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Published online 30 July 2007
Published in Crop Sci 47:1361-1366 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP BREEDING & GENETICS

Analysis of Seed Zinc and Other Minerals in a Recombinant Inbred Population of Navy Bean (Phaseolus vulgaris L.)

J. R. Gelin*, S. Forster, K. F. Grafton, P. E. McClean and G. A. Rojas-Cifuentes

Dep. of Plant Sciences, North Dakota State Univ., Fargo, ND 50105; J.R. Gelin, current address, Syngenta Seeds, Inc., Soybean Product Development, 9497 County Highway 10 West, Glyndon, MN 56547

* Corresponding author (robert.gelin{at}syngenta.com).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Mineral deficiencies in common bean (Phaseolus vulgaris L.) negatively affect plant growth and development. Genetic differences in seed mineral concentrations have been detected among landraces and genotypes for trace elements such as Zn and Fe, and major elements such as P and Ca, and these differences have been exploited for the genetic improvement of the crop. The objectives of this study were (i) to develop and evaluate a navy bean recombinant inbred population segregating for seed Zn, (ii) to measure other micronutrients in the seeds such as Fe and the major elements P and Ca, and (iii) to identify associations between microsatellite markers and seed mineral content. Transgressive segregants were observed for seed Zn and variation was also found for Fe, P, and Ca. Bean microsatellites associated with Zn, P, and Ca were identified, but there was no association with Fe. Our molecular data identified a locus associated with seed Zn accumulation in bean located on linkage group 9. Further studies would help to find the exact location of the gene. As more information becomes available, breeders will be able to combine techniques of molecular genetics with conventional breeding methods through marker-assisted selection to develop cultivars with higher seed Zn content.

Abbreviations: MAS, marker-assisted selection • QTL, quantitative trait loci • RILs, recombinant inbred lines


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
COMMON BEAN (Phaseolus vulgaris L.) is cultivated throughout the world as the most important grain legume grown for direct human consumption and an important source of dietary proteins, vitamins, and minerals (Broughton et al., 2003). Various races and market classes are grown in tropical, subtropical, as well as temperate regions of the world at elevations ranging from sea level to 3000 m (Broughton et al., 2003; Singh et al., 1991a). Most of the production takes place in Latin America and the Caribbean, but important regions also exist in eastern and southern Africa, Asia, Europe, and North America. In the United States, North Dakota is the largest producer of dry bean, with a total of 387,454 Mg with an estimated value of over $130 million in 2005 (USDA, 2006).

Genetic differences in seed mineral concentrations have been detected among landraces and genotypes for trace elements such as Zn and Fe and major elements such as P and Ca (Singh et al., 1991b; Graham et al., 1999; Blair et al., 2005). These differences have been exploited for the genetic improvement of the crop, especially since production is largely influenced by both biotic and abiotic factors (climatic and edaphic). Mineral deficiencies in beans are a type of edaphic stress that can play a negative role on growth and development (Ambler and Brown, 1969; Bath et al., 1992; Beebe et al., 2000). According to Thung and Rao (1999), Zn deficiency in beans can be induced by Fe deficiency in the soils, and yield losses of up to 75% have been associated with P-deficient soils. Moraghan and Grafton (1997) reported that navy beans accumulate more seed Ca than other large-seed market class beans, although the role of environmental and genetic factors cannot be neglected.

Apart from its negative effect on the quality of the seed in terms of its nutritive value, Zn deficiency in particular is associated with severe yield reductions (Cakmak, 2000) and delayed or irregular maturity even without visual symptoms (Boawn et al., 1969). Zn deficiency can cause nutritional concerns for people in developing countries and vegetarians in industrialized countries. The deficiency negatively affects human growth, sexual maturity, and the immune defense system (Frossard et al., 2000). Zinc has several biochemical functions in humans including the synthesis and degradation of proteins, carbohydrates, lipids, and nucleic acids, which makes it an important element for human growth and development. Therefore, increasing the level of Zn in edible parts of plants may increase the amount of Zn available for human consumption and may overcome many of the Zn deficiency–related problems in humans. Determining seed Zn content is particularly useful in bean breeding programs because genotypes can be evaluated in the absence of foliar Zn-deficiency symptoms. In addition, seeds can be stored for longer periods of time until testing compared with foliar tissues.

The majority of common bean production in North Dakota occurs on high pH soils that are frequently low in available Zn. According to the 1999 Northarvest Bean Growers Survey, Zn fertilizer was applied to 54% of North Dakota respondents' hectares (Lamey et al., 2000) as a way to alleviate deficiency in the soils. In general, Zn availability to the plant decreases when the soil pH is above 7.0 (Marschner, 1995). Navy bean cultivars tend to be more susceptible to Zn-deficient soil compared to other classes of common bean. However, Moraghan and Grafton (1999) reported that Zn-efficient genotypes possess more seed Zn than Zn-inefficient genotypes when grown on soils high in available Zn. Recent studies indicate that resistance to soil Zn deficiency in common bean is under the control of a single dominant gene (Singh and Westermann, 2002; Cichy et al., 2005). Therefore, improving seed Zn accumulation through plant breeding efforts should be an attainable goal. Identifying navy bean cultivars that are less susceptible to Zn deficiency may reduce the need for Zn fertilization where soil is naturally low in available Zn.

Although conventional breeding techniques have resulted in significant genetic improvement for common bean, molecular markers have recently been developed as a promising selection tool for marker-assisted selection (MAS) (Kelly and Miklas, 1999; Bliss, 2004; Kelly, 2004). Several molecular maps have been constructed for common bean (Nodari et al., 1993; Freyre et al., 1998), and molecular markers have been used to detect chromosomal regions or quantitative trait loci (QTL) associated with traits of importance including flower and seed coat color (Brady et al., 1998; Erdmann et al., 2002; McClean et al., 2002), disease resistance (Kelly and Miklas, 1998), and agronomic and quality traits (Rivkin et al., 1999; Tar'an et al., 2002; Jacinto-Hernandez et al., 2003). Quantitative trait loci associated with seed mass, Ca, Fe, Zn, and tannins were identified in a wide cross of common bean (Guzmán-Maldonado et al., 2003). Microsatellite markers, in particular, are among the most recent types of DNA markers used for molecular studies in common bean (Yu et al., 1999; Gaitán-Solís et al., 2002; Guerra-Sanz, 2004). They are abundant and codominant in nature and offer highly reproducible results. Blair et al. (2003) have recently developed microsatellites that cover every chromosome in the bean genome. The objectives of this study were (i) to develop and evaluate a navy bean recombinant inbred population segregating for seed Zn, (ii) to measure other micronutrients in the seeds such as Fe and major elements P and Ca, and (iii) to identify associations between microsatellite markers and seed nutrient content.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Plant Material
The navy bean cultivars Voyager (Rogers Seed Company, 1995) and Albion (Asgrow Seed Company, 1987) were crossed in 1997 to develop a recombinant inbred population. Voyager is a CIAT Type III Zn-efficient genotype, and Albion is a CIAT Type I Zn-inefficient genotype. The cross was used to study the inheritance of seed Zn accumulation in navy bean (Forster et al., 2002; Cichy et al., 2005). The recombinant inbred population was developed in the greenhouse by single seed descent, and lines were planted in the field at Erie, ND, for seed increase in 2000. In 2001, recombinant inbred lines (RILs) in the F5:8 generation were field evaluated at Erie and Hatton, ND.

Experimental Design and Field Evaluation
In 2001 73 RILs and 13 entries of each parent were planted in the field for evaluation. One Zn-inefficient navy genotype, ‘Mackinac’ (Kelly et al., 1998), was added as check to give a total of 100 entries. At Erie, the lines were sown on 6 June and harvested on 3 September. At Hatton, they were sown on 8 June and harvested on 4 September. A lattice design with two replicates was used at each location. Individual plots consisted of one 3.0 m row which was end-trimmed to 2.7 m to facilitate harvest, and spacing between rows was 0.76 m. Plots were hand-weeded as necessary. Fertilization, pest control, and common cultural practices were consistent with bean production in North Dakota.

Agronomic Performance
Seed weight and plot yield were recorded at each location. Seed weight (g) was calculated as the weight of 100 seeds. Plot yield (kg ha–1) was collected by hand-harvesting 2 m of each row at maturity and threshing plants using an Almaco (Model LPT-MRB-G, Nevada, IA) stationary plot thresher modified for use on common bean.

Seed Nutrients Analysis
Thirty mature pods were randomly collected from each plot at each location. Seeds were harvested, bulked, counted, washed with deionized water containing Joy detergent (Proctor and Gamble, Cincinnati, OH), and later rinsed with deionized water only. Samples were oven-dried at 70°C for 48 h, weighed, and ground in an agate mortar with an agate pestle (Brinkmann Instruments Co., Westbury, NY) to pass a sieve with 0.25-mm openings. Samples were then redried at 70°C and analyzed for Zn, Fe, Ca, and P, as previously described (Moraghan and Grafton, 1999).

Statistical Analysis
Data were analyzed using the Statistical Analysis System (SAS Institute, 1985). Analysis of variance (ANOVA) was conducted to determine differences among lines. Individual ANOVAs were calculated for each environment. Locations were considered homogeneous if the ratio of the effective error variances for each trait was less than 10-fold. The adjusted mean values of each line were used in the combined analysis and analyzed as a randomized complete block (RCBD).

Survey of Microsatellite Markers
A set of 68 microsatellite markers, developed by Blair et al. (2003) and mapped in two intergene pool crosses of common bean, was used in this study to detect putative chromosomal regions associated with the accumulation of seed nutrients. Genomic DNA was extracted from leaf tissues collected from bean seedlings grown in the greenhouse, as previously described (Doyle and Doyle, 1990). Each sample was quantified and diluted to 25 ng µL–1 before carrying out the reactions. The polymerase chain reaction was performed according to Blair et al. (2003), and DNA fragment analysis was performed using a high-throughput nondenaturing polyacrylamide gel electrophoresis system (Wang et al., 2003). Microsatellite markers that showed polymorphism between the two parents were used to screen the entire population. Each line was scored according to the banding pattern of the two parents, and heterogeneous lines were scored as missing values. Proc GLM was used in SAS to detect the individual and combined effects of loci on the accumulation of seed micronutrients. The amount of variation explained by each locus or combination of loci was calculated by multiplying the coefficient of phenotypic determination (r2 value) by 100. The value obtained was divided by the heritability estimate for seed Zn (Cichy et al., 2005) to give the coefficient of genotypic determination (rG2). Data recorded for loci that were previously mapped to the same linkage group were analyzed using MAPMAKER (Lincoln et al., 1992) to determine genetic distances between linked markers. The software NQTL (Tinker and Mather, 1995) was used with 1000 permutations to find the threshold value for type I error rate associated with the putative QTL (Churchill and Doerge, 1994).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Agronomic Performance
Across location means for seed weight and seed yield are presented in Table 1. There was a significant difference between the two parents for seed weight and seed yield (P < 0.05). Voyager had significantly higher seed weight and seed yield than Albion. Albion also had lower seed yield than the population. The control Mackinac had the lowest seed yield and was similar to Albion for seed weight.


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  		Table 1. Across location mean of parents and population for seed weight and seed yield in a navy bean (Phaseolus vulgaris L.) recombinant inbred population derived from a cross between ‘Albion’ and ‘Voyager’.

 
Seed Nutrients Analysis
Significant differences were detected among the across location means of two parents for Zn, Fe, and Ca, but not for P seed content (Table 2). Compared with the two parents, the population mean was intermediate for Zn and Fe content, similar for P content and greater than the mean of Albion for Ca content. Transgressive segregation was observed with some RILs exceeding the seed nutrients concentration of either parent. Means were sorted and the top and bottom 10% identified for each trait. The largest significant difference between the top 10% lines and the two parents was detected for Zn and Fe. These elite lines had a mean for seed Zn of 32.3 mg kg–1 compared with 21.2 mg kg–1 for Albion, 29.5 mg kg–1 for Voyager, and 25.5 mg kg–1 for the population. The mean of the top 10% for seed Fe was 86.9 mg kg–1, similar to that of Voyager, compared with 62.2 mg kg–1 for Albion, and 75.4 mg kg–1 for the population. Considering that the cross was originally made between two divergent navy genotypes for seed Zn, it is interesting from a breeding perspective to find transgressive segregants among the progeny. The superior seed Zn accumulating RILs, as well as the extremely low seed Zn accumulating RILs, have potential for future research. Further evaluation of these superior lines would reveal whether they can be used in breeding programs as a source of germplasm to generate improved cultivars for seed Zn accumulation.


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  		Table 2. Across location mean of parents and population for seed micronutrients in a navy bean (Phaseolus vulgaris L.) recombinant inbred population derived from a cross between ‘Albion’ and ‘Voyager’.

 
Phenotypic Correlations
Pearson's correlation coefficients were calculated for all phenotypic traits measured in the population (Table 3). Seed Zn was positively correlated (P < 0.05) with Fe (r = 0.40), Ca (r = 0.23), P (r = 0.39), and yield (r = 0.21). Using F2:3 generation for a cross between a cultivated and a wild bean genotype, Guzmán-Maldonado et al. (2003) found similar correlation coefficients for Zn and Ca (0.38) and for Zn and Fe (0.27). These findings indicate the possibility to combine these traits in selected genotypes through appropriate breeding programs. There was no significant correlation between Zn and seed weight, and a negative correlation was found between Ca and yield (r = –0.21). The highest correlation (r = 0.67) was found between Fe and P, suggesting that breeding for one trait may lead to significant progress in the other. As with other studies, Zn and Fe concentrations were correlated (Beebe et al., 1999; Welch et al., 2000; House et al., 2002). This provides further support for the suggestion that the same genetic and molecular mechanisms are controlling both Zn and Fe mobilization, uptake, distribution, and accumulation in the plant (Clemens et al., 2002).


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  		Table 3. Pearson's correlation coefficients (P < 0.05) for selected traits among 73 F5:8 navy bean (Phaseolus vulgaris L.) recombinant inbred lines derived from a cross between ‘Albion’ and ‘Voyager’ grown in 2001 at Erie and Hatton, ND.

 
Marker Association
Among the 68 primer pairs tested with the nondenaturing polyacrylamide gel electrophoresis system, five were clearly polymorphic between the two parents (Fig. 1 ). Among the five polymorphic markers, one (BM211) was not significantly associated with any micronutrient. The four markers that were associated with seed Zn, P, and Ca are presented in Table 4 with their location and individual and combined effects. It was a bit surprising that the Zn marker was not associated with Fe given the significant correlation between the concentrations of the two minerals. This is consistent with the results of Guzmán-Maldonado et al. (2003) who discovered two markers associated with Fe content and one with Zn content, yet none of these were linked.


Figure 1
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  		Figure 1. Polymerase chain reaction products showing polymorphism for the two parents, Phaseolus vulgaris L. ‘Albion’ and ‘Voyager’, and recombinant inbred lines. This example is from bean microsatellite marker BM184.

 

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  		Table 4. Microsatellite markers associated (P < 0.05) with seed nutrients in a navy bean (Phaseolus vulgaris L.) recombinant inbred line population derived from a cross between ‘Albion’ and ‘Voyager’.{dagger}

 
Three of these four markers were previously mapped to linkage group 9 and the other to linkage group 3 (Blair et al., 2003). BM159 mapped to linkage group 3 and accounted for 7.1% of the variation in seed Ca. BM141 mapped to linkage group 9 and accounted for 14.3% of the variation in seed P. BM154 and BM184 both mapped to linkage group 9, 5.2 cM apart. These accounted for 11.7 and 15.3% of the variation in seed Zn, respectively. The combined effect of BM154 and BM184 was 17.8%. The consistency of their effect and their genetic linkage suggest these markers define a single locus that affects seed Zn content in navy bean. With 1000 permutations conducted on the genotypic data, the threshold value for type I error rate associated with the locus is 0.0450, which is under the probability value of 0.05. Considering that Cichy et al. (2005) using the same Albion x Voyager cross estimated the heritability for Zn to be around 0.8, the coefficient of genotypic determination for Zn alone would be 22.2% with the combined effect of markers BM154 and BM184.

Using amplified fragment length polymorphism in a wide cross of common bean, Guzmán-Maldonado et al. (2003) found a locus that accounted for 15.2% of the phenotypic variation associated with Zn. The effect of the locus we discovered in different cross is of similar magnitude. The monogenic inheritance of seed Zn in dry bean has already been established (Forster et al., 2002; Singh and Westermann, 2002; Cichy et al., 2005), and the symbol Znd was proposed for the dominant allele controlling resistance to Zn deficiency in the soil (Singh and Westermann, 2002).

Our results represent an initial step in the identification of genes controlling zinc concentration in navy bean seed. The low polymorphism found in the navy bean population can be attributed to the fact that the two parental genotypes are from the same gene pool and the same seed class in contrast to previous mapping studies which have generally been conducted with wide crosses.


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Our data reveal that variation exists in navy bean for seed Zn, Fe, P, and Ca, and that the agronomic performance of the inbred lines was associated with seed nutrients to varying degrees. There was a positive correlation between Zn and Fe, Ca, P, and seed yield. Transgressive segregants were observed for seed Zn, which offers hope for significantly improving this trait in bean through appropriate breeding programs. Field evaluation of the lines belonging to the top 10% for seed Zn may help to identify superior genotypes that could potentially be released with high Zn content as a value added trait. Our molecular data suggest that a gene responsible for seed Zn accumulation in bean is located on linkage group 9. Further molecular studies would help confirm our finding and pinpoint the exact location of the gene. As more information becomes available regarding the specific location of this locus, breeders will be able to combine techniques of molecular genetics with conventional breeding methods through MAS to develop cultivars with higher seed Zn content.


   ACKNOWLEDGMENTS
 
Support for this study came from the Northarvest Bean Growers Association. The authors would like to also thank Mr. A. Vander Wal for his technical assistance during the field evaluation of the recombinant inbred population, and Mr. R. Lee for his technical assistance with the molecular analysis.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication August 4, 2006.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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