similarity of Bot1 and Bot1 analogues in B. oleracea (C genome) versus Brassica rapa (A genome), we concluded that Bot1 has encountered several rounds of amplification in the oleracea genome only, and has played a major role in the recent rapa and oleracea genome divergence. We performed in silico analyses of the genomic organization and internal structure of Bot1, and established which segment of Bot1 is C-genome specific. Our work reports a fully characterized Brassica repetitive sequence that can distinguish the Brassica A and C chromosomes in the allotetraploid Brassica napus, by fluorescent in situ hybridization. We demonstrated that Bot1 carries a host S locus-associated SLL3 gene copy. We speculate that Bot1 was involved in the proliferation of SLL3 around the Brassica genome. The present study reinforces the assumption that transposons are a major driver of genome and gene evolution in higher plants.

Contrasting evolutionary patterns and target specificities among three Tourist-like MITE families in the maize genome.

Miniature inverted-repeat transposable elements (MITEs) are short, non autonomous DNA elements that are widespread and abundant in plant genomes. The high sequence and size conservation observed in many MITE families suggest that they have spread recently throughout their respective host genomes. Here we present a maize genome wide analysis of three Tourist-like MITE families, mPIF, and two previously uncharacterized families, ZmV1 and Zead8. We undertook a bioinformatic analysis of MITE insertion sites, developed methyl-sensitive transposon display (M-STD) assays to estimate the associated level of CpG methylation at MITE flanking regions, and conducted a population genetics approach to investigate MITE patterns of expansion. Our results reveal that the three MITE families insert into genomic regions that present specific molecular features: they are preferentially AT rich, present low level of cytosine methylation as compared to the LTR retrotransposon Grande, and target site duplications are flanked by large and conserved palindromic sequences. Moreover, the analysis of MITE distances from predicted genes shows that 73% of 263 copies are inserted at less than 5 kb from the nearest predicted gene, and copies from Zead8 family are significantly more abundant upstream of genes. By employing a population genetic approach we identified contrasting patterns of expansion among the three MITE families. All elements seem to have inserted roughly 1 million years ago but ZmV1 and Zead8 families present evidences for activity of several master copies within the last 0.4 Mya.

Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE.

The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, beta-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.

Unraveling the Developmental and Genetic Mechanisms Underpinning Floral Architecture in Proteaceae.

Proteaceae are a basal eudicot family with a highly conserved floral groundplan but which displays considerable variation in other aspects of floral and inflorescence morphology. Their morphological diversity and phylogenetic position make them good candidates for understanding the evolution of floral architecture, in particular the question of the homology of the undifferentiated perianth with the differentiated perianth of core eudicots, and the mechanisms underlying the repeated evolution of zygomorphy. In this paper, we combine a morphological approach to explore floral ontogenesis and a transcriptomic approach to access the genes involved in floral organ identity and development, focusing on Grevillea juniperina, a species from subfamily Grevilleoideae. We present developmental data for Grevillea juniperina and three additional species that differ in their floral symmetry using stereomicroscopy, SEM and High Resolution X-Ray Computed Tomography. We find that the adnation of stamens to tepals takes place at early developmental stages, and that the establishment of bilateral symmetry coincides with the asymmetrical growth of the single carpel. To set a framework for understanding the genetic basis of floral development in Proteaceae, we generated and annotated de novo a reference leaf/flower transcriptome from Grevillea juniperina. We found Grevillea homologs of all lineages of MADS-box genes involved in floral organ identity. Using Arabidopsis thaliana gene expression data as a reference, we found homologs of other genes involved in floral development in the transcriptome of G. juniperina. We also found at least 21 class I and class II TCP genes, a gene family involved in the regulation of growth processes, including floral symmetry. The expression patterns of a set of floral genes obtained from the transcriptome were characterized during floral development to assess their organ specificity and asymmetry of expression.

A systems biology approach uncovers a gene co-expression network associated with cell wall degradability in maize.

Understanding the mechanisms triggering variation of cell wall degradability is a prerequisite to improving the energy value of lignocellulosic biomass for animal feed or biorefinery. Here, we implemented a multiscale systems approach to shed light on the genetic basis of cell wall degradability in maize. We demonstrated that allele replacement in two pairs of near-isogenic lines at a region encompassing a major quantitative trait locus (QTL) for cell wall degradability led to phenotypic variation of a similar magnitude and sign to that expected from a QTL analysis of cell wall degradability in the F271 × F288 recombinant inbred line progeny. Using DNA sequences within the QTL interval of both F271 and F288 inbred lines and Illumina RNA sequencing datasets from internodes of the selected near-isogenic lines, we annotated the genes present in the QTL interval and provided evidence that allelic variation at the introgressed QTL region gives rise to coordinated changes in gene expression. The identification of a gene co-expression network associated with cell wall-related trait variation revealed that the favorable F288 alleles exploit biological processes related to oxidation-reduction, regulation of hydrogen peroxide metabolism, protein folding and hormone responses. Nested in modules of co-expressed genes, potential new cell-wall regulators were identified, including two transcription factors of the group VII ethylene response factor family, that could be exploited to fine-tune cell wall degradability. Overall, these findings provide new insights into the regulatory mechanisms by which a major locus influences cell wall degradability, paving the way for its map-based cloning in maize.