Published online 16 January 2008
Published in Crop Sci 48:189-193 (2008)
© 2008 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Slow Darkening in Pinto Bean (Phaseolus vulgaris L.) Seed Coats Is Controlled by a Single Major Gene
Donna C. Junk-Knievel,
Albert Vandenberg and
Kirstin E. Bett*
Dep. of Plant Sciences, Univ. of Saskatchewan, Saskatoon, SK, Canada S7N 5A8
* Corresponding author (k.bett{at}usask.ca).
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ABSTRACT
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Postharvest seed coat darkening is a significant problem in pinto beans (Phaseolus vulgaris L.), resulting in product that is undesirable to consumers and that is discounted in the marketplace. There is a range in the rate and extent of darkening among pinto germplasm and recently, slow-darkening (SD) lines have been identified. Line 1533-15, an SD line from the University of Saskatchewan, was crossed to CDC Pintium, a regular darkening pinto, and seed of F1 and F2 individuals and F5:6 recombinant inbred lines were assessed for their darkening phenotype. Segregation data indicated that there is a single, recessive gene that controls the SD phenotype. All F2 individuals from a cross between 1533-15 and Pinto Saltillo, another SD line, were slow-darkening suggesting that the phenotype is controlled by the same gene in both lines. The simple genetics of this trait should facilitate the introduction of this trait into breeding programs, thereby increasing the quality of pinto beans being developed.
Abbreviations: CDC, Crop Development Centre • FDM, Flor de Mayo • RIL, recombinant inbred line • RD, regular-darkening • SD, slow-darkening • UVC, ultraviolet C
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ACKNOWLEDGMENTS
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Funding for this project was provided by the Saskatchewan Pulse Growers and the Saskatchewan Agriculture and Food Agriculture Development Fund.
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NOTES
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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 April 24, 2007.
Slow Darkening in Pinto Bean (Phaseolus vulgaris L.) Seed Coats Is Controlled by a Single Major Gene
Donna C. Junk-Knievel,
Albert Vandenberg and
Kirstin E. Bett*
Dep. of Plant Sciences, Univ. of Saskatchewan, Saskatoon, SK, Canada S7N 5A8
* Corresponding author (k.bett{at}usask.ca).
Postharvest seed coat darkening is a significant problem in pinto beans (Phaseolus vulgaris L.), resulting in product that is undesirable to consumers and that is discounted in the marketplace. There is a range in the rate and extent of darkening among pinto germplasm and recently, slow-darkening (SD) lines have been identified. Line 1533-15, an SD line from the University of Saskatchewan, was crossed to CDC Pintium, a regular darkening pinto, and seed of F1 and F2 individuals and F5:6 recombinant inbred lines were assessed for their darkening phenotype. Segregation data indicated that there is a single, recessive gene that controls the SD phenotype. All F2 individuals from a cross between 1533-15 and Pinto Saltillo, another SD line, were slow-darkening suggesting that the phenotype is controlled by the same gene in both lines. The simple genetics of this trait should facilitate the introduction of this trait into breeding programs, thereby increasing the quality of pinto beans being developed.
Abbreviations: CDC, Crop Development Centre • FDM, Flor de Mayo • RIL, recombinant inbred line • RD, regular-darkening • SD, slow-darkening • UVC, ultraviolet C
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INTRODUCTION
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SEEDS OF COMMON BEAN (Phaseolus vulgaris L.), like other pulses, are sold based on visual characteristics such as size, shape, and color. Darkening of the seed coat during storage is a significant problem in several market classes, particularly pinto and carioca beans. Merchants and consumers generally assume that beans that have darkened are older and more difficult to cook. This can result in a loss of economic value through discounted pricing. Some varieties darken more quickly than others and, as a result, are downgraded more often than those that retain their bright background color.
Seed coat darkening is accelerated by storage conditions with high relative humidity, high temperatures, and light (Park and Maga, 1999). Exposure to ultraviolet and cool-white light has also been linked to an increased rate of seed coat darkening (Hughes and Sandsted, 1975; Brackmann et al., 2002; Junk-Knievel et al., 2007). Sartori (1982) found that after 6 mo of storage at 24°C and 75% relative humidity, pinto beans that were stored in an enriched N atmosphere showed no change in color while pinto beans stored in a natural atmosphere began to darken significantly.
While environment plays an enormous role in the rate of darkening, within the pinto bean germplasm there is also a wide range in rate of darkening (Junk-Knievel et al., 2007) and some varieties are considered to have better color than others (e.g., Bill Z, CDC Pintium, and Maverick). Conventional pinto beans do darken after a few months in storage, and after a year will no longer have the desired bright background. Three new lines have been identified that appear to maintain their bright background color for much longer than even the best conventional varieties: 1533-15, from the Crop Development Centre (CDC), University of Saskatchewan; Pinto Saltillo, developed at the Saltillo Experiment Station of the Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias (INIFAP) in Mexico (Sanchez-Valdez et al., 2004); and SDIP-1, from the University of Idaho (Singh et al., 2006). This phenotype is described as slow-darkening (SD) as the seeds do eventually darken although to a lesser extent than regular-darkening (RD) pintos.
During storage, compounds in the seed coat can undergo oxidation or other chemical changes that lead to novel compounds which may change the color of the seed. Polyphenols such as proanthocyanidins (syn. condensed tannins) are colorless products that are converted to visible pigments during dehydration and oxidation (Stafford 1990). Seed coats of CDC Pintium (RD) contain higher concentrations of polyphenols than those of 1533-15 (SD) (Beninger et al., 2005), suggesting that darkening in pinto bean may be related to the presence of polyphenolic compounds. Polyphenol oxidase and peroxidases have also been implicated in seed coat darkening in bean (Moura et al., 1999; de Oliveira Rios et al., 2002).
The inheritance of the SD trait was studied in progeny of crosses between the SD pinto lines 1533-15 and Pinto Saltillo and RD pinto lines.
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MATERIALS AND METHODS
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Plant Material
CDC Pintium is an early-maturing RD pinto bean cultivar from Saskatchewan that has good color at harvest but, like most conventional pintos, darkens in storage. HR99 is a pinto bean line from the breeding program at Agriculture and Agri-Food Canada, Harrow, ON, which also darkens in storage. Breeding line 1533-15 is an early-maturing SD line from the CDC breeding program that darkens much more slowly than other pintos, retaining its color for more than a year. 1533-15 was developed from a cross between CDC Pintium and a breeding line, SC11743, from the International Centre for Tropical Agriculture (CIAT), and selected for early maturity, high yield, and improved seed quality. Two F2 populations were developed from the cross CDC Pintium x 1533-15. These populations were advanced through single seed descent then pooled to develop a single population of 105 F5:6 recombinant inbred lines (RILs). A population of F2 plants and 64 F5:6 RILs were also developed from the cross HR99 x 1533-15. F2 plants of all three populations plus the parents were grown in the field at Saskatoon, SK, in 2003. Single rows of each RIL from all three populations plus the parents were grown in the field at Saskatoon in 2006. Two additional F2 populations from the cross CDC Pintium x 1533-15 and its reciprocal were also grown in the field in Saskatoon in 2004 (Table 1
).
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Table 1. Segregation for seed coat darkening phenotype in F2 populations derived from crosses among regular-darkening (CDC Pintium and HR99) and slow-darkening (1533-15 and Pinto Saltillo) lines.
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Pinto Saltillo is a photoperiod sensitive, SD pinto cultivar from Mexico (Sanchez-Valdez et al., 2004). F2 populations were developed from the cross Pinto Saltillo x CDC Pintium, and Pinto Saltillo x 1533-15 to study the allelism of the two SD genotypes. F2 populations of CDC Pintium x Pinto Saltillo, Pinto Saltillo x CDC Pintium, Pinto Saltillo x 1533-15, and the parents were grown in the field in 2004 at Saskatoon. Due to the daylength sensitivity of Pinto Saltillo and some of the F2 progeny, two additional F2 populations from 1533-15 x Pinto Saltillo and its reciprocal were developed and grown, along with F1 and parent plants, in the Agriculture Greenhouses at the University of Saskatchewan in 2005. Plants were grown on shade benches to restrict the daylength to 12 h to ensure flowering of the photoperiod sensitive plants.
Color Assessments
Initially, it is very difficult to distinguish RD from SD phenotypes. Using the accelerated darkening protocol developed by Junk-Knievel et al. (2007), samples of 15 to 20 seeds of each parent and F2 plant or RIL were artificially darkened by exposing them in a single layer to UVC light (
= 254 nm; model G40T10, Ushio America, Inc., Cypress, CA) for 72 h. Each seed sample was then visually classified as RD or SD based on the extent of darkening relative to the unexposed half of the seed coat. In addition, the seed coat color was quantified following UVC treatment by recording L* and a* color values of the exposed side of the seeds using a Hunter Lab colorimeter (model No 45/0-L MiniScan XE, Hunter Associates Lab Inc., Reston, VA). The L* value indicates brightness with an L* value of 100 being perfectly white while an L* value of 0 is perfectly black. The a* value indicates redness with higher values indicating redder color. Chi-square tests were conducted to determine the genetic control of the SD trait.
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RESULTS
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When classified as RD or SD based on whether or not the seed coat darkened following exposure to UVC light, all F1 seed coats from crosses involving SD and RD parents were RD. The seed from plants of the F2 populations derived from crosses between the SD parent, 1533-15, and the RD parents, CDC Pintium or HR99, segregated 3 RD:1 SD for all crosses (Table 1). Of the 97 F5:6 RILs from the cross CDC Pintium x 1533-15 that were homozygous for the darkening trait, 55 were RD and 42 were SD (
21:1 = 1.74, P = 0.19). Homozygous lines from the HR99 x 1533-15 RIL population segregated 30 RD:33 SD (21:1 = 0.14, P = 0.71). L* and a* values were bimodally distributed with a clear separation between RD and SD individuals for both F2 and RIL populations (Fig. 1
and 2
). These results clearly indicated single gene control of the darkening phenotype with SD being controlled by a recessive allele.
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Figure 1. Frequency distribution of L* value (top) and a* value (bottom) of seed of F2 individuals from the cross CDC Pintium x 1533-15 and its reciprocal cross grown and harvested from the field in Saskatoon, SK, in 2004. Parental color values: CDC Pintium L* = 32 and a* = 11; 1533-15 L* = 39 and a* = 7.5.
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Figure 2. Frequency distributions of L* value (top) and a* value (bottom) of seed of recombinant inbred lines from the cross CDC Pintium x 1533-15 grown and harvested from the field in Saskatoon, SK, in 2006. Parental color values: CDC Pintium L* = 28, a* = 6.5; 1533-15 L* = 37, a* = 4.
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There was variability within both RD and SD phenotypes for L* and a* values (Fig. 1 and 2) suggesting further influences on the level of darkening within the two phenotypes. One possibility may be variability in maturity resulting in variability in the extent of darkening before harvest. Alternatively, there may be modifying genes that influence the full extent of darkening among both the RD and SD phenotypes.
Because Pinto Saltillo is photoperiod sensitive, it begins flowering just before the first killing frost at Saskatoon. Progeny from crosses with this parent segregate for photoperiod sensitivity and therefore the number of F2 plants that set seed is reduced. The seed from F1 plants of crosses between CDC Pintium and Pinto Saltillo, grown indoors under short days, had the RD phenotype. Field-grown F2 plants, pooled from two reciprocal crosses and that managed to set seed, segregated 22 RD:6 SD which is not significantly different from 3:1 (2 = 0.19, P = 0.66; Table 1) suggesting single gene control. Similarly, not all F2 plants of the crosses between 1533-15 and Pinto Saltillo grown in the field reached maturity. Of the 18 that did set seed, all were SD. All plants from the F2 populations grown in the greenhouse under short days also displayed the SD phenotype suggesting the same gene controlled the SD trait in 1533-15 and Pinto Saltillo and that daylength sensitivity did not influence the darkening phenotype in the field. The L* and a* values for the seed coats from the F2 plants of a 1533-15 x Pinto Saltillo cross grown under short days both followed a normal distribution (Fig. 3
) suggesting some quantitative or environmental influence on the results.
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Figure 3. Frequency distribution of L* value (top) and a* value (bottom) of seed of F2 individuals from the cross 1533-15 x Pinto Saltillo grown and harvested in the greenhouse under short days in 2005. Parental color values: 1533-15 L* = 48 and a* = 6; Pinto Saltillo L* = 44 and a* = 8. F1 color values: L* = 49 and a* = 6. For reference purposes, CDC Pintium L* = 40 and a* = 12.
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DISCUSSION
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Since the SD trait is controlled by only one recessive gene, breeding for this trait in pintos is straightforward. The trait can also be transferred to genetically related market classes such as carioca. Carioca beans have the same cream-colored background as pinto beans and a single gene controls the striped vs. spotted pattern phenotype that distinguishes these two classes (Lamprecht, 1947; Bassett, 2007). Flor de Mayo (FDM) is another market class with a cream-colored background and a tendency to darken post-harvest. The pink splash pattern of FDM is also allelic with the stripe and spots of the cariocas and pintos (Bassett, 2007). Interestingly, crosses between 1533-15 and several different FDM lines have always resulted in F1 seed coats with gray rather than pink pattern colors, and all SD FDM phenotypes that arise in the F2 have the gray pattern. It would appear that the gene controlling darkening is somehow interfering with the pattern color.
The presence of the recessive allele of bean seed coat color gene J has been associated with reduced levels of postharvest darkening and a dulling of seed coat color (Prakken, 1970; Bassett 1996). Crosses with lines carrying j/j will have to be studied to ascertain if the recessive allele identified in these SD lines is indeed j. Some lines within other market classes of dry bean, such as yellow and red, also appear to resist after-darkening. Crosses between 1533-15 and these lines would indicate whether there is only the one major gene controlling post-harvest darkening in common bean or several different genes depending on seed coat color chemistry.
The identity of the gene involved in controlling post-harvest darkening can be further elucidated through a combination of biochemical and molecular characterization of the RILs. Biochemical analyses of CDC Pintium and 1533-15 (Beninger et al., 2005) have shown higher levels of kaempferol and proanthocyanidins in the RD CDC Pintium compared to the SD 1533-15. Should levels of these two compounds also correlate with the darkening phenotypes in the individual RILs from CDC Pintium x 1533-15, it would suggest that this gene controls a step early in the flavonoid pathway.
The association between color change and cooking quality is not yet understood because it is easily confounded with aging. It is likely that other genes that affect seed coat properties also affect cooking time. The ability to control the darkening phenomenon genetically may help to elucidate the other factors affecting cooking quality.
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CONCLUSIONS
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Evidence from crosses using two sources of SD pintos and two different RD pintos demonstrated the single gene control of post-harvest darkening with SD being controlled by a recessive allele. It also appears that the gene in 1533-15 is likely the same as that in the genetically unrelated line Pinto Saltillo, suggesting that the SD phenotype may be present in other breeding populations but has yet to be identified. This genotype is of commercial interest as it allows producers to retain the value of their beans for an extended period. Merchants also appreciate the ability to store beans for a longer time without experiencing color deterioration.
Funding for this project was provided by the Saskatchewan Pulse Growers and the Saskatchewan Agriculture and Food Agriculture Development Fund.
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 April 24, 2007.
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REFERENCES
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- Stafford, H.A. 1990. Flavanoid metabolism. CRC Press Inc., Boca Raton, FL.
TOP
ABSTRACT
INTRODUCTION
