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Published online 22 January 2007
Published in Crop Sci 47:162-167 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP BREEDING & GENETICS

A Quantitative Analysis of Resistance to Mycosphaerella Blight in Field Pea

Roger Zhanga, Sheau-Fang Hwangb,*, Bruce D. Gossend, Kan-Fa Changc and George D. Turnbulle

a Alberta Research Council, Vegreville, AB Canada T9C 1T4
b Alberta Agriculture, Food and Rural Development, Crop Diversification Centre North, Edmonton, AB Canada T5Y 6H3
c Alberta Agriculture, Food and Rural Development, Field Crop Development Centre, Lacombe, AB Canada T4L 1W1
d Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK Canada S7N 0X2
e Univ. of Alberta, Edmonton, AB Canada T5B 4K3

* Corresponding author (Sheau-Fang.Hwang{at}gov.ab.ca)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Mycosphaerella blight, caused mainly by Mycosphaerella pinodes (Berk. and Blox.) Vestergren, is the most important foliar disease on field pea (Pisum sativum L.) in western Canada. A quantitative trait analysis of resistance to M. pinodes was undertaken on five crosses with reciprocals of P. sativum to examine broad-sense (H2) and narrow-sense (h2) heritability, minimum number of genes involved (MNG), midparent heterosis (MPH), cytoplasmic inheritance, and epistasis. Mean H2 was 0.75 (range 0.67–0.80) and mean h2 was 0.59 (range 0.41–0.70), indicating that additive variance is important and that improvement in resistance can be achieved through breeding. Mean MNG was 2.16 genes (range 0.06–6.22), indicating that genes for resistance differed among parent lines. Mean MPH was 50% (range 47–57%), indicating that heterosis did not influence the expression of resistance to M. pinodes. There was no difference between the mean of any F1 population and its reciprocal, indicating lack of maternal inheritance. The mean of the epistatic points was –0.01 (range –0.1 to 0.12), indicating that epistasis was not important in these crosses. These results will further the understanding of the natural genetic diversity for disease resistance to M. pinodes in P. sativum.

Abbreviations: ADD, average degree of dominance • H2, broad-sense heritability • h2, narrow-sense heritability • MNG, number of genes involved • MPH, midparent heterosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
MYCOSPHAERELLA BLIGHT can cause substantial yield reduction in field pea in western Canada (Xue et al., 1998; Chang et al., 1999). The most important pathogen in the disease complex is M. pinodes (anamorph: Ascochyta pinodes L.K. Jones) (Bretag and Ramsey, 2001), although A. pisi (Lib.) and Phoma medicaginis Malbr. & Roun. in Roun. var. pinodella (L.K. Jones) Boerema (syn. A. pinodella) have also been reported (Bretag, 1989).

Resistance to M. pinodes has been demonstrated in lines of P. sativum (Wroth, 1998). Considerable effort has been directed to develop cultivars with resistance to M. pinodes (Wallen, 1965; Durieu and Ochatt, 2000), because this would be a cost-effective and environmentally sound management strategy (McPhee, 2003). Resistance is generally believed to be multigenic (Timmerman-Vaughan et al., 2002) and quantitatively inherited (Roger et al., 1999; Hwang et al., 2006), but there are a few reports of resistance controlled by major genes (e.g., Clulow et al., 1991). Evidence of a strong environmental influence on blight severity provides support for the complex inheritance of resistance (Meiners, 1981). Breeding efforts have been hampered by deficiencies in our understanding of the inheritance of resistance (Kraft et al., 1998). Thus, a detailed study of the heritability of resistance is needed to speed the development of cultivars with improved resistance to Mycosphaerella blight (Fondevilla et al., 2005; Mahuku et al., 2004).

In this study, the disease reaction of five crosses with reciprocals, including F1 and F2 generations, F1 backcross progenies, and the parent lines, were used to quantify a range of general indices of inherited resistance to M. pinodes in P. sativum, which include broad-sense (H2) and narrow-sense (h2) heritability, MNG, MPH, and the impact of cytoplasmic inheritance and epistasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Plant Materials and Field Design
The seed source and disease reaction for each of the six parental lines in the study are listed in Table 1, based on disease assessments for resistance to M. pinodes in a previous study (Hwang et al., 2006). No completely resistant genotype has been discovered in P. sativum (Zhang et al., 2006), but various levels of genotypic resistance to M. pinodes have been identified through consistent variation in infection levels. The six parental genotypic lines were selected to represent a wide range of susceptibility levels from moderately resistant to highly susceptible, to ensure that the crosses represented an extensive assortment of potential interactions. Five pairs of reciprocal crosses, representing different segregating combinations, were made in 2004 in the field (Table 2). The F1 plants were both selfed and backcrossed to their parents in a greenhouse over the winter of 2004–2005. The blight reaction of F1, F2, backcross, and parental populations were assessed in a field trial in the spring of 2005. A field trial was conducted at the research farm of the Alberta Research Council, Vegreville, AB, in a field where P. sativum had never been grown. Each plot consisted of one row, 10 m in length, with plants spaced approximately 30 cm apart within the rows. Rows were spaced 90 cm apart. The plots of each cross were planted as a block, arranged in the order of parental lines, F1 and reciprocal F1, backcrosses, and F2 lines. Plots of the susceptible line PI 179449 and moderately susceptible cv. Radley were planted with every cross as controls. The seed was planted in mid-May. The seedbed had been chisel-plowed, disked, and harrowed before seeding.


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  		Table 1. Seed sources of Pisum sativum used as parents and Mycosphaerella blight reactions (Hwang et al., 2006).

 

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  		Table 2. Variance analysis of genetic effects for resistance to Mycosphaerella pinodes in five pairs of reciprocal crosses of Pisum sativum.

 
Inoculum and Inoculation
A highly aggressive isolate of M. pinodes (Iso-1) was collected from an infected crop of P. sativum in Alberta in 2003 (Hwang et al., 2006) and maintained on potato dextrose agar medium. To induce sporulation, the isolate was grown on oat water agar medium (water agar plus 1% oat powder) for 4 wk at 20/15°C, under a 16-h photoperiod and a light intensity of 300 µmol m–2 s–1. After incubation, the oat water agar dishes were flooded with sterile water and scraped to release the conidia. The spore suspension was filtered through cotton gauze, the numbers of conidia were assessed using a hemacytometer, and the spore concentration was adjusted to 100 conidia µL–1 using sterile water.

One liter of fresh spore suspension was mixed evenly into an inoculum medium consisting of 5 kg peat moss, 1 kg autoclaved soil, and 0.5 kg sand (Zhang et al., 2006). This inoculum was broadcast in the field plots at approximately 1 x 107 spores m–2 at the early flowering stage (June 26) (Zhang et al., 2006). All of the individual plants of each generation were rated once for Mycosphaerella blight in mid-August at the late flowering stage, using a 0 to 9 scale (Xue et al., 1996). We chose to inoculate this test, rather than relying on background inoculum, to guarantee uniform distribution of primary inoculum under conditions that were suitable for blight development.

Analysis
Analysis of variance for each trial was performed using PROC GLM, and variance components were calculated using PROC VARCOMP of SAS (SAS Institute Inc., Cary, NC). The variance of genetic effects ({sigma}2Gen) within F2 populations ({sigma}2F2) and the associated mean square expectations were derived (Lande, 1981) as:

Formula 1[1]

Formula 2[2]
where {sigma}2Env is the variance of environmental effects estimated from {sigma}2F1, {sigma}2P1, and {sigma}2P2, which are the variances of blight severity in F1 and parental lines, respectively. The variance among generations was analyzed using generation means (Hayman, 1958).

The estimates of H2 and h2 were calculated following Mather and Jinks (1971) as:

Formula 3[3]

Formula 4[4]
where {sigma}2P1, {sigma}2P2, {sigma}2F1, {sigma}2F2, {sigma}2BC1F1, and {sigma}2BC2F1 are variances of blight severities in parental, F1, F2, BC1F1, and BC2F1 generations, respectively.

The average degree of dominance (ADD) estimate was calculated (Mather and Jinks, 1971) as:

Formula 5[5]
where M P1, M P2, and M F1, are the mean of blight severity in parental and F1 generations, respectively.

The MNG controlling the resistance of each cross was estimated (Lande, 1981) as:

Formula 6[6]
where MP1 is the mean of Parent 1, MP2 is the mean of Parent 2, {sigma}2F2 is the variance of the F2 generation, and {sigma}2(P1, P2, F1) is the pooled variance of P1, P2, and the F1 generation, respectively.

The maternal inheritance pattern of extranuclear genes contributing to resistance to M. pinodes was estimated from the comparison of reciprocal F1 progeny of the five paired combinations (Sincich, 1992):

Formula 7[7]
with degrees of freedom equal to (n1 + n2) – 2; X1 and X2 are the means of the reciprocal crosses (e.g., Radley x Highlight and Highlight x Radley); s21 and s22 are the variances of these crosses, respectively; n1 and n2 are the number of plants in each cross; and md is the mean difference in the null hypothesis. In this case, the null hypothesis was that there was no difference between population means, so md = 0.

Midparent heterosis was calculated as the performance of the F1 compared with the mean performance of its parents (Fehr, 1991):

Formula 8[8]
where MF1, MP1, and MP2 are mean blight severity in the parents and F1 generation, respectively.

Epistasis estimates were calculated as a comparison of generalized least squares means of the population (LSMPopulation) with that of the midparent (LSMParent) (Goodwill, 1975). The relative parent values were calculated as follows (Burnham et al., 2003):

Formula 9[9]
Epistatic gene action was demonstrated if the population mean was significantly different from the midparent based on a t-test (Upadhyaya and Nigam, 1998).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Genetic Variation of Resistance
Generation mean squares from the analyses of variance of Mycosphaerella blight ratings are presented in Table 2 and the population distribution of the five combinations are shown in Fig. 1 . Significant differences in disease severity were detected in 8 of 10 crosses, but there were no differences in the crosses between PI 179449 x JI 716; both lines were highly susceptible. This pattern of response indicates that differences in disease severity among the generations and among the crosses were due to genetic differences among the parental lines for resistance to M. pinodes. These results are consistent with our previous reports (Hwang et al., 2006; Zhang et al., 2006).


Figure 1
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  		Fig. 1. Frequency distribution of F2 populations and the range of parental and F1 populations from five reciprocal crosses of P. sativum of resistance to M. pinodes (ranged by mean Mycosphaerella blight infection severity from low to high).

 
Heritability
Mean H2 over the 10 populations was 0.75, with a range of 0.67 to 0.80 (Table 3). This value indicates that resistance to M. pinodes is moderately heritable and also indicates the degree of difference between the parental lines. This result is substantially lower than the 0.9 reported in a recent study (Wroth, 1999). This difference in heritability between the studies may be due to differences in environment or genotypes used. For a self-pollinated crop like P. sativum, h2 represents a direct measure of additive variance. Mean h2 over the 10 populations was 0.59, with a range from 0.41 to 0.70 (Table 3). The results indicate that a substantial portion of this genetic variation is additive in nature and improvement in resistance to M. pinodes can be realized through breeding (Rodriguez-Herreraa et al., 2000; Zhao et al., 2005). This relatively high value of additive variance (van Oeveren and Stam, 1992) also indicates that conventional pedigree and early generation selection methods should be effective for improving resistance (Cross et al., 2000; Navabi et al., 2004; Stelling et al., 1990).


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  		Table 3. Broad-sense heritability (H2), narrow-sense heritability (h2), minimum number of genes (MNG), midparent values, average degree of dominance (ADD), reciprocal effects, and epistasis of resistance to Mycosphaerella pinodes in Pisum sativum.

 
Heterosis, Average Degree of Dominance, Reciprocal Effects, and Epistasis
The F1 disease severity was slightly above the average of both parents, indicating that some effects of heterosis may exist (Table 2). However, disease severity was close to the average in all F2 generations. Mean midparent heterosis was 50%, with a range from 47 to 57% (Table 3), so heterosis was not a major factor in the expression of resistance to M. pinodes in this study. These data also indicate that there is no difference between alleles that cause a heterotic effect at a locus or that there is a low level of dominance among those alleles in the F1 generations. This appears to be the first study to examine heterosis in P. sativum for resistance to M. pinodes. However, this result is similar to that for resistance to a root parasite, broomrape (Orobanche crenata Forsk.) in P. sativum, where heterosis was not significant (Valderrama et al., 2004). In contrast, heterosis in certain cross-pollinated crops was reported to increase resistance to diseases (Lamb et al., 1993; Maertens et al., 2004).

Mean ADD for resistance to M. pinodes in these crosses was 0.11, with a range from –0.28 to 0.80 (Table 3); a value of 1.0 corresponds to complete dominance (Edwards and Lamkey, 2002). Average degree of dominance in the cross Tara x 273605 and its reciprocal were both 0.01, indicating that the gene(s) controlling M. pinodes were predominantly additive (Comstock and Robinson, 1948). Similarly, in the pair of crosses with Highlight x Radley (ADD = 0.11, –0.28), Radley x PI 273605 (–0.28, 0.40), and JI716 x PI 179449 (0.12, 0.80), resistance exhibits a low degree of incomplete dominance or predominantly additive gene action.

There was no difference between mean blight severity on the F1 and its reciprocal F1 in any cross (Table 3). This indicates that the maternal lines (which contribute the cytoplast) had no additional impact on resistance. This result supports previous reports in which genes for resistance to M. pinodes in Pisum were mapped to the nucleus (Timmerman-Vaughan et al., 2004; Prioul et al., 2004; Takahara et al., 2005).

Epistasis describes a phenotype where the deviation cannot be explained by the combined additive effect of two or more loci, but by the interaction between or among those involved genes (Falconer and Mackay, 1996). Mean epistasis for resistance to M. pinodes was estimated to be –0.01, with a range of –0.1 to 0.12 (Table 3). Neither negative values nor other results of the t-tests indicated a significant difference between each generalized least squares mean of an F2 population and its midparent (Upadhyaya and Nigam, 1998), indicating that directional epistasis was not present. This test is sensitive only to cumulative epistatic effects that tend to work in the same direction but not for specifically paired loci (Burnham et al., 2003). This result is similar to observations from a previous study on Mycosphaerella blight conducted in Australia (Wroth, 1999). The impact of epistasis and dominance can inflate values of heritability (Fernandez and Miller, 1985). However, our results indicate that epistasis and dominance are not important factors in resistance to M. pinodes in P. sativum. Therefore, we conclude that the calculated values of H2 and h2 for resistance likely result primarily from the additive impact of quantitative resistance genes.

Minimum Number of Genes
The mean MNG controlling M. pinodes resistance was 2.2 genes, with a range of 0.1 to 6.2 (Table 3). In crosses between the most resistant parental lines (Radley x PI 273605 and its reciprocal), MNG was 1.0. In crosses between the most susceptible parents (PI 179449 x JI 716 and its reciprocal), MNG was only 0.1, indicating that no segregation for resistance occurred. However, in crosses between a moderately resistant and the highly susceptible line (PI 273605 x PI 179449 and its reciprocal), MNG was 5.9. Thus, between the most resistant (cv. Radley) and most susceptible lines (PI 179449), the number of segregated factors conferring M. pinodes resistance was estimated to be 5.9 + 1.0 = 6.9, indicating that seven genes were possibly involved. This result is consistent with gene numbers reported from other host–pathogen systems, such as head blight [caused by Fusarium culmorum (W.G. Smith) Sacc.] on wheat (Triticum aestivum L.) (Snijders, 1990) and ascochyta blight [caused by Ascochyta rabiei (Pass.) Lab.] in chickpea (Cicer arietinum L.) (Tekeoglu and Santra, 2000), where gene numbers for resistance varied substantially among crosses.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
In this study, parental lines that varied widely in resistance to Mycosphaerella blight were selected to ensure that the crosses represented a wide range of potential interactions. As expected, the number of resistance factors segregating in the F2 generation differed substantially among the crosses; this had a large impact on blight severity in the F2 progeny. Quantitative analysis indicated that a simple additive model accounted for most of the genetic resistance in the 10 populations. However, inoculation with a single isolate of the pathogen created a simple system (Hwang et al., 2006). Where inoculum comes from a genetically diverse natural population (Xue et al., 1998), genetic resistance to M. pinodes may be more complex. These results will help to further our understanding of the genetics of resistance to M. pinodes in P. sativum.


   ACKNOWLEDGMENTS
 
We thank the Alberta Agricultural Research Institute, Alberta Crop Industry Development Fund, Western Grain Research Foundation, and Alberta Pulse Growers Commission for partial funding of the study, M. Hiltz for assistance with SAS, and C. Hundeby for technical assistance.

Received for publication May 9, 2006.


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




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R. X. Zhang and B. D. Gossen
Heritability Estimates and Response to Selection for Resistance to Mycosphaerella Blight in Pea
Crop Sci., November 7, 2007; 47(6): 2303 - 2307.
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