Phenotypic variation, correlations and heritability
A total of 301 SDP lines were successfully characterized for the 17 morphological traits across both replications. The results show a wide and continuous variation for all evaluated traits (see Figure S1 and Table S1). For instance, PL and NSP, two traits related to yield, ranged between 7.1 to 26.4 cm and 2.2 to 8.3 seeds, respectively. The SDP exhibited wide variation for color with green (241 lines), yellow (38), purple (3), green mottled (16) and yellow mottled (2) pods. Pod color measured by the CIE scale exhibited wide variation as well for the L*, a*, and b* vectors. For example, b* varied from -4.38 to 40.8. The H2 estimations for the 16 quantitative traits were high ranging from 0.31 for PSW to 0.91 for PLW (see Table S1).
Correlation analyses indicated significant relationships between many evaluated traits (Figure 1). There were significant and positive correlations among the six pod section traits and a significant negative correlation for PSH/PSW, PSC and PSW. Most of the six pod length variables were significantly correlated except PL/PLC with PLP and PLA. Correlation analyses also revealed significative correlation among section and length traits except in five cases; PSC with PLP, PL and PLC and PSW with PLW and PL/PLC. NSP was significantly correlated with four pod length traits (PLA, PLP, PL and PLC). Finally, the three pod color variables (L*, a*, b*) were also significantly correlated.
Characterization and detection of SNPs
Sequencing of the GBS libraries yielded approximately 418 million reads in total for the 301 SDP lines. About 76.3 % of the reads were successfully aligned to the common bean reference genome, 21.5 % of the reads mapped to more than one locus, and 23.7 % were unmapped. The NGSEP genotyping pipeline produced 346,819 biallelic SNPs in the 11 chromosomes and scaffolds of the reference genome. 32,812 SNPs distributed across the eleven bean chromosomes were retained after filtering parameters (Figure S2). Most of these SNPs were present in coding regions (51.1 %) and represented 46.1% silent mutations, 32.6% missense, 5.4% non-sense and 15.9% prime UTR regions. While intronic and intergenic regions contained 31.2% and 17.8%, respectively. A genome‐wide transition/transversion (Tr/Tv) ratio of 1.17 was observed.
GWAS
SL-GWAS (MLM) revealed 63 significant QTNs, 57 of them grouped in 9 QTI: 7 for pod length, 1 for cross-section, and 1 for pod color. Six QTN showed a single association (Table S2). QTNs were not detected for PCOL and NSP. Interestingly, twenty-eight QTNs for pod morphological traits were detected at the distal end of chromosome Pv01 (45,582,871 to 48,454,962) and 9 QTNs for the color vector b* in the telomere of chromosomes Pv07 (32026373-32413401).
ML-GWAS, using the six multi-locus models in the mrMLM package, revealed 103 QTN (Tables S3, S4, S5). QTNs were not detected for the index PSC. The mrMLM method detected the most associations (37) while FASTmrEMMA detected the fewest associations (21). In all, 14 significant QTNs were found for pod section traits (Table S3) and the QTN number per character ranged from 5 for PSH and only one for PSA and PSW. For pod length traits, 52 QTN were detected (Table S4), with 18 of them identified by at least two different GWAS methods. The number of QTNs ranged from 2 for PL/PLC a and 10 for PLP, PLA, PLC and NSP. These QTNs were mostly located in the telomeric regions of Pv01 and Pv02. Concerning pod color measured by CIE space, a total of 27 QTNs were detected (11 for the vector L*, 9 for a* and 7 for b*) while 10 QTNs were detected for pod color measured visually as a qualitative character (Table S5). These QTNs were mostly located in telomeric regions of chromosomes Pv02 (7) and Pv08 (5). 31 QTNs revealed by ML-GWAS were grouped in eleven QTI. Three QTI were identified by both methods (SL-GWAS and ML-GWAS): Chr01:48090873- 48454962; Chr02: 47302543-47669811; Chr07:32026373-32413401.
The 166 QTNs detected 62 QTL, 23 for pod length, 6 for cross-section, 18 for pod color and 6 for number of seeds per pod as well as 9 QTL associated with multiple characters (Table 2). Most QTL were located on chromosomes Pv02 (12), Pv04 (7), Pv08 (7), and Pv10 (11) (see Figure 2).
Co-location of QTL
Genomic positions for 96 previously reported QTL [10, 12, 14, 15, 19] for pod morphological traits in common bean were examined for overlap with the QTL identified in this work (Figure 2). There were 15 genomic regions where a reported QTL and QTL detected in this study for pod traits overlapped (Table 3; Figure 2). These regions were located on seven chromosomes (Pv01, Pv02, Pv03, Pv04, Pv05, Pv06 Pv08 and Pv11). The beginning of chromosome Pv02 (542087-959169) only co-located QTL for pod color, whereas the other overlapping QTL were associated with pod morphological traits or both morphological and pod color traits.
In silico genome exploration
In silico analysis of the annotated genes underlying the 62 QTL for pod traits revealed 25 candidate genes in 16 QTL (Table 4). There were 12 candidate genes underlying six QTL for pod size traits. Five of these candidate genes were Cytochrome P450 and five were WRKY or MYB transcription factors, proteins involved in multiple processes like responses to biotic and abiotic stresses, development, differentiation, metabolism, defense, and pigment synthesis [23, 24]. Seven of these genes were involved in controlling pod development: Phvul.001G229900, Phvul.001G221500, Phvul.002G016100, Phvul.004G144900, Phvul.006G076800, Phvul.006G077200 and Phvul.010G010200 [25]. Four genes, homologous with genes controlling silique size in A. thalina [26] were detected underlying QTL associated with both size and color traits: Phvul.001G262600 with SPL10 gene in QTL NSPCol01_51; Phvul.002G141800 with FIS2 gene near QTL PodL02_29.1; Phvul.006G074600 with TTG2 gene in QTLPodLSN06_18.4; and Phvul.008G019500 with AML4 gene in QTL NSPCol08_1.7. A DELLA protein gene Phvul.001G230500, controlling various aspects of plant growth and development, including flowering, and pod setting and development [27], is a candidate for QTL PodLCol01_48.4.
Concerning QTL for pod color traits, six candidate genes encoding Cytochrome P450 proteins underlie the QTL PodCol02_2.4, PodCol02_43.6, PodLCol02_47.6 and PodCol10_38.7, and five candidate genes encoding MYB TRANSCRIPTION FACTOR underlie the QTL PodLCol01_48, PodCol07_32 and PodCol08_60.2 (Table 4). Also, Phvul.001G261500 encoding a Flavonoid 3'-hydroxylase is a candidate gene for QTL PodColN01_51, and a cluster of genes encoding Flavone/flavonol 7-O-beta-D-glucoside malonyltransferase bordered QTL PodLCol08_2.7.