Inbreeding and Gene Action in Butternut Squash (Cucurbita moschata) Seed Starch Content
Endocría y Acción Génica para el Contenido de Almidón en Semilla de Zapallo (Cucurbita moschata)
Sanín Ortiz Grisales1; Lucy Viviana Bastidas Burbano2; Ginna Alejandra Ordoñez Narváez3; Magda Piedad Valdés Restrepo4; Diosdado Baena García5 and Franco Alirio Vallejo Cabrera6
1 Associate Professor. Universidad Nacional de Colombia -Sede Palmira - Facultad de Ciencias Agropecuarias. Apartado Aéreo 237, Palmira, Colombia. <sortizg@unal.edu.co>
2 Agroindustrial Enginner. Universidad Nacional de Colombia -Sede Palmira - Facultad de Ciencias Agropecuarias. Apartado Aéreo 237, Palmira, Colombia. <lvbastidasb@unal.edu.co>
3 Master Student. Universidad Nacional de Colombia -Sede Palmira - Facultad de Ciencias Agropecuarias. Apartado Aéreo 237, Palmira, Colombia. <gaordonezn@unal.edu.co>
4 D.Sc. Student. Universidad Nacional de Colombia -Sede Palmira - Facultad de Ciencias Agropecuarias. Apartado Aéreo 237, Palmira, Colombia. <mpvaldesr@unal.edu.co> ]]>
5 Full Professor. Universidad Nacional de Colombia -Sede Palmira - Facultad de Ciencias Agropecuarias.Carrera 32 No. 12-00, Palmira, Valle, Colombia. <dbaenag@unal.edu.co>
6 Full Professor. Universidad Nacional de Colombia -Sede Palmira - Facultad de Ciencias Agropecuarias. Carrera 32 No. 12-00, Palmira, Valle, Colombia. <favallejoc@unal.edu.co>
Received: February 21, 2013; accepted: September 13, 2013.
Abstract. The effect of inbreeding and gene action on butternut squash (Cucurbita moschata Duch.) seed production and seed starch content was evaluated at two locations in the department of Valle del Cauca, Colombia, using six accessions (S0) and their inbred lines S1 and S2. Significant differences were found between the accessions and inbred lines, but not between localities. The seed production and seed starch content showed no significant effects of inbreeding depression. An additive-type gene action predominated, suggesting that recurrent selection could be the best strategy to increase the frequencies of genes promoting seed production and seed starch content.
Key words: Butternut squash, genetic improvement, nutrition, genes.
Resumen. Se evaluó el efecto de la endocría y la acción génica en la producción de semilla y en el contenido de almidón en la semilla de zapallo (Cucurbita moschata Duch.) en dos localidades del departamento del Valle del Cauca, Colombia, utilizando seis accesiones (S0) y sus respectivas líneas endocriadas S1 y S2 . Se detectaron diferencias significativas entre accesiones y líneas endocriadas pero no entre localidades. La producción de semilla y el contenido de almidón no presentaron efectos significativos de depresión endogámica. Predominó la acción génica de tipo aditivo, sugiriendo que la selección recurrente podría ser la mejor estrategia para incrementar las frecuencias de los genes que favorecen la producción de semilla y contenido de almidón en la semilla.
Palabras clave: Ahuyama, mejoramiento genético, nutrición, genes.
]]> Only the pulp of butternut squash (Cucurbita moschata Duch.) is used in human and animal nutrition. The seeds are discarded, which could represent a potential loss of valuable nutrients because little information is available on the potential value of these seeds as a source of nutrients (Karaye et al., 2012).
Each squash fruit can contain between 30-150 g of seed, adding up to 500-1000 kg/ha (Ortiz, 2009). These chewable seeds have a sweet, nutty flavor, attributable to their ethereal extract content, which also confers a seed oil content higher than 45% (Applequista et al., 2006; Ortiz et al., 2009). The only commercial value generally given to squash seeds is, however, its use as grain (Criollo et al., 1999). Squash seed cakes have 50% crude protein (CP) and a gross energy (GE) above 4.5 megacalories per kg (Ortiz et al., 2009).
Seed starch yield should be measured quantitatively to identify the S0 populations and their inbred S1 and S2 lines, presenting greater consistency in overall seed starch production and their response to inbreeding (Ortiz, 2009).
Inbreeding has been used in breeding programs to fix phenotypes of agronomic interest, identify favorable genotypes and reduce the percentage of heterozygotes in populations (Ortiz et al., 2008). Inbreeding, however, not only reduces the population mean, causing a loss of vigor (fitness), particularly in allogamous plants, but also increases genetic variance between families and reduces it within families (Falconer and Mackay, 1996), with a gradual increase of additive variance (sA) at the expense of dominance (sD) in completely homozygous lines (Ceballos, 1998).
In the case of butternut squash, inbreeding should be considered as mandatory when selecting good parental material. Although inbreeding depression may occur, as in most allogamous species, it is almost imperceptible in cucurbits (Ortiz et al., 2008) but has been recorded in advanced lines of the cucumber, squash, melon and watermelon (Robinson, 2000; Cardoso, 2004).
In cucurbits, particularly in butternut squash, inbreeding depression depends on the genetic structure of each population, so it is possible to select inbred lines So, S1 and S2 that outperform S0, attributable to the accumulation of favorable, homozygous genes (Ortiz et al., 2009), or lines that can be used as parental material in hybridization breeding programs.
Although some information is available on the effect of inbreeding on traits related to fruit yield and quality (dry matter, starch, carotene and protein in pulp), little is known about the impact it has on seeds and seed components.
This research aimed to study the performance of six accessions of butternut squash in three generations of inbreeding (So, S1 and S2) and the resulting impact on seed production and seed starch content.
MATERIALS AND METHODS
]]> Location. The study was conducted at two locations in the department of Valle del Cauca, Colombia. The first, the Experiment Center of the Universidad Nacional de Colombia-Palmira Campus (CEUNP, its Spanish acronym), is located in the municipality of Candelaria (03°25'N latitude, 76°25'W longitude) at an altitude of 973 m above sea level, with a mean annual temperature of 26 °C, an annual precipitation of 1,100 mm, and 76% relative humidity (Ortiz, 2009). The second, a facility of the Servicio Nacional de Aprendizaje (SENA), is located in the municipality of Buga (3°53'N latitude, 76°18'W longitude) at an altitude of 969 m above sea level, with a mean annual temperature of 23 °C, an annual precipitation of 980 mm, and 74% relative humidity (Ortiz, 2009).The macromolecular analysis of the squash fruits was performed at the Animal Nutrition and Agricultural Prospective Laboratory of the Universidad Nacional de Colombia-Palmira campus.
Genetic material. Six open-pollinated So accessions were used as well as their S1 and S2 inbred lines (Ortiz et al., 2009) and the commercial check variety UNAPAL-Bolo Verde (Table 1).
Experimental methodology. The accessions and their inbred lines were planted in a field using a randomized complete block experimental design with four replicates and five plants per replicate. At harvest, three plants were gathered from the center of each plot and one fruit was selected from each of these plants to submit to laboratory analysis.
Test variables. Seed production per plant (in grams) and seed starch content (%) were measured using the method proposed by the American Association of Cereal Chemists (BeMiller and Low, 1998; Peris-Tortajada, 2000).
Statistical analysis. The 1-ws/w0 model was used to estimate inbreeding depression, where ws is the mean of the trait in inbred plants and w0 the mean trait in non-inbred plants (Hayes et al., 2005). The positive or negative relationship determines the type of gene action that controls the expression of the quantitative trait (Fox, 2005). The statistical significance of inbreeding depression in S1 and S2 was estimated using Student's t-test at the 0.05 and 0.01 levels of probability.
The model proposed by Mather and Jinks (1982) was used to estimate the genetic effects associated with additivity and dominance, as described below:
Mean of the zero generation of inbreeding (
):
): 
Based on the above expressions, it can be deduced that:
The model proposed by Gardner and Lonnquist (1959) was used to estimate the average degree of dominance (a.d.d.):
RESULTS AND DISCUSSION
]]> Table 2 shows the mean for each trait in the three generations of inbreeding (S0, S1 and S2) and at both localities.No significant differences were found between the generations of inbreeding or between the locations. Inbreeding had no significant effect on the average performance of the accessions. Its effect depended on the genetic complexity of the trait being studied, the geographical origin of the accession and the level of heterozygosity, combined with the ability of each accession to respond differently to the inbreeding process, which could be attributed to the fact that the starch synthesis pathway is not only controlled genetically but is also influenced by the metabolic phase during which sugar conversion occurs (Tofiño et al., 2006). This corresponds to a physiological response of the genotype to the environment as indicated by Falconer and Mackay (1996), where inbred individuals are highly sensitive to the effects of environmental variation.
Response to inbreeding. The significant negative values obtained by the model ID= [1-(Ws/W0)]100, where ID is inbreeding depression, are associated with favorable responses to the inbreeding process. Significant positive values correspond to evident depressive effects while non-significant values, both positive and negative, indicate insensitivity to inbreeding (Table 3).
ID values, such as -75.7** (S0-S1) and -60.4** (S0-S2) for the trait seed production/hectare in the case of accession 2, indicate a favorable response to inbreeding as a result of a higher concentration of favorable homozygous genes, which increases seed production/hectare, as compared with values of 39.7** (S1-S2) and 37.1** (S0-S2) in the case of accession 34, which are associated with depressive effects and could be attributed to the manifestation of homozygous genes that reduce seed production/hectare. This indicates that there is no pattern of response to inbreeding in the case of these accessions and traits (Ortiz et al., 2009).
According to the differential response of S0 accessions and their inbred lines, three groups were defined:
G1 (-, -) Negative response to inbreeding in S1 and S2. Inbreeding depression is moderate in S1 and S2, for example in the case of the trait seed production/plant in accession 34 (Table 4).
G2 (+, -) Positive response to inbreeding in S1 but presenting inbreeding depression when passing from S1 to S2, as occurs in accessions 6, 34 and 80 for seed starch content and in accessions 2, 28, 34, 79 and 80 for seed production/hectare. The positive response to inbreeding assumes highly significant negative values; in other words, inbreeding does not suppress a trait per se, which characterizes the performance of butternut squash (Ortiz, 2009).
]]> G3 (+, +) Positive response to inbreeding in S1 and S2. Inbreeding helped improved the mean performance of accessions in S1 and S2, for example seed production/plant in accessions 2 and 6 and seed production/hectare in accession 6 (Table 4).The information given in Tables 3 and 4 indicates that, unlike other self-pollinated species such as maize, inbreeding depression in cucurbits is uncertain, at least for the traits being evaluated in this study, and depends not only on the accession's degree of heterozygosity in S0, but also on the "remnant" genetic load manifested in S1 and S2. As a hypothesis, it can be assumed that, in the case of accessions that have been geographically isolated for a long time, undesirable genes responsible for inbreeding depression may have been removed through a natural process of inbreeding in related crosses so that these accessions, contrary to what is assumed, have a high degree of homozygosity when collected and entered into the program's work collection. As a result, when these accessions are submitted to inbreeding they are either insensitive or respond favorably or unfavorably to the process, as found by Espitia et al. (2006) regarding fruit traits.
Type of gene action. If S0 accessions are heterozygous, inbreeding is expected to depress their performance because a certain number of heterozygous loci acquire the recessive homozygous condition. If these recessive homozygous genes are part of the genetic load of the inbred accession, then the depressive effect on the population mean will be evidenced (Sahagún and García, 2009).
When S1< S0, parameters d and D/A, which are associated with deviations in dominance and a.d.d., are less than zero as a result of loss of heterozygosity, which, in addition to the residual heterozygosity in S1, reduces the mean value of the inbred accession (effect of reverse dominance) as in group G (-, -) (Table 4).
When S1≈S0, parameters d and D/A are approximately zero and the gene action that controls trait expression is additive in nature or, if not, then there is a balance between positive and negative deviations of dominance (d+≈d-), for example in the case of the trait seed production/hectare in accession 80.
When both d and D/A present positive values, the partial dominance significantly increases the means of the evaluated traits in inbred accessions as occurred for most of the traits under study, with a.d.d up to 0.55, as occurred for the variable seed production/hectare in accession 79 (Table 5).
The parameter m + a was positive in all accessions (Table 5), attributable to a higher proportion of alleles with favorable additive effects for trait expression in loci acquiring the homozygous condition after selfing.
The performance of the trait seed production/plant in accession 80, with a.d.d. of -1.11, should be highlighted because inbreeding in this particular case creates an imbalance in the dominance relationships in heterozygous loci so that intralocus interactions favor the alleles depressing the expression of said trait.
Different breeding strategies for seed production and starch content can be derived from the above results such as the following: advancing to S2 those accessions suffering inbreeding depression for subsequent crossbreeding between inbred lines to obtain commercially valuable hybrids; selection of S1 or S2 inbred populations presenting a mean performance superior to that of the S0 accession; or simply selection of S0 accessions for traits insensitive to inbreeding.
]]>CONCLUSIONS
The parameter D/A indicates that, with the exceptions already mentioned, inbreeding in butternut squash helps increase additive gene action as a result of the increase in the number of homozygous loci after selfing, which in turn significantly favors the mean response of inbred accessions. Similarly, the loci remaining heterozygous in S1 (residual heterozygosity) express levels of dominance in favor of traits associated with seed production and seed starch content.
The response to inbreeding in butternut squash varies among accessions, generations of inbreeding (S1, S2) and traits, with no clearly defined pattern.
The predominance of additive gene action over the dominance type for the traits under study suggests that a recurrent selection program could serve as a strategy to increase the frequencies of genes that promote the expression of traits associated with seed production and starch content in butternut squash.
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