Genome-wide identification and characterization of germin-like protein family in Brassica juncea reveals their role against biotic stress.
Provisional — an automated or curator check raised a specific concern and points reviewers here. This is NOT a final assessment and not a determination about the authors.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- 🔴A deviation arose in the data or preprocessing
- 🔴A deviation was attributed to the published material
- 🔴Reported values were not (fully) derivable from the shared data
- 🔴The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🔴Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Plant gene-family identification paper; code = third-party TBtools (P16). Genome pinned to BRAD Braju_tum_V2.0 (BjuT84V2); the paper's BjuVA/BjuVB gene IDs resolve via the BRAD per-gene API (coords match the supplement exactly). Reproduced ProtParam (==Biopython) + cupin_1 PF00190 (==hmmer) on 98/101 family proteins on «our HPC» («job»). NOT a clean 1:1: the deposited Supplementary Table 1 physicochemical values do NOT map to their gene IDs -- only 17/98 protein lengths (15/98 MW, 18/98 pI) match the gene printed beside them. The numbers are genuine ProtParam outputs but row-misaligned (demonstrable -1 row shift across blocks, e.g. BjuGLP03-14 where gene N's real value == gene N-1's printed value), and ~9 large values (1800-1900 aa / 196-205 kDa) match no listed locus -> DATA-INTEGRITY / possible-fabrication flag (provisional, for human audit). What DOES reproduce: cupin_1 membership 98/98 (31 double) = exact; chromosomal max/min (B02=9, B08=1) = matches 'Chr02=9/Chr08=1'. Count discrepancy: text says 102 GLPs, the table lists 101 (40+/61- vs 41+/61-). NOT attempted: full BLASTp re-identification (no bulk proteome in mirror) and the RNA-seq DESeq2 expression pipeline on PRJNA860050 (heavy 12-library, the hard 20%).
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessment Score 58assessed: 2026-06-16 ⛓ c5707e6767d9
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusGermin-like proteins (GLPs) were poorly characterized in Brassica juncea and its progenitor species; this study aimed to identify and characterize the GLP family in B. juncea, B. nigra, and B. rapa and to determine candidate B. juncea GLP genes conferring resistance against biotic stress, using Alternaria leaf spot disease (A. alternata) as the model biotic stress.
- ★ 102 GLPs were identified in B. juncea, 51 in B. nigra, and 48 in B. rapa via genome-wide in-silico analysis finding
- ★ This is the first identification and characterization of the GLP family in B. juncea and its parental species B. nigra and B. rapa resource
- ★ Ten differentially expressed BjuGLPs were validated by RT-qPCR: 5 upregulated (BjuGLP06, BjuGLP23, BjuGLP34, BjuGLP70, BjuGLP97) and 5 downregulated (BjuGLP04, BjuGLP33, BjuGLP71, BjuGLP72, BjuGLP91) under A. alternata infection finding
- ★ In-silico RNA-seq expression trends of B. juncea GLPs were confirmed by in-vitro RT-qPCR, indicating GLPs contribute to defense against Alternaria leaf spot finding
- ★ Phylogenetic analysis of GLPs across B. juncea, B. nigra, B. rapa and A. thaliana revealed 4 major clades divided into 21 subclades/members finding
- Synteny analysis showed GLPs are conserved and transferred from both progenitor genomes into B. juncea, with gene duplication events observed in B. juncea and B. nigra finding
- All GLPs possess a conserved cupin_1 domain and conserved germin box motif, with 20 conserved motifs predicted across the three species finding
- Ka/Ks analysis revealed mixed purifying and positive selection acting on GLP gene pairs in B. juncea and B. nigra finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Genome-wide in-silico GLP identification (orthology to AtGLPs) | B. juncea, B. nigra, B. rapa genomes | none | number and physiochemical properties of GLP genes/proteins | — |
| Phylogenetic analysis | B. juncea, B. nigra, B. rapa, A. thaliana GLPs | none | clade/subclade structure | IQ-TREE v2.1.2 (1000 bootstrap) |
| Motif/domain/gene structure analysis | BjuGLPs, BniGLPs, BraGLPs | none | conserved motifs, cupin_1 domain, exon-intron structure | TBtools v2.210, MEME (E=1e-5), Pfam |
| Synteny analysis | B. juncea (AABB), B. nigra (BB), B. rapa (AA) | none | collinear/syntenic gene relationships | MCScanX in TBtools v2.210 (E-value 1e-10) |
| Cis-regulatory element (CRE) analysis | BjuGLP/BniGLP/BraGLP promoters (1500 bp upstream) | none | CRE types in promoters | PlantCARE |
| Ka/Ks selection analysis | GLP paralog pairs in B. juncea and B. nigra | none | Ka/Ks ratios (selection pressure) | — |
| In-silico RNA-Seq differential expression analysis | B. juncea under Alternaria brassicae infection (2 DPI, 4 DPI) | Alternaria infection | differentially expressed GLPs (|log2FC|=1) | Galaxy (usegalaxy.org); SRA PRJNA860050 |
| RT-qPCR expression validation | Super raya (B. juncea) plants, diseased vs control | A. alternata inoculation at 2-3 leaf stage | relative expression of 10 selected BjuGLPs (ref gene UBQ9) | — |
- – 102 GLPs identified in B. juncea, 51 in B. nigra, 48 in B. rapa 102/51/48
- ▲ 5 BjuGLPs upregulated (BjuGLP06, BjuGLP23, BjuGLP34, BjuGLP70, BjuGLP97) under A. alternata infection ≥2-fold
- ▼ 5 BjuGLPs downregulated (BjuGLP04, BjuGLP33, BjuGLP71, BjuGLP72, BjuGLP91) under A. alternata infection ≥2-fold
- – 25 GLPs at 2 DPI and 30 GLPs at 4 DPI identified as differentially expressed in RNA-seq |log2FC|=1
- – Phylogenetic tree resolved 4 major clades (GLP1, GLP3, GLP4, GLP5) and 21 subclades 4 clades / 21 members
- – Highest number of GLPs predicted in extracellular space (66 BjuGLPs, 31 BniGLPs, 34 BraGLPs)
- – In-silico expression trends confirmed in in-vitro RT-qPCR for all 10 validated B. juncea GLPs
- – No duplicated GLP genes found in B. rapa; duplications found in B. nigra and B. juncea
- count 102 GLPs in B. juncea, 51 in B. nigra, 48 in B. rapa (genome-wide GLP identification)
- fold_change 2-fold (|log2FC| = 1) (threshold for DEG selection and RT-qPCR validation)
- count 25 DEG GLPs at 2 DPI, 30 DEG GLPs at 4 DPI (RNA-seq differential expression (45 detected at 2 DPI, 42 at 4 DPI))
- other isoelectric point 4.77 to 10.2 (physiochemical properties of GLPs)
- other protein length 160–2000 aa; molecular weight 12–206 kDa (physiochemical properties)
- count 47 distinct CRE types (light 7, auxin 6, defense/stress 2) (cis-regulatory element analysis of promoters)
- count B. juncea strand: 41 positive, 61 negative (45% positive, 55% negative) (strand position analysis)
- other up to 47% yield losses worldwide (Alternaria leaf spot disease impact)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This genome-wide study combined in-silico bioinformatics analyses (physiochemical characterization, maximum-likelihood phylogenetics, synteny, cis-regulatory element prediction, and Ka/Ks evolutionary analysis) with RNA-Seq differential expression screening to identify candidate GLP genes in Brassica juncea and its progenitor species responsive to biotic stress. Differentially expressed genes were filtered by a 2-fold change threshold (|log2FC| ≥ 1) from publicly available RNA-Seq data processed via Galaxy, and 10 representative genes were selected for RT-qPCR validation in plants inoculated with Alternaria alternata. Results were reported primarily as descriptive gene-family characterizations, fold-change categories, and qualitative expression trends (upregulated/downregulated), without explicit reporting of p-values or dispersion measures for most analyses.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Maximum-likelihood phylogenetic inference (IQ-TREE) with bootstrap resampling | Phylogenetic tree of BjuGLPs, BniGLPs, BraGLPs, and AtGLPs (Fig. 4) | 102 BjuGLPs + 51 BniGLPs + 48 BraGLPs + AtGLPs (exact AtGLP n not stated) | not stated |
| Log2 fold-change threshold filter (|log2FC| ≥ 1, i.e., ≥2-fold change) for RNA-Seq differential expression | In-silico expression analysis of BjuGLPs at 2 DPI and 4 DPI (PRJNA860050, Fig. 7 heat map) | 45 BjuGLPs detected at 2 DPI; 42 at 4 DPI; no biological replicate n stated | not stated |
| Ka/Ks (nonsynonymous-to-synonymous substitution rate) ratio analysis | Evolutionary selection analysis of duplicated BjuGLP and BniGLP gene pairs | 6 BjuGLP pairs; multiple BniGLP pairs (exact n not stated) | not stated |
| MEME motif discovery (E-value threshold 1e-5) | Conserved motif identification in BjuGLPs, BniGLPs, BraGLPs (Figs. 1–3) | 102 BjuGLPs, 51 BniGLPs, 48 BraGLPs | not stated |
| MCScanX collinearity/synteny analysis (E-value cut-off 1e-10; similarity threshold >90%) | Synteny analysis between B. juncea, B. nigra, and B. rapa (Fig. 5) | Whole-genome gene complements of three species | not stated |
| RT-qPCR relative quantification (statistical test not explicitly stated in available text) | Validation of 10 selected BjuGLPs in A. alternata-inoculated vs. control Super raya plants | 6 RNA samples used (2 of 8 excluded due to faint bands); exact biological replicate n not stated | not stated |
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Differentially expressed BjuGLPs were identified using a fold-change threshold (|log2FC| ≥ 1) without a reported statistical test or false discovery rate correction↳ Could also: A negative binomial model-based DEG caller such as DESeq2 or edgeR, with Benjamini-Hochberg FDR correction, could also have been applied to the same Galaxy-processed read counts — Combining a fold-change threshold with an adjusted p-value (e.g., FDR < 0.05) controls the expected proportion of false positives across the family of ~100 tested genes, which is a widely recommended practice when the number of simultaneous comparisons is large
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RT-qPCR results were described qualitatively as 'upregulated' or 'downregulated' without a stated statistical test or measure of dispersion across biological replicates↳ Could also: A Student's t-test or one-way ANOVA (with Tukey or Bonferroni post-hoc correction for multiple genes) on ΔΔCt values across biological replicates, reported with mean ± SD and individual replicate n, could also be used — Formal testing and dispersion reporting allow readers to assess whether observed expression differences exceed random biological variation and are standard for RT-qPCR validation studies in plant biology
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Phylogenetic confidence was assessed using 1000 bootstrap replicates with IQ-TREE (maximum-likelihood framework)↳ Could also: Bayesian inference (e.g., MrBayes or BEAST) with posterior probability support could also be applied to the same aligned sequences — Bayesian posterior probabilities and ML bootstrap values both quantify node support but differ in interpretation; reporting both or choosing one with an explicit model-selection step (e.g., ModelTest-NG) is a common complementary approach in plant gene-family studies
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Ka/Ks ratios for duplicated gene pairs were interpreted descriptively by comparing the ratio to thresholds (< 1, = 1, > 1)↳ Could also: Branch-site or site models in PAML (codeml) could also be used to test statistically whether positive selection (Ka/Ks > 1) is significant via a likelihood ratio test — A likelihood ratio test against a null model adds a formal probability statement to the selective-pressure inference, complementing the ratio-threshold description used here
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Cis-regulatory elements were identified in 1500 bp upstream sequences using PlantCARE and counted descriptively↳ Could also: Motif enrichment analysis (e.g., AME from the MEME suite or Homer) comparing GLP promoters to a background set of non-GLP promoters could also be applied — Enrichment testing identifies which CRE types are over-represented specifically in GLP promoters relative to genome-wide expectation, adding statistical context to the observed CRE catalogue
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Two RNA samples (JD3, JC4) were excluded from RT-qPCR analysis due to faint RNA bands, reducing the effective n without a stated imputation or sensitivity analysis↳ Could also: Re-extraction of those two samples, or a sensitivity analysis comparing results with and without the excluded samples, could also be performed — Documenting whether conclusions hold after exclusion of low-quality samples, or attempting re-extraction, helps readers assess whether the exclusion affected the directional findings
Citation network
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Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
Downstream reach in the literature
1 downstream papers · 1 datasetsHow widely the datasets deposited by this paper are reused across the whole literature (Europe PMC), beyond our assessed set. This is a factual dependency map — reusing a public dataset is normal, good science. It is not a judgement on the downstream papers; the only verdict here is this paper's own, with its cited rationale.
- Transcriptome Analysis of the Necrotrophic Pathogen... 2023 · 6 cites
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-41327045
Paper: Genome-wide identification and characterization of germin-like protein (GLP) family in Brassica juncea reveals their role against biotic stress. Abdul Sattar G, Rana IA, Atif RM, Niazi AK. BMC Plant Biol 2025. PMC12763953 · DOI 10.1186/s12870-025-07805-y.
"Code" (P16): TBtools v2.210 (third-party GUI) + ExPASy ProtParam, CDD/Pfam, MEME v5.5.0, IQ-TREE v2.1.2, MCScanX, BUSCA, PlantCARE, Galaxy (HISAT2/StringTie/ DESeq2). No authors' own scripts — applying standard tools to the paper's data is P16-valid and reproducible.
Genome (pinned): BRAD V3.0 (http://brassicadb.cn/). B. juncea assembly
Braju_tum_V2.0 (server internal name BjuT84V2; var. tumida T84-66). Gene-ID
format BjuVA##G##### (AA subgenome), BjuVB##G##### (BB subgenome), Contig##G#####.
The BRAD download mirror («ip»:82) only ships juncea V1.1/V1.5; the V2
proteins are served per-gene by the API
http://«ip»:8001/api/search-gene-test/?geneID=<ID> (returns protein_fa,
cds_fa, coords). NOTE: paper Methods says "Bju_tum_V2"; the gene IDs are the
Varuna-style BjuV* tags but the API confirms species=BjuT84V2/Braju_tum_V2.0,
coordinates matching the supplement exactly → genome correctly identified.
In scope (pipeline-derived, attempted)
| ID | Result | Source | Pipeline | Plan |
|---|---|---|---|---|
| C1 | Physicochemical properties of the BjuGLP family (protein length PL, MW kDa, pI) per gene | Suppl. Table 1 (MOESM2 .docx), 101 rows BjuGLP01–101 | ExPASy ProtParam | Fetch each gene's protein from BRAD API; recompute length+MW+pI with Biopython ProteinAnalysis (== ExPASy Bjellqvist/average-mass); compare per gene |
| C2 | Family membership criterion: cupin_1 (PF00190) domain present in all members | Methods + Results | CDD/Pfam (hmmer) | hmmsearch PF00190 against the 101 proteins; count members with domain; single vs double cupin |
| C3 | Chromosomal + strand distribution | Results: "Chr02=9 highest, Chr08=1 lowest"; strand 41+/61− | TBtools gene-location | Count per (sub)chromosome and strand from coords; compare |
| C4 | Family size = 102 BjuGLPs (abstract/text) | Abstract/Results | BLASTp(AtGLP×36)+CDD | Consistency check vs Suppl. Table 1 (which lists 101); full re-BLAST deferred (bulk V2 proteome not in mirror) — method-sensitive 20% |
Out of scope / not attempted (with reason)
- RNA-seq expression (DESeq2 on PRJNA860050) — heavy multi-sample align/quant/DE (12 libraries, HISAT2+StringTie+DESeq2 via Galaxy). The hard 20%; deferred for budget. Reported numbers (45/42 genes detected, 25/30 DEGs) not regenerated.
- Phylogeny (IQ-TREE), conserved motifs (MEME, "20 motifs"), gene structure, synteny/MCScanX, cis-elements (PlantCARE) — figure-level/visual, no single pinnable scalar to grade 1:1; not attempted.
- RT-qPCR validation — wet-lab, out of scope.
Auditability notes / possible-fabrication flags (provisional)
- F1 (count): Abstract/text say 102 BjuGLPs; Suppl. Table 1 enumerates only 101 (BjuGLP01–BjuGLP101). Strand split in the table is 40+/61− (=101); the paper states 41+/61− (=102). Off-by-one between prose and the deposited table.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
The deterministic, scriptable parts of the pipeline reproduce cleanly — cupin_1/PF00190 membership is exact (98/98, 31 double) and chromosomal max/min match (Chr02=9, Chr08=1) — and the method is validated by exact anchors (BjuGLP01/02 length/MW/pI 1:1), so input-data identity and comparability are sound. The defect is on the authors' side: Suppl. Table 1's physicochemical columns are demonstrably row-shifted against the gene IDs (only ~17/98 match as printed) and include ~9 large orphan values (up to 1897 aa) that map to no listed gene, plus a 102-vs-101 family-count discrepancy. The underlying numbers are genuine ProtParam outputs but mis-tabulated, making per-gene lookups unreliable and triggering a possible-fabrication flag. The core family/domain conclusion survives, but the characterization table's integrity failure makes this a critical reproduction outcome.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
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