Pod indehiscence in common bean is associated with the fine regulation of PvMYB26

Linkage mapping and histological and expression pattern analyses in pods identified PvMYB26 as the best candidate gene for pod indehiscence, mediated by a non-functional abscission layer in the pod.


Supplementary tables
Table S1.Segregation of pod shattering on a subset of the BC4/F2 lines.Phenotyping was performed on four days (19,29 September,13,23 [full ripening] October, 2016), for the identification of totally indehiscent plants and dehiscent plants.Shattering modulation (% of twisting pods per plant) was scored for the dehiscent lines.

Figure S5 .
Figure S5.Schematic representation of the pod anatomy, depicting the main tissues putatively involved in the pod shattering modulation.Right, schematic representation of a cross-section of a pod in common bean.V, valve; DoS, dorsal suture; VeS, ventral suture; VS, ventral sheath; DS, dorsal sheath; AZ, abscission zone; LVL, internal lignified valve layer; VB, vascular bundles.

Figure S6 .
Figure S6.Densities of the 19,420 SNP markers identified within a 1-Mb window size using genotyping by sequencing.

Figure S7 .
Figure S7.Genome-wide association study for occurrence of pod shattering on the IL population.Left: Manhattan plots to show the associations between the SNP markers and the phenotypic scores for pod shattering.Dashed red line, fixed threshold of significance for the 19,420 SNP markers physically distributed across the 11 common bean chromosomes.Right: QQplots of the distributions of the observed p values compared to the expected distribution.The following traits were mapped: (a) 'Sh y/n' (only clear phenotypic data, no intermediate phenotypes);

Figure S9 .
Figure S9.Decay of the linkage disequilibrium within the major locus for pod indehiscence qPD5.1-Pv.Intrachromosome distance is expressed in Kilobases (Kb).

Figure S10 .
Figure S10.Gene expression (RNA-seq) in common bean pods for candidate genes at the major locus for pod indehiscence.Data are presented for five and ten old-days pods in a panel of wild and domesticated Mesoamerican and Andean genotypes.MW, Mesoamerican wild; MD, Mesoamerican domesticated; AW, Andean wild; AD_Snap, Andean snap beans (domesticated); AD, Andean dry beans (domesticated).Gene expression for candidate genes: a, Phvul.005G156700;b, Phvul.005G156900;c, Phvul.005G157000;d, Phvul.005G157600;e, Phvul.005G163800;f, Phvul.005G163901;g, Phvul.005G164800.The vertical dashed line separates the expression data for five old-days pods (left box) and for ten old-days pods (right box) in each panel.Data are means across TMM (Trimmed Means of M-values) ±standard deviation of the biological replicates.

Figure S11 .
Figure S11.Physical positions of the putative structural genes for lignin biosynthesis on the common bean chromosomes.Red, left, position of the major locus qPD5.1-Pv on chromosome Pv05; green, right, genomic locations of the genes with putative functions in lignin biosynthesis.Centromeric regions are indicated for each of the chromosomes.Maps constructed using the online tool MapGene2Chrom Web2 (http://mg2c.iask.in/mg2c_v2.1/).

Figure S12 .
Figure S12.Gene expression by qRT-PCR for Phvul.005G157600 for the pods of the three highly dehiscent ILs (as indicated, blue) and for the indehiscent pods of variety Midas (MIDAS, red) across the eight developmental stages from 2 DAP to 13 DAP.Mean pod expression is shown.*, p <0.05; **, p <0.01; versus MIDAS.Data are means ±standard deviation of the biological replicates (n = 3 for each highly dehiscent line; n=4 for Midas).T.test for detection of significant differences, homoscedastic, two tails.

Figure S13 .
Figure S13.Structure of the GBS library.Samples-specific barcode, pool-specific Illumina index and P5/P7 annealing sites are indicated.

Phenotypic description 0
Extremely indehiscent pods that do not open along the sutures (extremely indehiscent plant) 1 Pods that hardly open along the sutures (putative indehiscent plant) 2 Pods that can be opened along the sutures (putative dehiscent plant) 3 Extremely dehiscent pods that open easily, with a snap when subjected to pressure (extremely dehiscent plant)

Table S4 . Differential gene expression by qRT-PCR of the target candidate genes at the major locus qPD5.1- Pv for pod indehiscence.
Comparisons as fold-changes (shattering/ indehiscent) between the pods of the three high shattering lines, 232B, 244A/1A and 038B/2A2 (both individually and combined), and the indehiscent variety Midas across the different developmental stages (DAP).The best candidate Phvul.005G157600(orthologue to AtMYB26) is reported first, followed by the other target candidates according to genomic location.Significance: yellow shading, p ≤0.05; red shading, ≤0.01.

Table S5 . Orthologous genes putatively involved in pathways associated to pod shattering modulation across different species.
Correlation with lignin pattern deposition in the sclerenchyma of pod valves and pod shattering modulation by promoting torsion of dried pod walls(G.max;Funatsuki et al.,   2014); the presence of a putative causal polymorphism in the gene is associated with low pod shattering susceptibility(P.vulgaris;Parker et al., 2020), selection signatures (P.vulgaris; Schmutz et al., 2014) and expression data (P.vulgaris; this research) PvIND (P.vulgaris), AtIND (A. thaliana) Valve margin lignification and silique shattering modulation (A.thaliana; Liljegren et al., 2004); co-mapping with the St locus for the presence of pod string (P.vulgaris; Koinange et al., 1996) and expression data (P.vulgaris; this research)

Table S6 . Sequences of the single-stranded oligos for the adapters used for GBS library preparation
. _F, _R, forward and reverse oligo sequences of the 24 barcoded-adapters are provided.In bold, barcodes are highlighted.

Table S7 . Sequences of the primers used for the amplification, indexing and quantification of the GBS library.
In bold, Illumina indexes are highlighted.