Yield and antiyield genes in common bean (Phaseolus vulgaris L.)

High yield is the primary criterion for the development of new cultivars at the University of Guelph common bean breeding program. As a complex trait, yield is encoded by a number of genes contributing minor effects while also being significantly affected by environmental factors. Genes that increase yield with fixed resources have their effects by increasing input use efficiency. When suppressed, the BnMicEmUP gene has a positive effect on seed production in Arabidopsis. Preliminary work has shown that ortholog of this gene (Phvul.009G190100) exists in common bean, and its expression was negatively correlated with yield in a field test of 10 navy bean cultivars with different yield potentials. The aim of this research was to characterize the Phvul.009G190100 gene and to develop gene‐based marker(s) to test for alleles associated with high yield in common bean. A database search identified a second yield‐related gene (Phvul.009G202100) on the same chromosome (Pv09), which is a homolog to Phvul.009G190100. Both genes contain a DUF1118 protein domain, which has the molecular characteristics of a basic leucine zipper (bZIP) transcription factor, based on in silico analysis. Temperature switch polymerase chain reaction (PCR) markers, which were developed for both genes, were significantly associated with yield and maturity in 42 bean genotypes belonging to different market classes. The work will benefit bean breeding programs by making them more efficient in selecting high yielding cultivars, and it will directly benefit bean producers through accelerated access to new, high yielding cultivars.

High yield is the major objective of most plant breeding programs.
The average yield of Canadian beans has increased by 59% from an average of 1,415.8 kg ha −1 during 1961-1970 (10-year average) to 2,250.9 kg ha −1 for period 2009-2018 (10-year average), which is a 1.6%  or 1. 0% (1961-2018) annual increase (FAOSTAT, 2020; available at: http://www.fao.org/faostat/en/#data/QV). Globally, there is renewed interest in increasing the yield potentials of crops to meet demands from population growth that might double by 2050. In addition, there is a concern that shifts to meat-rich diets, increased biofuel production, and climate change will put additional pressure on crops to yield more with the same or decreasing inputs (Tilman et al., 2011). Studies have estimated that yields will need to increase by 2.4% annually, rather than the current average yield increase of approximately 1.2% for the major crops such as corn, soybean, wheat, or rice (Ray et al., 2013).
Common bean improvement programs have been successful in using conventional plant breeding techniques in the development and release of cultivars with greater yield potential (Kelly et al., 1998). The use of molecular markers may permit selection in the absence of selection pressure. However, they have been most effective for relatively simple traits, like disease resistance. The application of markerassisted selection (MAS) for more complex traits, such as yield, has recently shifted to genomic selection approaches (Desta & Ortiz, 2014) that are based on genome-wide association studies (GWAS) and do not require knowledge of the underlying genes that control the trait (Atwell et al., 2010).
As a quantitative trait, yield is encoded by a number of genes, which usually have small effects on trait expression and can be significantly modified by environment. From a crop physiology perspective, a number of morphological characteristics and physiological processes contribute to yield, including flowering time, time to maturity, number of pods per plant, number of seeds per pod, and seed weight. To achieve maximum yield, all these components should be well-balanced considering that grain yield can be influenced by altering any one of them (Chapman et al., 2003). In addition, because the yield loss is usually caused by biotic and abiotic stresses (Fageria, 2002;Lynch, 2007), an improvement of resistance to these stressors should be included in breeding for high yielding cultivars. For example, the improvement of disease resistance has led to extended duration of photosynthesis in many crops but selection for high yield "has not resulted in genetic increase in photosynthesis per unit leaf area" (Richards, 2000).
Because yield determinations are labor-intensive and dependent on the environment, it would be beneficial to have a constant parameter correlated with the trait that can be used as a selection tool. Genes that increase yield with fixed resources have their effects by increasing input use efficiencies. There are some promising leads with genes associated with yield in a number of crops (Tian et al., 2017;Xu et al., 2018). Two mutants, reduced induction of nonphotochemical quenching 1 (riq1) and 2 (riq2), were identified in Arabidopsis (Yokoyama et al., 2016). They are encoded by paralogous AT1G74730 (RIQ2) and AT5G08050 (RIQ1) genes, which code for proteins of unknown function (DUF1118) and "affect the organization of lightharvesting complex II and grana stacking in Arabidopsis." The BnMicEmUP gene, homologous to AT1G74730, was identified in a screen of upregulated genes in canola microspore cultures . It was shown that in Arabidopsis RNAi transgenics in which BnMicEmUp expression was suppressed, seed production was reduced (Shahmir, 2014). Bioinformatic analyses of the structure of the protein encoded by BnMicEmUP suggest that it is a leucine zipper type transcription factor localized in the chloroplast . A model for a dual targeting of BnMicEnUP protein and its involvement in abscisic acid (ABA)-mediated transcriptional regulation has been proposed. Because of the negative correlation between the gene expression and yield related characteristics, this gene was characterized as an antiyield gene.
A homolog to this gene was identified in the common bean genome sequence annotation v1.0 (Phvul.009G190100) on chromosome Pv09. Preliminary results identified that the level of expression of this gene was negatively correlated with yield potentials in a field test of 10 navy bean cultivars (Qi, 2015). This observation is consistent with the suggestion that Phvul.009G190100 can also be characterized as an antiyield gene. The objectives of this research were to characterize in silico orthologs of AT1G74730 (antiyield) and its homolog, AT5G08050 genes, develop gene-based markers for these genes and test their usefulness for predicting the yield potential of beans from different market classes.  (Goodstein et al., 2012). The best matches were analyzed using an ab initio gene prediction and Arabidopsis/common bean/soybean specific gene-finding parameters in Fgenesh (Salamov & Solovyev, 2000).
Gene structure was visualized in Gene Structure Display Server (GSDS; Hu et al., 2015).
Expression data for antiyield genes in Arabidopsis, common bean, and soybean were retrieved from Phytozome.
Extended untranslated regions (UTRs; 1,500-bp upstream flanking sequence and 1,000 bp downstream flaning sequence, respectively) of the genes were downloaded from Phytozome and analyzed in silico for potential 5 0 UTR cis-regulatory sequences using the PlantCARE database (Lescot et al., 2002) and MEME suites 5.2.0 (Multiple Em for Motif Elicitation; Bailey et al., 2009). Drawings of the UTR elementsbased maps were done with MapChart (Voorrips, 2002).
Final MSA outputs were produced in BoxShade 3.21 at ExPASy (Swiss Institute of Bioinformatics) and edited manually.
Conserved motif and domain scans were performed on several platforms, including Conserved Domain Database (CDD, using default parameters) at NCBI-NIH (Marchler-Bauer et al., 2015), MEME suite 5.2.0 (with searches for 20 motifs of 3-100 amino acids wide), and the MyHits at the SIB (Pagni et al., 2007).
Genomic DNA was isolated from young leaves from the plants grown in a controlled environment room (16-h photoperiod, 22 C) with DNeasy Plant Mini Kit (Qiagen, Canada) following manufacturer's instructions. PCRs were performed in 20-μl volumes containing 1× PCR buffer and 3 mM MgCl 2 (both supplied with enzyme), 0.1 mM each of dNTPs (Invitrogen, Fisher Scientific, Canada), 0.6 U JumpStart Taq DNA Polymerase (Sigma, Burlington, ON, Canada), 0.25 μM each of the forward and reverse primer, and 25 ng of common bean genomic DNA, with a Bio-Rad MyCycler™ Thermal Cycler System (Bio-Rad, Mississauga, Canada). The amplification program consisted of an initial 3-min denaturation step at 94 C, followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 45 s, and extension at 72 C for 1 min, with a final extension at 72 C for 10 min.
The PCR products were separated on 1% (w/v) agarose gel (with ethidium bromide added) in 1× TBE buffer at 100 V for 1-2 h. Single-band PCRs were purified (QIAquick PCR purification kit, Qiagen, Canada; Diffinity RapidTip, Sigma, Canada) and used as a template for in-house sequencing (3500 Series Genetic Analyzer [Applied Biosystems, Thermo-Fisher Scientific-CA] using a BigDye™ Terminator chemistry). Sequences were processed with BioEdit sequence alignment editor (Hall, 1999) and assembled using a CAP3 sequence assembly software (Huang & Madan, 1999). Complete sequences were aligned (Clustal Omega) and searched for single nucleotide polymorphism (SNP) differences among five genotypes in two yield/antiyield genes. Gene sequences were submitted to GenBank under accession numbers MW341245-MW341254.

| Development of yield/antiyield gene-based TSP markers
SNPs identified in AYD1 and AYD2 gene sequences were visualized using a three-primer temperature switch PCR (TSP) technique Tabone et al., 2009). The assay consisted of a pair of a gene (locus)-specific primers (GS) and a nested allele-specific (AS) primer that is designed to assay for SNP. Forward and reverse GS primers were designed with melting temperature between 60 C and 65 C (63 C optimum), PCR product size greater than 400 bp, and located at least 100 bp from the SNP. A single AS primer (forward) was designed with the melting temperature between 43 and 48 C (45 C optimum) with 3 0 end ending at the SNP. A short noncomplementary stretch of 2 bp was added to the 5 0 -tail to increase melting temperature to 53 C. The amplification was completed with a final extension at 72 C for 10 min. The TSP PCR products were separated on a 1% ultra-pure agarose (Invitrogen) gel (with ethidium bromide added) at 100 V in 1× TBE buffer for 1-2 h. The amplicons were visualized on ChemiDoc™ XRS system with the Image Lab™ software (BioRad).

| Testing usefulness of yield/antiyield genebased TSP markers
The usefulness of the TSP markers was tested with 42 bean genotypes from the University of Guelph bean registration performance trials including 14 large seeded color (Andean gene pool) and 28 navy (small seeded, Mesoamerican) beans ( Figure S1). Beans were evalu- Maturity (DM) was reported as the number of days from planting to physiological maturity. Yield (YD) was expressed as seed weight measured for each plot in kg ha −1 and adjusted to 18% moisture content. Harvestability (HR) was determined as standability of plants in plot at maturity using a scale of 1-5 (1, erect plants suitable for combining; 5, prostrate plants not suitable for machine harvesting). A derived trait, yield gain per day (YGD) or economic growth rate (EGR), was calculated from the yield and maturity measurements (YGD = YD/DM).
Average yield gain (kg day −1 ha −1 ) describes the seed weight accumulated per growing day from a unit area for each genotype (Scully & Wallace, 1990).

| Weather conditions during 2014 bean growing season
Daily weather data for the two Ontario locations (Elora and Woodstock) in 2014 were collected at the nearest weather stations (available at: https://climate.weather.gc.ca/; Figure S2). Environmental variation was observed between the experimental locations during the growing season (June-October). However, due to the incomplete weather data for the Woodstock site, direct comparison between the two locations was not possible for the whole bean growing seasons.
In the current annotation of the common bean genome sequence (P. vulgaris v2.1), the antiyield gene AYD1 (Phvul.009G190100) is located close to a yield quantitative traits locus (QTL) SY9.1, while its homolog, gene AYD2 (Phvul.009G202100), is located in the region of the second yield QTL SY9.2 (LIS). Similarly, four antiyield genes in the soybean genome sequence annotation Wm82.a2.v1 are located in the regions of two seed yield (SY) QTL on chromosome Gm04 and eight yield QTL on chromosome Gm06 (SoyBase; Figure 1).
F I G U R E 1 Genome locations (Mb) of yield/antiyield genes AYD1 and AYD2 in common bean, Arabidopsis, and soybean. Arabidopsis chromosomes At1 and At5 are shown as patterned (cross hatch lines) bars, common bean chromosome Pv09 as a black bar, and soybean chromosomes Gm04 and Gm06 are shown as clear bars. Yield/antiyield genes are presented in red (larger font); AYD1 genes are underlined. Genome regions associated with previously identified yield QTL (black bars) in common bean and soybean are indicated (see text for details). [Color figure can be viewed at wileyonlinelibrary.com] 3.1.2 | Gene structure All eight yield/antiyield genes have a similar structures (Figure 2a; Table S2), consisting of two exons and one intron with sizes varying from 84p (Phvul.009G190100) to 1,399 bp (Glyma.06G204200). The only exception is the soybean gene Glyma.04G174400 on chromosome Gm04, which has an additional intron in the 3 0 UTR region. In all three species, Exon 2 in the AYD1 genes is the same size (444 bp), whereas the Exon 1 in the AYD2 gene is longer in Arabidopsis and common bean (153 bp) compared with the two soybean genes (141 bp). However, the AYD2 genes are more variable. Exon 2 is longer in common bean and soybean (423 bp) compared with Arabidopsis (378 bp). The size of Exon 1 is different in all three species (Table S2).
The analysis of the 5 0 UTR sequences (1,500-bp upstream of ATG) identified a moderate degree of divergence in these regions of the eight yield/antiyield genes (36-66% identity). The degree of identity between the 3 0 UTR (1,000 bp from the stop codone) regions ranged from 35% to 52%.
Based on the transcript sequence similarity, the yield/antiyield genes in the three species separate into two groups (Figure 2b). At the level of the coding regions (CDSs), the percent identity between the two groups was 43-45%. However, sequence conservation was higher within each group and ranged from 63% (Arabidopsis and legumes) to 93% (soybean).
3.1.3 | Potential cis-regulatory elements in structurally different upstream regions of yield/ antiyield genes An in silico analysis in PlantCARE database of the promoter regions (1,500-bp upstream of ATG) of the AYD1 and AYD2 genes in Arabidopsis, common bean, and soybean genomes identified 69 potential cis-regulatory elements. In particular, the analysis of the upstream sequence of yield/antiyield genes identified core promoter elements, including CAAT and TATA boxes, as well as a large number of light responsive elements (27), elements associated with defense and stress responsiveness (7), hormonal responsiveness (9), and tissue-specific expression (4). However, the functions of a significant number (12) of predicted cis-regulatory elements are unknown (Table S3).
Only six of the regulatory elements were found in all eight genes.
In addition to the two core elements (TATA and CAAT boxes), they include a G-box (light responsiveness element), a TC-rich repeat (for defense and stress responsiveness), an ABRE box (ABA response), and Unnamed_4 (unknown function). Fifty-five elements were identified in AYD1 genes, and 50 regulatory elements were predicted in 5 0 UTR sequences of AYD2 genes.
Forty-five promoter elements were predicted for the common bean yield/antiyield genes: 21 were common, and 12 were specific for the AYD1 (Phvul.009G190100) and AYD2 (Phvul.009G202100) genes, respectively (Table S3; Figure 3). A few elements were unique to the common bean genes. They include a MYB binding site involved in light responsiveness (MRE), which was present in both genes, as well as four cis-regulatory elements that were specific for AYD1 gene

| Yield/antiyield proteins
Based on the protein similarities, the yield/antiyield gene homologs in three species separated into two groups that corresponded with the groupings based on transcript sequence similarities. There was 26-33% amino acid identity between the groups and 65-94% identity within the groups. The protein encoded by the common bean AYD1

| Development of yield/antiyield gene-based TSP markers
The two types of yield/antiyield genes were sequenced in five common bean genotypes, including two small seeded Mesoamerican beans (white cultivars Lightning and T9905) and three large seeded Andean genotypes (Dynasty [DRK], Yeti [WK], and ACUG 13-C1 [cranberry]).

| AYD1 gene (Phvul.009G190100) sequence
In total, 2,807 bp of the AYD1 genes (Phvul.001G190100) in five bean genotypes were sequenced. There were no differences in the coding and intronic sequences for this gene among the analyzed genotypes, and they were identical to the Phvul.009G190100 sequence in the reference genome (G19833, Andean gene pool). The only differences that were detected were three single base substitutions in the untranslated regions. A single T to G change (at position 839 nt in the reference) was identified in 5 0 UTR region of the two Mesoamerican white beans compared with the reference G19833 and other three Andean genotypes (Table 1). Two single base changes were identified in the 3 0 UTR: an A to T change in the white bean T9905 (2,558 nt) and a T to C change in the cranberry bean ACUG 13-C1 (2,599 nt).

| AYD2 gene (Phvul.009G202100) sequence
Sections, 3,130 bp in length, of the AYD2 genes (Phvul.001G202100) were sequenced in five bean genotypes and compared with the reference genome (G19833, Andean gene pool). Fourteen single base changes were identified in the AYD2 gene sequences. Eight of the sequence differences were consistently found between Andean and Mesoamerican beans (one in 5 0 UTR, one in Exon 1 and six in 3 0 UTR).
A single base change (Exon 1) was found in all five sequenced genotypes when compared with the reference G19833 sequence. Five additional changes were identified only in one of the two white genotypes (one in Lighthouse [5 0 UTR] and four in T9905 [two in intron, one in Exon 2 and one in 5 0 UTR]) and therefore not specific to the Mesoamerican gene pool ( Table 1).
Three of these substitutions were identified within CDSs of the AYD2 gene. A single C to T change (2,916 nt in G19833) in Exon 2 of the AYD2 gene in the white genotype T9905 resulted in a conservative codon change (ACC to ACT) for threonine (T, Thr) at position 61 but did not change AYD2 protein in this genotype. However, two T to A changes that were observed in Exon 1 could affect the F I G U R E 3 The locations of potential cis-acting regulatory elements identified in the 5 0 regulatory sequences (5 0 UTR) of common bean yield/antiyield genes Phvul.009G190100 (AYD1, left) and Phvul.009G202100 (AYD2, right) using the PlantCARE database (available at: http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/) properties of the proteins in the analyzed beans. Variation at position 1,844 nt (position in G19833) that was found in Exon 1 of the five  (Table 1 and Figure S4). These substitutions resulted in slight changes in protein properties in analyzed beans compared with the reference G19833 (Table S5).

| Yield/antiyield gene-based TSP markers
A gel-based TSP marker (AYD1m) was developed on the basis of the single T to G substitution identified in the 5 0 UTR (position −871 in G19833) of the Phvul.009G190100 (AYD1) gene sequence. The AYD1m PCR product of 497 bp was amplified from sequences with the reference (G19833) allele (T) and colored, large-seeded genotypes (Table 2), whereas the alternate allele (G), resulting in a 340-bp fragment (SNP), was observed in the navy beans ( Figure 5).
The second TSP marker (AYD2m) was developed for the T to A change identified in Exon 1 (position 35 in G19833) of the AYD2 gene (Phvul.009G202100). The marker was developed for the T allele observed in the reference (G19833) genotype. The AYD2m PCR F I G U R E 4 Comparison of yield/antiyield proteins in common bean, Arabidopsis, and soybean. Multiple sequence alignment for AYD1 (AT1G74730, Phvul.009G190100, Glyma.04G174400, and Glyma.06G190300) and AYD2 (AT5G08050, Phvul.009G202100, Glyma.04G161600, and Glyma.06G204200) proteins was performed in Clustal Omega (EMBL-EBI) and BoxShade (ExPASy, SIB product of 255 bp (T allele) was identified in reference (G19833) and large seeded colored genotypes, whereas the 407-bp amplicon (A allele, SNP) was present in the navy beans ( Figure 5).

| Validation of yield/antiyield gene-based TSP markers
As a part of Ontario bean registration trials, a set of 42 diverse bean genotypes were evaluated for yield and a number of yield-related traits in trials at two locations (Elora and Woodstock) in 2014. Significant variation was identified for all analyzed traits in navy and colored bean genotypes. For example, yield (averaged over two locations) in navy beans ranged from 2,650 (OAC Thunder) to 3,609 kg ha −1 (Lighthouse) ( Table S6) and in large seeded colored beans from 2,119 (ACUG 12-D1) to 3,111 kg ha −1 (OAC Inferno) ( Table S7).

Amplification of both yield/antiyield gene-based TSP markers
was successful in most of the analyzed genotypes (except AYD1m in genotype ACUG 14-2 and AYD2m in three genotypes [HMS Medalist, ACUG 14-2, and ACUG 12-D1]). In marker AYD1m (AYD1 gene, T A B L E 1 Sequence polymorphisms in yield/antiyield genes identified between Andean and Mesoamerican beans Phvul.009G190100 Phvul.009G190100), G allele (340 bp, T to G mutation) was associated with a high yield (Mesoamerican, navy beans), whereas the T allele (497 bp, wild type G19833) was linked to a low yield (Andean, large seeded colored beans). In AYD2m (AYD2 gene, Phvul.009G202100) marker, the T allele (255 bp, wild type G19833) was associated with a low yield (large seeded colored beans, Andean gene pool), and the A allele (407 bp, mutation T to A) was connected to a high yield (navy beans, Mesoamerican gene pool) (Table S8).
Both TSP markers were significantly associated with yield, maturity, YGD (derived from yield and maturity measurements), and HR (Table 3).
Significant correlations were identified between analyzed traits as well as traits and yield/antiyield gene-based TSP markers (Table 4).
Yield was negatively correlated with HR (a lower value indicates better standability) but was in positive association with maturity. There was negative correlation between two TSP markers, and they showed opposite associations with the analyzed traits. The AYD1m (AYD1 gene, Phvul.009G190100) marker was positively correlated with HR but negatively with maturity, yield, and YGD (trait derived from the maturity and yield measurements). Conversely, AYD2m (AYD2 gene, Phvul.009G202100) marker was negatively correlated with HR but was positively association with maturity, yield, and YGD (Table 4). Therefore, Phvul.009G202100, AYD2 gene can be characterized as a yield gene.    Traits: HR, harvestability (scale 1 to 5); DM, days to maturity (days); YD, yield at 18% moisture (kg ha −1 ); YGD, yield gain per day (YGD = YD/DM, kg day −1 ha −1 ). c TSP markers: AYD1m, temperature switch PCR (TSP) marker for the AYD1 gene, Phvul.009G190100; AYD2m, TSP marker for the AYD2 gene, Phvul.009G2020100. improvement programs have successfully used conventional plant breeding techniques to develop and deploy dry bean cultivars with increasing seed yield potentials at a steady rate for several decades (Kelly et al., 1998). However, the development of new high yielding cultivars is challenging because of the complexity of the trait. Yield is controlled by many genes that affect a number of related traits, including maturity, seed number, and seed size. In addition, trait expression is highly influenced by environment. The use of MAS permits selection in absence of selection pressure and has become a common tool in many common bean breeding programs (Kelly et al., 2003;Miklas et al., 2006), especially for disease resistance.

T A B L E 4 Correlations between analyzed traits and yield/antiyield genebased temperature switch PCR (TSP) markers identified in a set of 42 common bean genotypes evaluated in two locations
Arabidopsis transformed with an antisense construct derived from the canola BnMicEmUP gene had enhanced seed production (Shahmir, 2014). Preliminary work showed that an ortholog to BnMicEmUP exists in dry bean, and the expression level of this gene (Phvul.009G190100) was negatively (but not significantly) correlated with the yield in a selected set of 10 navy beans (Qi, 2015). Therefore, both genes can be defined as antiyield genes. Because the genes that increase yield with fixed resources have their effects by increasing input use efficiencies, the purpose of this work was to characterize (in silico) antiyield gene in bean and develop gene-based markers.

| Yield/antiyield genes identified on common bean chromosome Pv09
The rationale for selecting soybean (G. max Wm82.a2.v1) and Arabidopsis (A. thaliana TAIR10) genomes for comparative analyses with common bean (P. vulgaris v2.1) is that, in addition of being a model dicot plant, Arabidopsis mutants of AT1G74730, the homologous gene to BnMicEmUP, were used to characterize the function of BnMicEmUP (Shahmir, 2014;, and soybean is closely related to common bean, and the genomes of the two species are highly syntenic (Galeano et al., 2009;McClean et al., 2010;Reinprecht et al., 2013).
A search for a homologous sequence confirmed the location of the Phvul.009G190100 (AYD1) gene on the chromosome Pv09 but in a slightly different location compared with its placement in the common bean genome v1.0 (Schmutz et al., 2014), which was initially used to identify and characterize it as an antiyield gene (Qi, 2015). Its position would likely change with the future updates to high-quality genome assembly/annotations. The current finding that the region on bean chromosome Pv09 containing the yield/antiyield genes is syntenic with regions on soybean chromosomes Gm04 and Gm06 agrees with previous studies of syntenic relationships between common bean and soybean chromosomes (Galeano et al., 2011;McClean et al., 2010;Reinprecht et al., 2013). In particular, the relationship between AYD1 (Phvul.009g190100) and AYD2 gene (Phvul.009g202100), which are located 1.8 kb apart on chromosome Pv09 in bean, is preserved in the two syntenic regions in soybean chromosomes Gm04 an Gm06, which agrees well with the current understanding that soybean underwent a whole genome duplication event after the divergence of beans and soybeans (Schmutz et al., 2010).
Six genes (two in common bean and four in soybean) examined in the current study are located in regions to which Quantitative Trait Locus/Loci (QTL) associated with yield or yield-related traits were previously mapped. In bean, the physical locations of the yield/ antiyield genes on chromosome Pv09 correspond with the locations of yield QTL previously identified on this chromosome Tar'an et al., 2002). In a more recent QTL study, Mukeshimana et al. (2014) mapped two yield QTL to the same location on chromosome Pv09. In particular, the AYD1 gene Phvul.009G190100 of the current study is close to the yield QTL SY9.1 (yield reduction), whereas its homolog Phvul.009G202100 (AYD2 gene) is located near to the second yield QTL SY9.2 (yield increase), which agrees with their classification as antiyield (Phvul.009G190100) and yield (Phvul.009G202100) genes, respectively. Similarly, the four yield/antiyield genes in the soybean genome sequence (Wm82.a2.v1) are located in the regions of two SY QTL on chromosomes Gm04 (SY23-4 [Guzman et al., 2007] and SY25-1 [Palomeque et al., 2009]) and eight overlapping yield QTL on chromosomes Gm06 (SY5-1 [Renyna & Sneller, 2001], SY7-1 [Orf et al., 1999], SY19-1 and SY19-2 [Zhang et al., 2004], SY21-1 [Reinprecht et al., 2006], SY22-12 [Du et al., 2009] The phylogenetic analysis, which grouped the four proteins encoded by the AYD1 genes, including Arabidopsis At1G74730 (RIQ2), bean Phvul.009G190100, soybean Glyma.04G174400, and Glyma.06G190300, and grouped the four AYD2 proteins, including Arabidopsis At5G08050 (RIQ1), bean Phvul.009G202100, soybean Glyma.04G161600, and Glyma.06G202400 in separate clusters, is in agreement with the previous division of the Arabidopsis RIQ genes into two clusters by Yokoyama et al. (2016). They suggested that the division corresponded to the ancient gene duplication event in Arabidopsis and further proposed that because these genes are unique to land plants, they have a conserved function "to optimize the efficiency of light harvesting and to determine grana structure, which might play an advantageous role in survival under the severe growth conditions on land." The in silico modeling and bioinformatic analyses of the structure of the proteins encoded by canola BnMicEmUP genes and the homologous Arabidopsis gene (AT1G74730) indicate that they are specific leucine zipper type transcription factors called PEND proteins, first described in pea (Terasawa & Sato, 2009). Transgenic Arabidopsis plants, which overexpress AT1G74730 or BnMicEmUP, had lower SY than nontransformed control plants, and transgenics, which had reduced levels of gene expression, had increased yield (Shahmir, 2014).
The yield/antiyield genes are highly expressed in leaves (Phytozome). In all three species, the expression of the AYD1 gene was higher compared with the AYD2 gene. In beans, the expression of the AYD1 gene Phvul.009G190100 was 77% higher compared with its homologous AYD2 gene Phvul.009G202100 (Phytozome). Qi (2015) identified a negative trend between the expression of AYD1 gene Phvul.009G190100 and yield in a study with 10 navy beans evaluated over two locations. However, the correlation was not significant. That study was restricted to a small number of genotypes belonging to the same gene pool (Mesoamerican)/market class (navy). Because of its high expression in leaves and localization in chloroplasts, the previous quantitative PCR (qPCR) study was performed with leaf tissue. However, if seed were used instead of leaf tissue for the qPCRs, different results might have been obtained.
The multiple TATA and CAAT box sequences found upstream of the transcription start site in the yield/antiyield genes are core promoter elements important for accurate initiation of transcription (Kozak, 1981) that may function in promoting high levels of transcription of these genes and may also facilitate differential regulation of their activity in response to different environmental conditions (Doyle & Han, 2001). In particular, the stress responsiveness of the yield/antiyield genes is supported by the occurrence of different types of stress-related elements (ABRE elements) in the promoters of these genes. ABRE cis-acting elements are found in ABA-responsive genes and regulate gene expression in response to abiotic stress (Mundy et al., 1990;Xu et al., 1996). Shahmir and Pauls (2021) found that BnMicEmUP3 overexpression in Arabidopsis increases the sensitivity of seedlings to exogenous ABA. The environmental sensitivity of the yield/antiyield genes is further supported by the occurrence of the heat-shock cis regulatory elements (HSEs) in their promoters. Thermoinducibility of the BnMicEmUP gene homolog of Phvul.009G190100 was observed during embryogenesis in induced B. napus microspore cultures (Shahmir, 2014). In addition, cis elements identified in the promoter of the Phvul.009G190100 gene, such as the TGACG-motif and ERE, suggest that it is also regulated by jasmonic acid and ethylene. Stress-related and many defense-related genes respond to jasmonates (Creelman & Mullet, 1997) and ethylene (Müller & Munné-Bosch, 2015). Interestingly, transcription factors, such as basic leucine zipper (bZIPs) (Shinozaki et al., 2003), with domains similar to the Phvul.009G190100 protein, are involved in regulating the expression of genes with stress-related cis-acting elements in their promoters, making it possible that the yield/antiyield genes are autoregulatory. Shahmir and Pauls (2021) found that Arabidopsis transformed with 35S::BnMicEmUP had enhanced sensitivity to ABA inhibition of seed germination, whereas transgenics with anti-BnMicEmUP were less sensitive to ABA.
The current study also shows that the upstream sequence of Phvul.009G190100 gene contains many light responsive cis elements, such as the G-Box, suggesting that its expression may be regulated by light, which may be related to the chloroplast targeting of the protein.
Interestingly, some regions in the promoter may bind transcription factors in a competitive manner because some of the cis-acting regu-

| Yield/antiyield gene-based TSP markers are associated with yield in common bean
The selection of bean genotypes from both gene pools was an appropriate approach to discriminate sequence variability of the two yield/ antiyield genes. The analyses also showed that the AYD1 gene (Phvul.009G190100) was more conserved than the AYD2 gene. The only SNP that was discovered in the AYD1 gene was a single T to G substitution at the 5 0 UTR (at position −847 nt from ATG in G19833) in navy beans (Mesoamerican) compared with the reference G19833 and three large seeded beans (Andean). The polymorphism is located in a light responsive Box I (TTTCAAA) element and an ERE (ATTCAAA) motif that is involved in ethylene responsiveness of genes in plants. The sequence of the AYD2 gene (Phvul.002G202100) was more variable among five genotype, with eight single nucleotide changes identified including a T to A substitution in Exon 1 that would result in the replacement of a hydrophobic amino acid (isoleucine) found in the large seeded beans and normally buried inside the protein core with a polar amino acid (asparagine) in two white (small seeded) beans. This change would likely affect protein properties.
Most of the lower yielding large seeded colored beans (Andean) had a wild-type (G19833) allele in both yield/antiyield genes. The only exception were two Andean kidney genotypes (ACUG 12-D1 and Yeti), which had mutation in AYD1 gene and wild-type allele in AYD2 gene and lower yield. This study showed that the mutation in both yield/antiyield genes (AYD1, antiyield gene Phvul.009G190100, and AYD2, yield gene Phvul.009G202100) contribute to the high yield in navy beans (Mesoamerican).
The SNPs were visualized using a gel-based TSP approach that consists of a biphasic PCR with three primers, including a set of locusspecific primers and a nested AS (SNP) primer. The original TSP assay Tabone et al., 2009) was slightly modified to use a 1% agarose gel with added ethidium bromide. The sample DNA was dissolved in water, and standard PCR reagents were used in a threeprimer system with only one AS primer. A 10-fold concentration of the AS primer compared with the GS primers was found to be optimal for the yield/antiyield gene-based TSP markers. Both markers provided accurate codominant differentiation of reference and SNP allele.
Wild type (reference G19833) alleles were associated with the low yield of the large seeded colored beans (Andean gene pool), whereas the SNPs were linked to the high yield in navy beans (Mesoamerican gene pool).
The current measurements of yield in beans support many studies that show that this trait is highly variable and that absolute values fluctuate from year to year and among plots established in different locations. However, the positive correlation identified between the yields in two locations in the current study indicates that, although the genotype × environment is often significant, the overall trends and yield ranks of the lines were relatively stable.
The development of functional molecular markers for candidate genes underlying significant QTL for traits of interest can speed up and simplify breeding for traits. For example, Reinprecht et al. (2009) developed gene-based markers for mutations in two ω-3 desaturase genes (Fad3A and Fad3B) that explained significant variation in seed linolenic acid level in soybean seed. In barley, Watt et al. (2020) developed a functional marker based on 9-bp indel at the position −84 in the 5 0 UTR of HvDEP1 gene associated with the length and weight of the seed. This gene encodes a γ-subunit of the heterotrimeric G-protein complex and was identified previously as a candidate gene for a major QTL underlying grain length in barley.
In the current study, both yield/antiyield gene-based markers that are near yield QTL (marker AYD1m for AYD1 gene, Phvul.009G190100) or in yield QTL (marker AYD2m for AYD2 gene, Phvul.009G202100) identified previously on chromosome Pv09 (Mukeshimana et al., 2014) were significantly associated with yield in a set of 42 diverse common bean genotypes. A number of flowering and maturity QTL are also located in the same region on the chromosome Pv09 . In addition, a seed weight QTL (SW9.1) is located in the top arm of this chromosome .
Therefore, the continuation of the research should include evaluation of these traits (in addition to the yield) in replicated trials with a larger set of more diverse bean genotypes.

| CONCLUSIONS
Genes that increase yield with fixed resources have their effects by increasing input use efficiencies. We have discovered genes in bean (Phvul.009G190100 and Phvul.009G202100), which are homologous to a canola antiyield gene that are associated with yield and yieldrelated traits in bean. Both genes are near or in yield QTL regions identified previously in bean.
In silico analyses performed in this work suggested that Phvul.009G190100 and Phvul.009G202100 encode cbZIP transcription factors targeted to chloroplasts involved in stress responses. This study is an example of how examinations of genes with unknown function in crop plants may lead to discoveries that are agronomically relevant and ultimately to the crop improvement.
In addition, simple codominant molecular markers were developed for both yield/antiyield genes based on the TSP technique that can be used by any lab equipped for basic PCR and electrophoresis equipment. Thought from University of Guelph is highly appreciated. We acknowledge the assistance of a number of summer students with field measurements.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest regarding the publication of this study.

AUTHOR CONTRIBUTIONS
Karl Peter Pauls conceptualized this research and supervised the work. Thomas H. Smith designed and conducted field experiments.
Yarmilla Reinprecht conducted lab experiments, analyzed data, and prepared the original manuscript draft. Fariba Shahmir did initial work in canola (PhD dissertation) and assisted in Yanzhou Qi's bioinformatic analysis in bean. Yanzhou Qi did initial work in beans (MSc thesis). All authors were involved in the manuscript revision.

ETHICAL STATEMENT
This research does not contain any studies with human or animal subjects.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon request.