Abbreviations: AG: Population A55 x G122; BJ: Population BAT93 x Jalo EEP558; BTS: Black Turtle Soup; CDRK: California Dark Red Kidney; CY: Population CDRK x Yolano; GH: Greenhouse; LG: linkage group; MG: Population Midas x G12873; NIL: Nearly-isogenic lines; RIP: recombinant inbred population; QTL: quantitative trait locus; SDS/PAGE: polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

Karl Sax (1923) presented the first evidence for linkage between genes controlling qualitative traits (seed color or color patterns) and a quantitative trait (seed weight). Subsequent studies have shown that the P locus, which was one of the seed color genes segregating in the common bean (Phaseolus vulgaris) populations used by Sax, is linked to the Phs locus on linkage group D7. The Phs locus codes for phaseolin, the most abundant seed storage protein in common bean. Variation at this locus had previously been correlated with seed weight in one population. Our goal was to examine wether the Phs locus could be a possible candidate locus for the Plocus-linked seed weight QTL identified by Sax. If the association between the Phslocus and seed weight would be observed consistently across a wide range of environments and populations, inside and outside P. vulgaris, this consistency would provide strong circumstantial evidence for our hypothesis. An association between seed weight and phaseolin type was observed through univariate ANOVA's in the five populations that were analyzed here, each of which segregated for seed size and phaseolin type, in addition to two populations analyzed previously. Three populations were grown in more than one season (in a greenhouse) or environment (in the field). Across these seasons or environments, the Phs locus remained consistently associated with seed weight. Depending on the population or the environment, the Phs locus accounted for 18-33% of total phenotypic variation for seed weight. Mapping on the common bean linkage map of probes linked to seed weight QTLs in mung bean or cowpea showed that the QTL associated with the Phs locus appeared to be conserved in mungbean, whereas results were inconclusive for cowpea. Other QTLs for seed weight in the three species appeared not to be conserved. The phaseolin locus is an attractive experimental system to investigate the molecular basis of quantitative variation based onextensive amount of information available on genetic diversity and molecular basis for gene expression at this locus.

INTRODUCTION

In his ground breaking 1923 paper, Karl Sax proposed a method to locate and enumerate quantitative trait loci (QTLs) on a linkage map through association of the trait with qualitative trait (marker) loci. Specifically, he was able to identify QTLs for seed weight in common bean through associations between seed weight (a quantitative trait showing continuous segregation) and seed color or color patterns (qualitative traits showing a discontinuous segregation).

One of the qualitative traits considered by Sax (1923) was seed color, colored vs. whie corresponding to the presence vs. absence, respectively, of anthocyanins (Feenstra 1960). The major seed weight QTL identified by Sax was associated with seed color. The P locus, which controls seed color, has recently been linked to the Phs locus on linkage group D7 at a distance of approximately 10 cM (Gepts et al. 1993).

The Phs locus codes for phaseolin, the major globulin storage protein, which constitutes about 35-50% of the total seed nitrogen in common bean seeds (Ma and Bliss, 1978). Two major and several minor phaseolin types have been identified on the basis of their banding patterns in one-dimensional SDS polyacrylamide gels (reviewed by Osborn, 1988). The major types found in cultivated germplasm are S (for Sanilac, the cultivar in which S type phaseolin was originally characterized), and T (Tendergreen) (Brown et al. 1982). Together, the 'S' and 'T' types are found in 80% of common bean cultivars (Gepts and Bliss 1988; Gepts et al. 1988). Phaseolin type has been used as a tool to differentiate between both wild and cultivated common beans belonging to the Mesoamerican (S phaseolin type) and Andean (T type) gene pools (Gepts and Bliss, 1986; Debouck et al., 1993; reviewed in Gepts, 1993). The linkage between the P and Phs loci, the nature of the gene product and the strict developmental regulation of the Phslocus (an abundant seed protein) suggested that Phs could be a candidate gene for the major seed weight factor identified by Sax (1923).

Hartana (1983) developed near-isogenic lines (NILs) for different phaseolin types in a Sanilac genetic background to analyze the effects of phaseolin genotype on the agronomic and biochemical characteristics of cultivars. He demonstrated that the T type (and C type) phaseolin was significantly correlated with higher seed weight, increased overall seed protein, and an increase in the percent phaseolin compared to S type phaseolin. Seed yield, however, was not significantly affected by phaseolin type. These findings raised the question whether the same association between phaseolin type and seed weight would also be displayed in other populations and environments.

In this paper, we provide additional evidence supporting the hypothesis that Phsis a candidate gene for a seed weight QTL. We compared previously published and unpublished information} on intraspecific variation for seed weight and phaseolin type in P. vulgaris and in the Phaseolinae subtribe, which includes cowpea (Vigna unguiculata) and mung bean (V. radiata), in addition to common bean. We observed, with only few exceptions, a consistent association between allelic variation at the Phs locus and seed weight across a broad range of genetic backgrounds and environments.

MATERIALS AND METHODS

Plant Materials and Field or Greenhouse Experiments

Among the populations studied by Sax (1923), the Improved Yellow Eye 1317 (PP;Phs^TPhs^T) x White 1228 (pp;Phs^SPhs^S) population (Table 1) is highlighted because it was the only F₂ population for which F3 families were observed - thus allowing a separation of the F₂ classes into homozygous dominant PP and heterozygous Pp classes. As far as we are aware, however, the genotypes used by Sax (1923) are not available anymore. Analyses of related materials in the Improved Yellow Eye and White classes showed these materials to have 'T' and 'S' type phaseolins, respectively (Gepts and Bliss 1988).

Hartana (1983) developed six pairs of near isogenic lines (NILs) differing at the Phs locus. BBL 240 (Bush Blue Lake 240: PP;Phs^TPhs^T) was backcrossed to Sanilac (pp;Phs^SPhs^S), donating T type phaseolin to six BC₅S₅ lines. These six lines were then backcrossed again to Sanilac and an analysis was performed on the F₃ progeny of homozygous F₂individuals grown near Hancock, Wisconsin (Table 1).

Koinange (1992) developed a recombinant inbred population (RIP) between Midas (an Andean derived snap bean cultivar: pp;Phs^TPhs^T) and the wild Mesoamerican accession G12873 (PP;Phs^SPhs^S) (population MG). Agronomic evaluation of this population was performed in Popayán, Colombia, as described in Koinange et al. (1996). (Raw data in file saxmgf8.htm.)

Four additional populations were studied by the authors. These were grown in various field and greenhouse locations (Table 1). The F₂ generation of population BTS (PP;Phs^SPhs^S) x Peru34 (PP;Phs^TPhs^T) was grown in a Davis greenhouse. Because of space limitations, this population was grown in two batches in different seasons. Hundred-seed weights were taken on an individual plant basis. (Raw data for phaseolin segregation and seed weight in filessaxbpf21.htm and saxbpf24.htm.)

The F₂ generation of cross BAT93 (PP;Phs^SPhs^S) x Jalo EEP558 (PP;Phs^TPhs^T) (population BJ) served as the initial mapping population for common bean in Davis. A linkage was established in this population (Nodari et al. 1993a) and a QTL analysis was conducted among F₂-derived F₃ families for resistance to common bacterial blight and Rhizobium nodulation (Nodari et al. 1993b). This population was also grown in a Davis greenhouse. Hundred-seed weights were taken on an individual plant basis from the F₂generation grown in a Davis greenhouse. (Raw data in file saxbjf2.htm.)

We developed the CY RIP from a cross between the cultivars California Dark Red Kidney (CDRK, Andean origin: PP;Phs^TPhs^T) and Yolano (Mesoamerican origin: PP;Phs^SPhs^S) through single seed descent (Table 1, pop. 6). Evaluation of seed weight was performed on greenhouse grown F4:5 families. (Raw data in file saxcyf5.htm) In addition, we analyzed seed weight in a subset (50 F4:6 RILs) of this population in replicated field trials at Davis and Salinas CA in 1995. Ten seeds for each of the 50 RILs were space planted per 1.02 m plot, with a two plant in-row border between plots. Hundred-seed weight was measured on a per-plot basis by taking a sample of the harvested seed in each plot. Standard agronomic practices were maintained at each site. (Raw data in file saxcyf6.htm.)

The AG RIP was developed by Steve Magnusson of Harris Moran Seed Company (San Juan Bautista, CA) from a cross between the Mesoamerican breeding line A55 (PP;Phs^SPhs^S) and the Andean-derived cultivar G122 (also known as Jatu Rong: PP;Phs^TPhs^T). A set of 57 RILs was replicated three times in each of the three field locations - at Davis, Salinas, and the Westside Field Station (near Five Points, CA) (Fig.1). Field conditions were as above for the CY population. (Raw data in file saxagf6.htm.)

Laboratory and Statistical analyses

RESULTS

Phaseolin segregation

Association between phaseolin and seed weight segregations

The Phs locus accounted for between 18 and 46 percent (R²) of the variation in seed weight depending on the population and the environment from which data was collected (Table 1). Substitution of a lower-weight allele by a higher-weight allele resulted in a seed weight increase of 1 to 6 g/100-seed, depending on the genetic background and the environment.

In the F₂ populations studied (the first three populations in Table 1), there was evidence for dominance at the Phs locus. In all three populations, the average 100-seed-weight for heterozygotes was significantly different from that of the TT homozygotes but was not significantly different (Fisher's LSD, 0.05 level) from the average for SS homozygotes. Thus, it appears that the S haplotype, associated with lower seed weight, exhibits some degree of dominance over the T haplotype. Sax (1923) came to a different conclusion (albeit using a different population and a different marker) in his original study (Table 1), where he found evidence for additivity for seed weight associated with the P locus.

When averaged across locations, population A55 x G122 exhibited seed weight phenotypes approximating a normal distribution, with the parents falling towards the upper and lower ends of the curve (Fig. 2). At each location, outliers were observed with average 100-seed-weight greater than or less than the high and low parents, suggesting that both parents carry alleles for both high and low seed weight.

Environmental effects

Population 4 was developed from a cross between the same two parents, and was grown at different times in a greenhouse at Davis. Genotypes at the Phs locus accounted for 18% of the variation in seed weight in season 1, but then accounted for 32% of the variation in season 2, presumably due to environmental differences interacting with the same type of population.

Notable G x E interactions were observed for seed weight in population 7 and its parents. Particularly interesting was the behavior of the A55 parent, which averaged 17 g at Davis and 20 g at Five Points. Most genotypes, however, had larger seeds at Davis than at Five Points.

Stability of seed weight across environment was analyzed according to Lin et al. (1986). They suggested that stability can be viewed as a) (Type I) a lack of variability across environments (quantified by Francis and Kannenberg (1978) as the coefficient of variation, CV = Variance/Mean, reflecting the biological concept of homeostasis) or b) (Type II) a response to environmental variations parallel to that of the average of the genotypes under consideration. In the replicated field trials of population 7, several genotypes were notably more stable (Type I) than others: for example, CV (AG 54) = 0.01, while CV (AG 57) = 4.60 [CV(mean) = 1.14, CV(standard deviation) = 0.76). However, an analysis of variance found no evidence for differential Type I stability based on Phs locus genotype (i.e. 'S' vs. 'T' phaseolin genotypes).

Finlay and Wilkinson (1963) quantified Type II stability as the regression coefficient of the trait on an environmental index for the trait (the average of all genotypes across the set of experimental environments). The values for Type II stability of the RILs lead to different conclusions - the R²value for AG 54 was the lowest at 0.58, while the R² value for AG57 was among the highest at 0.99 (average R² = 0.92, R²(std. dev.) = 0.09). An analysis of variance found no significant relationship between Type II stability and genotype at the Phs locus.

Association between seed weight and yield.

Closer examination of the data revealed that most of the largest-seeded RILs in Salinas were also lines exhibiting symptoms of partial infertility (extremely low harvest index, production of parthenocarpic pods). Problems with viability and fertility in the progeny of crosses between the gene pools of common bean are well documented (Coyne, 1965 and 1969; Finke et al.,1986; Gepts and Bliss, 1985; Singh and Gutiérrez, 1984; Singh and Molina, 1991).

Because reduced seed set can lead to increased seed size through yield component compensation, we hypothesized that the negative association of seed yield and seed weight in Salinas was due to statistical outliers (low yield / high seed weight) caused by the relatively few lines exhibiting reduced fertility. To test this hypothesis, we removed the five lines exhibiting the most severe symptoms of infertility at Salinas from the data set (those with an adjusted yield per plot of less than 1.5 grams per day) and performed the analysis again. In the second analysis, no significant associations between yield and seed weight could be observed (R² = 0.008, P = 0.517).

Relationship with seed weight factors in related legume genomes

In P. vulgaris, seed weight QTLs have been identified in two populations, BAT93 x Jalo EEP 558 and Midas x G12873. In the latter population (population 3) (Koinange et al. 1996), two QTLs for seed weight explaining 27 and 18% of the variation for this trait were mapped to linkage groups D7 and D1, respectively. Two major QTLs mapped to the same regions on linkage groups D1 (accounting for 17% of the variance) and D7 (28%) in the BAT93 x Jalo EEP558 population (5) (Nodari 1992). The location of the overlapping QTLs on linkage group D7 on the common bean map span the phaseolin locus. Two additional QTLs with smaller effects were detected on linkage groups D7 (15%) and D11 (16%) in population Midas x G12873 (Koinange et al. 1996) and on linkage groups D3 (11%) and D4 (10%) in population BAT93 x Jalo EEP558 (Nodari 1992) (Table 2).

To correlate the QTLs identified in P. vulgaris with those identified in V. unguiculata and V. radiata (Fatokun et al. 1992), probes linked to QTLs in mungbean and cowpea were mapped in a P. vulgarisrecombinant inbred population derived from the cross BAT93 x Jalo EEP558 (population 5). Their map location was then compared with that of the previously identified seed weight QTLs in P. vulgaris (Nodari 1992; Koinange et al. 1996).

Marker Bng199, closely linked to markers A955 and A515, which mark a seed weight QTL on mung bean linkage group 4 (Fatokun et al. 1992; N. Young, pers. comm.), mapped to common bean linkage group D7 at approximately 10 cM of the Phs locus. While the common bean QTL on linkage group D7 was the major seed weight QTL in that species, it was only a minor QTL in mung bean (Fatokun et al. 1992). There was no apparent QTL matching this seed weight QTL in cowpea (Table 2).

Marker O103 located near a major QTL for seed weight in cowpea (36.5%) and mungbean (32.5%) (Fatokun et al. 1992) mapped to common bean linkage group D3. The mapping distance was 45 cM between O103 and D1132, the marker most closely linked to a common bean seed weight QTL (Nodari 1992). The large linkage distance suggests that these may actually be different QTLs.

The other two QTLs identified in mung bean, on linkage groups 3 and 11, mapped to common bean linkage group D2 and D5 or D7, respectively. No common bean QTLs for seed weight have so far been identified in these regions (Table 2). Likewise, an additional QTL in cowpea for seed weight on linkage group 7, mapped to common bean linkage group D8, to which no seed weight QTL had been mapped in that species thus far.

Inspection of Table 2 revealed that generally QTLs for seed weight were not conserved across the populations used for these three species with the possible exception of the QTL associated with the Phs locus.

DISCUSSION

Phs as the candidate locus for a seed weight QTL identified by Sax (1923)

The P locus, initially identified by Sax (1923) as marking a genomic region carrying a QTL for seed weight, was segregating in only three out of 8 populations listed in Table 1 (IYE 1713 x White 1228, Sanilac x BBL 240, and Midas x G12873). This suggests strongly that some other gene or genes in the vicinity of P rather than P itself is involved in seed weight differences. The results of Hartana (1983) indicating a correlation between phaseolin type and seed weight, suggest that Phs may be the QTL for seed weight that was segregating in Sax's (1923) populations. Alternatively, any locus tightly linked to Phs could also be a candidate locus. This latter suggestion is perhaps less likely because it assumes that the linkage disequilibrium between Phs and the QTL for seed weight would have been maintained throughout the evolutionary history of common bean. Indeed, the relationship between Phs and seed weight was observed also in the cross between G12873 and Midas, representing a wild common bean from the Mesoamerican gene pool and a cultivar from the Andean gene pool, respectively. In that cross, both the Phs and P loci were segregating. The magnitude of the effect (R²) on seed weight associated with the Phslocus was slightly higher (27%) than that associated with the Plocus (22%) consistent with the hypothesis that the seed weight factor is not associated with the P locus but rather with the Phslocus.

More definitive evidence on the role of the Phs locus as a candidate locus for a seed weight QTL should come from transformation experiments. For example, S phaseolin genotypes could be transformed with T phaseolin genes under control of the appropriate regulatory sequences. The transformants could then be observed for any increase in seed weight.

Beavis et al. (1991) suggested that most QTLs are in close proximity to mapped qualitative genetic loci and that "qualitative genetic loci are the same loci that affect quantitative traits." This statement was based on the observation that a number of QTLs affecting plant height in maize mapped to chromosomal regions known to carry genes conditioning dwarfism. Mansur et al. (1993) also implicitly speculated on the association of qualitative and quantitative genes, noting that "the markers associated with the QTL explaining the major portion of the phenotypic variance for seed oil mapped to linkage group 3, which also contains the structural gene for thiol protease, a protein associated with seed oil bodies." The association between seed weight in common beans and the phaseolin locus is additional evidence that, in some cases, genes conditioning quantitative traits may indeed be genes with discreet qualitative effects.

A discrepant observation

Evolutionary conservation (or the lack thereoff) of seed weight QTLs

A possible exception to the lack of conservation is the QTL associated with thePhs locus, which was observed in all P. vulgarispopulations and in mung bean but not cowpea. The absence of any detectable QTL in cowpea around the locus equivalent to phaseolin may be due to absene of segregation at or near that locus for seed weight factors, or the presence of a QTL below the detection limits of the experiment conducted by Fatokun et al. (1992).

Seed weight in common bean as a quantitative trait model system

Acknowledgements We are grateful to Robert Lewellen of the USDA Agricultural Research Station, Salinas, CA, and Steve Temple and Donald Helms of UC Davis, for providing space, maintenance, and agronomic advice for the field tests. Additional field help was kindly provided by David Posner, Leslie Goldberg, Denise Flanahan, Christhiam Cano, Asgar Shirmohamadali, Rosanna Freyre, Christa Haney, and Hau Truong. This work was supported in part by the US AID Bean/Cowpea CRSP and the California Dry Bean Advisory Board.

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________________________________________________________________
 
                                   ________Seed Weight________        
 
  Population                             PP  Pp    pp       p
 
  (phaseolin                  Pop.       or  or    or   R2  ->
 
     types)     Location Gen  Size P1 P2 PhsS PhsST
 
 PhsT (%) P
 
 _______________________________________________________________
 
                                   --------g/100 seeds---------
 
 Sax (1923):
 
 IYE 1317(T)    Orono,    F2   179 48 21  31   28  26   na  2.5
 
 x White 1228(S) ME
 
 (1)b
 
 
 
 Hartana (1983):
 
 Sanilac(S)     Hancock,  NIL    6 19 38  19   na  21   na  1.0
 
 x BBL 240 (T)  WI
 
 (2)c
 
 
 
 Koinange (1992):
 
 Midas(T)       Popayán,  F8    60 20  4   7   na   9.0 27  1.0
 
 x G12873(M)    Colombia                   7   na   8.8 22   
 
 (3)d
 
 
 
 Present results:
 
 BTS (S)        Davis     F2    66 na na  35   37  45   18  5.0
 
 x Peru34 (T), 1 GH 1
 
 (4)e
 
 
 
                Davis     F2    56 na na  27   26  34   30  3.5
 
                 GH 2
 
 
 
 BAT93 (S)      Davis     F2    75 14 29  18   22  28   30  5.0
 
 x Jalo EEP558  GH
 
 (T) (5)e
 
 
 
 CDRK (T) x     Davis     F4:5 149 48 36  34   na  43   31  4.5
 
 Yolano (S)(6)e GH
 
      
 
                Average   F4:6  50 58 38  42   na  32   43  5.0
 
                for 2
 
                field 
 
                sites
 
                Davis     F4:6  50 50 36  36   na  28   32  4.0
 
                Salinas   F4:6  50 66 42  36   na  48   46  6.0
 
 
 
 A55 (S) x      Average   F8    57 21 39  26   na  30   33  2.0
 
 G122 (T)       for 3
 
 (7)e           field
 
                sites
 
                Five-     F8    57 20 35  21   na  25   26  2.0
 
                Points
 
                Davis     F8    57 17 37  23   na  28   33  2.5
 
                Salinas   F8    57 25 46  30   na  38   28  4.0
 
 ________________________________________________________________
 
 
 
 a  Gen: Generation; Size: population size; P1 and P2: female and 
 
 male parent, respectively; PP, Pp, and pp refer to the
 
 genotype 
 
 at the P locus, and numbers are the average 100-seed-weight (g) of 
 
 the P or Phs loci genotypes depending on the population (see 
 
 footnotes below). R2: proportion of seed weight variation accounted 
 
 for by phaseolin type. p->P: average number of grams 100-seed-weight 
 
 is increased by converting a p allele to a P allele or a
 
 PhsS to a
 
 PhsT allele. na = not available or not applicable.
 
 
 
 b  Average 100-seed weight and P locus summary for the most
 
 thoroughly studied population of Sax (1923).
 
 
 
 c  Average 100-seed-weight and phaseolin data summary for the
 
 six pairs of NILs of Hartana (1983). Instead of the P locus
 
 genotypes, the genotypes at the Phs locus are used (i.e. p is linked
 
 in cis to the 'S' allele, P is linked in cis to the'T' allele).  From
 
 Hartana (1983).  
 
 
 
 d  Average 100-seed-weight and phaseolin data summary for the
 
 population of Koinange (1993) and Koinange et al. (1996).  The 
 
 phaseolin type of G12873 is an 'M' type instead of an  'S' type as 
 
 for other Mesoamerican genotypes in this study. Other variables are 
 
 as for population (2) except that the first set of values for p->P 
 
 and R2 correspond to the Phs locus and the second set to the
 
 P locus. 
 
 
 
 
 
 e  Average 100-seed-weight and phaseolin data summary for the
 
 five populations used in the current study. Variables are as for
 
 population (3).  In addition, populations 6 and 7 were grown in 
 
 replicated field trials to examine the effects of environment on
 
 100-seed-weight.  See text for details.
 
 
 
 

Table 2. Putative seed weight QTL correspondence among common bean, cowpea, and mung bean^a

_________________________________________________________________
 
           Common bean        
 
 ________________________________________     Cowpea      Mungbean
 
 LG   Markers                MG      BJ         LG           LG
 
 _________________________________________________________________
 
 
 
 D1   D1492-3, Pal-1        Yes      Yes         No           No
 
 D2   M78                   No       No          No           3
 
 D3   D1132                 No       Yes         No           No
 
 D3   D1009,O103            No       No          2            1
 
 D4   D1011                 No       Yes         No           No
 
 D7   Phs,Bng199            Yes      Yes         No           4
 
 D7   Uri-2                 Yes      No          No           No
 
 D8   Bng7, Bng138, A816    No       No          7            No
 
 D11  D0252                 Yes      No          No           No
 
 D5 (Bng166) or D7 (Bng222) No       No          No           11 
 
 _________________________________________________________________
 
 a Based on results of Koinange et al. (1996) and Nodari (1992)
 
 for common bean and Fatokun et al. (1992) for cowpea and mungbean