Crop Science 43:329-332 (2003)
© 2003 Crop Science Society of America
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
Genetic Analysis and Random Amplified Polymorphic DNA Markers Associated with Cooking Time in Common Bean
Carmen Jacinto-Hernandeza,
Susana Azpiroz-Riveroa,
Jorge A. Acosta-Gallegosa,
Humberto Hernandez-Sanchezb and
Irma Bernal-Lugo*,c
a CEVAMEX, Apdo. 10-56230 Chapingo México
b Departamento de Graduados e Investigacion en Alimentos, ENCB, IPN
c Facultad de Química, UNAM, Ciudad universitaria, D.F. 04510, México
* Corresponding author (irmofel{at}servidor.unam.mx)
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ABSTRACT
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Cooking time is an important trait in the breeding of common beans (Phaseolus vulgaris L.), especially in Mexico where 96% of beans consumed are prepared in the household. Because of the characteristics of the cooking time trait, a method of indirect selection would increase selection efficiency. The objective of this study was to identify random amplified polymorphic DNA (RAPD) markers associated with the trait and estimate genetic parameters of cooking time. For that purpose, 104 recombinant inbred lines (RILs), derived from contrasting cooking time bean cultivars were evaluated for three consecutive generations (F5 to F8). In each generation, cooking time was determined and plants in the F7 generation were genotyped. One marker was associated with cooking time. The polymorphic UNAM-16 of 310 base pairs (bp) explained 23% of the variation in cooking time of the lines studied. Narrow sense heritability (h2) was estimated for cooking time, as was the number of genes involved in the trait. A high value of h2 (0.74) was estimated for cooking time. Also, it was estimated that two genes control the cooking time trait.
Abbreviations: bp, base pairs • BM, cultivar Bayo Mecentral • BV, cultivar Bayo Victoria • CEVAMEX, Centro Valle de México • h2, heritability • masl, meters above sea level • PCR, polymerase chain reaction • RAPD, random amplified polymorphic DNA • RILs, recombinant inbred lines
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INTRODUCTION
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FOR MANY Latin American countries, common beans are an important source of dietary protein. However, bean consumption is limited by the long cooking time required to achieve the tenderness acceptable to the consumers. Because of the long time required for cooking, the amount of fuel needed for bean preparation is greater than for other foodstuffs.
The biochemical basis of the bean cooking phenomenon has been studied and shown to result from pectin solubilization (Moscoso et al., 1984; Hincks and Stanley 1986; Rozo et al., 1990; Shomer et al., 1990; Liu, 1993). As a consequence of pectin solubilization, the middle lamella softens and allows the separation of adjacent whole cells (Jones and Boulter 1983; Hincks and Stanley 1986). Cell separation contributes, in a very important way, to the acquisition of palatability (texture and flavor) acceptable to consumers. Therefore, rate of cooking might be related to the thermal solubility properties of pectic substances which in turn depend on pectin composition (BeMiller, 1986). A major drawback to selection for beans with short cooking time is that screening a large number of bean experimental lines for cooking time and/or pectin composition is expensive and time consuming.
Since cooking time is important to consumers, while yield is important to producers, genotypes combining higher seed yield per unit land area with short cooking times would be useful. This combination would increase value to both producers and consumers. Knowledge of heritability of seed short cooking time is important to bean breeders for developing short cooking time and high yielding cultivars. Because of the time and cost considerations in evaluating cooking time, marker-assisted selection for this trait could be useful.
The objectives of this research were to determine the inheritance of cooking time in a population of bayo type dry bean and to identify RAPD markers associated with this trait.
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MATERIAL AND METHODS
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Plant Material
The experimental material was a set of 104 F5 RILs from a cross between two P. vulgaris cultivars. Bayo Mecentral (BM) selected for its short cooking time and Bayo Victoria (BV) for its long cooking time. Both cultivars are highly productive and resistant to anthracnose, a disease caused by Colletotricum lindemuthianum (Sacc. & Magnus) Lambs.-Scrib.]. RILs and parental cultivars were sown in the spring of 1996 at the Valle de Mexico Experimental Station (INIFAP), located at 19°29' N, 98°53' W and 2240 masl. The soil type was eutric cambisol (FAO classification). The experiments were planted in a randomized complete block design with two replications. Plots size were four 5-m rows each. The plant stand was 10 plants per meter of row, and the distance between rows was of 0.8 m. In addition to the rainfall that occurred during the growing season, the crop received a supplementary irrigation (±50 mm) at the flowering stage. At the time of planting 40 kg ha-1 of N and 40 kg ha-1 P2O5 fertilizer was preplant incorporated. Weed control was achieved with cultural practices and by hand at the preflowering stage in the beginning of pod formation in every year. For disease control, an application of copper oxychloride (Cupravit, Bayer) in a 3 kg ha-1 dose at the beginning of pod formation was done. When plants were at harvest maturity, two plants from each row in a plot were separately threshed and seed used for cooking evaluations. During the spring of 1997 and 1998 the RILs were advanced to the F7 and F8 generation. The respective parents were sown with each population each year. Cooking time of each RIL was performed two times in each generation (F6, F7, F8) on replicated plants.
Evaluation of Cooking Time
Bean cooking time was determined on 25 seeds selected from each replication of each RIL and each parent. Seeds were soaked in distilled water for 18 h at 25°C. After the beans were soaked each was positioned in a cylindrical well of 25-well Mattson pindrop cooker (Jackson and Varriano-Marston, 1981). The piercing tip of a 200-g rod was placed in contact with the surface of each bean. Cooking time was calculated as the elapsed time from the initiation of cooking until 18 of the 25 pins (80%) of the instrument had dropped and penetrated seeds in the cooker. Data were taken from duplicate samples of each RIL in each generation.
DNA Extraction and RAPD Marker Analysis
From the 1997 trial, one young trifoliate leaf was harvested from each of 10 plants of each parent and each of the selected plants (97) in the F6:7. Tissue was lyophilized, ground, and stored at -80°C. DNA was extracted by means of the method described by Llaca (1992). The 80 random 10-base primers (decamers) used in this study were previously shown to amplify bean genomic DNA. Polymerase chain reaction (PCR) reaction conditions, in the final volume of 25 µL, were as follows: 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.0 mM MgCl2, 0.2 mM of dNTPs, 1.0 unit Taq polymerase (GIBCO-BRL), 50 ng template DNA, and 0.6 µM of primer. Amplification was performed in a Perkin Elmer GeneAmp DNA thermal cycler (Perkin Elmer, Foster City, CA) programmed one cycle of 94°C for 3 min; three cycles of 1 min at 94°C, 1 min at 36°C and 2 min at 72°C; 36 cycles of 10 s at 94°C, 20 s at 40°C, and 2 min at 72°C. The 36 cycles were followed by 5 min final extension at 72°C. Amplification products were resolved by electrophoresis at 3 V/cm in a 1.4% (w/v) agarose gel and stained with 1 µg/mL ethidium bromide. PCR reactions showing polymorphism were repeated 1 to 3 times to control for amplification artifacts.
To detect polymorphic RAPDs associated with cooking time, only 34 F7 RILs (each represented by an individual plant) showing the most extreme phenotypes (17 RILs with long and 17 RILs with short cooking time) were genotyped with parental polymorphic RAPD (Lander and Botstein, 1989). Primers associated with cooking time were subsequently screened in the remaining of RILs.
Statistical Analysis
Analyses of variance for cooking time of the F6, F7, and F8 lines were performed with the general linear model procedure (VAR COM) of SAS (SAS Institute Inc., 1988). Since in each growing season the weather was different, each crop year was considered a separate environment. Genotypes and environments were considered to be random.
Heritability on line mean basis (Hallauer and Miranda, 1988) was calculated as follows: h2 = 
2G/
, where 2G = genetic variance, 2 = experimental error, 2GE = genotype x environment interaction variance, r = number of replicates, and e = number of environments. Values were derived from variance components estimated by means of expected mean squares (Dudley and Moll 1969).
The number of genes was estimated by Wright's Eq. (1968), where n = (GR)2/4.27(2GF) and n = gene number (GR)2 = genotypic amplitude among lines 2GF = genetic variance in Fn.
In each generation, observed segregation ratios for short cooking time/long cooking time phenotypes were compared with the respective segregation ratios expected from one-, two-, and three-gene models by chi-square analyses.
The association between individual markers and the phenotypic data was detected by simple regression analyses by means of the procedure PROC REG of SAS (SAS institute, 1998). The coefficient of determination (R2) resulting from the multiple regression analysis indicated the level of phenotypic variation accounted by the selected markers.
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RESULTS AND DISCUSSION
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The two parents contrasted in cooking time while BM exhibited short cooking time (53 ± 15 min) in all three years; BV exhibited long cooking time with a large standard deviation (153 ± 46 min). Nonetheless, the parental cultivars had nonoverlapping distributions.
In all generations tested, the frequency distribution curve was continuous and skewed in favor of the short cooking parent (Fig. 1). This distribution indicated that cooking time is an oligogenic-inherited trait, and thus a few genes may be involved in the expression of seed cooking time. The skewness of the distribution in favor of the short cooking parent may suggest that the trait has maternal effect, as found by Elia et al. (1997), or that the trait is governed by dominant genes. To decide between these possibilities, it will be necessary to study cooking time by means of the proper mating design.
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Fig. 1. Frequency distribution of the RILs from BM xBV for cooking time. Arrows indicate the cooking time for the parents.
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Analyses of variance of data collected on cooking time in 1996, 1997, and 1998, showed significant effects (P 
0.0001) of environment and genotype (Table 1). The variance component for the genotypic effect was larger than the one for the environmental effect. This indicates that genetic differences among BM x BV RILs accounted for most of the variability in bean cooking time.
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Table 1. Mean squares (MS) and variance components (VC) from an analysis of variance of data on cooking time of 104 F5:8 RILs from a cross of B. Mecentral x B. Victoria and combined over three years (1996, 1997, and 1998) in Mexico.
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The variance component of the genotype x environment interaction on bean cooking time in three environments was also significant (P < 0.0001). This conclusion is further supported by the fact that the mean cooking time for genotypes varied among environments from 60 min in 1996 to 82 in 1998 (Table 2). These data agree with Proctor and Watts (1987) who evaluated cooking time in different growing locations and indicated the environmental influence on cooking time.
The narrow sense heritability estimate for bean cooking time was derived from formula provided by Hallauer and Miranda (1988) using variance components from the combined analysis of variance of 104 F5:8 progeny lines grown in 1996, 1997, and 1998 (Table 1). The magnitude of the calculated heritability was 0.74. According to Stansfield (1995), traits with a value of h2 higher than 0.5 are considered to have a high heritability. The value reported here is lower than the one reported by Elia et al. (1997) who reported a value of h2 = 0.9; however, our value is very close to the value (h2 = 0.76) reported for the same trait in Vigna unguiculata (L.) Walp. by Nielsen et al. (1993). The difference in the magnitude of h2 in the current study and that of Elia et al. (1997) study could be due to the fact that each study was conducted in a vastly different environment (Mexico vs. Tanzania). The value of a heritability estimate depends both on population and environment (Stansfield 1995).
Since bean cooking time in this study behaved as an oligogenic trait, the phenotypic data (Fig. 1) were subjected to a quantitative genetic analysis. The RILs in each generation were separated into long and short cooking time phenotypes. In each generation, classification was based on the range in cooking time observed in each parent; RILs exhibiting cooking times less than 80 min in F6 and less than 100 min in F7 and F8 were classified as short cooking time. RILs above those values were classified as long cooking time lines.
The progeny of each generation segregated for short and long cooking times in a ratio approaching 3:1 (Table 3), which corresponds to a two dominant gene model. Chi-square analysis indicated that for the F7 and F8 generations a relatively high level of probability occurred between the observed and expected values (Table 3). This suggests that cooking time was controlled by two genes. The hypothesis of two genes being responsible for bean cooking time was confirmed by the use of the genetic variance and genotypic amplitude among lines (Cockerham, 1963). The average number of genes estimated by this method for generation F6 and F7 was 1.9, a good fit to a two dominant gene model. The highly consistent results by the two methods used to calculate number of genes confirmed that two genes were responsible for bean cooking time in this study.
Identification of RAPD markers
Of the 80 decamer primers used, only 14 were polymorphic when analyzed against the parents (BM and BV). Each polymorphic primer resulted in the amplification of 1 to 4 discernible fragments, with a total of 23 discernible DNA fragments, ranging from 2320 to 310 bp. The polymorphic primers were screened in 34 individuals of the F7, showing the most extreme phenotypes (17 RILs of long and 17 RILs of short cooking time).
Out of the primers screened, three generated polymorphic DNA fragments that were apparently associated with cooking time. These RAPDs were scored against 70 RILs from the BM x BV population (Fig. 2). One of these RAPDs, UNAM 16, 310 bp (generated by a 5'GGCTGCAGAA 3' decamer), was found to be associated with the short cooking time phenotype (R2 = 0.21, P = 0.0001).
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Fig. 2. RAPD amplification of a RILs population of BM x BV. Parents BM and BV, lanes 1-2 respectively; lanes 3-21, different RILs. Key to individuals: RILS showing short cooking time, 4 to 8, 10, 12 to 15, 16 to 17, 19 (A), 1 to 2, 4 to 6, 8 to 11, 14 to 17 (B), 1 to 7, 10 to 17, 19 (C). The others are RILS showing long cooking time. The arrow indicates the RAPD band with molecular weight of approximately 310 bp (UNAM-16) which is only amplified in the short cooking parent. Lanes m1 through m2 molcular size markers ( X174 and  HindIII, respectively; size of band indicated in bp).
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Although the association between the marker and the trait is low, these findings will serve as starting point to investigate further the application of DNA markers in marker-assisted selection for short cooking time in common beans. Since the heritability of cooking time is high (this study and Elia et al., 1997) and the genotype x environment interaction small, it is worth the effort to pursue the search for a marker(s) to increase the precision in selecting genotypes with short cooking times. Until marker-assisted selection for cooking time becomes feasible, breeders should continue to select for short cooking genotypes on the basis of progeny means.
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CONCLUSIONS
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The low relationship between the associated RAPD marker and cooking time at the present do not support its use as an indirect selection tool. The large genotype effect of cooking time coupled with the high heritability for this trait (0.78) suggest that selection based on the trait itself may allow for progress in breeding.
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge the expert technical assistance in growing the RILs of Dr. R. Garza-Garcia, and the critical review of the manuscript to Dr. J. Lynch and Prof. AC Leopold. We appreciate the help of Dr. F. Castillo in the genetic analysis of the data. This work was supported by Alianza para el Campo Estado de Mexico and by grant 208998 from DGAPA, UNAM.
Received for publication December 21, 2001.
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ABSTRACT
INTRODUCTION
