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Crop Science 40:1251-1256 (2000)
© 2000 Crop Science Society of America

CROP BREEDING, GENETICS & CYTOLOGY

Ascochyta Blight Resistance Inheritance in Three Chickpea Recombinant Inbred Line Populations

Mucella Tekeoglua, Dipak K. Santraa, Walter J. Kaiserb and Fred J. Muehlbauera

a USDA-ARS, 303 W. Johnson Hall, Washington State University, Pullman, WA 99164-6434 USA
b USDA-ARS, 59 Johnson Hall, Washington State Univ., Pullman, WA 99164-6402 USA

muehlbau{at}wsu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 
Ascochyta blight (caused by Ascochyta rabiei [Pass] Labr.) is a devastating and widespread disease of chickpea (Cicer arietinum L.). Studies of the genetics of resistance to blight have generated inconsistent reports due to year to year and between location variation in screening trial results. Most previous studies have relied on F2 or backcross populations for segregation analyses; however, inheritance patterns have been difficult to confirm because of the inability to repeat the evaluations in time and space. The objective of this study was to determine the inheritance of resistance to ascochyta blight in chickpea using recombinant inbred line (RIL) populations. The RILs were derived from two intraspecific crosses, PI 359075(1) x FLIP 84-92C(2), `Blanco Lechoso' x `Dwelley', and one interspecific cross, FLIP 84-92C(3) x C. reticulatum Lad. (PI 599072). The resistant parents, FLIP 84-92C and Dwelley, had a common source of resistance derived from ILC-72. Disease reactions of the parents and RILs were scored using a 1 to 9 scale and also by using the area under the disease progress curve (AUDPC). Segregation among RILs indicated that three recessive and complementary major genes with several modifiers conferred ascochyta blight resistance. Absence of one or two of the major genes confers susceptibility, whereas the presence of the modifiers determines the degree of resistance.

Abbreviations: AUDPC, area under the disease progress curve • QTL, quantitative trait loci • RIL, recombinant inbred line


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results and discussion
 REFERENCES
 
CHICKPEA is a self-pollinated diploid annual, with . It is the third most important food legume worldwide (FAO, 1996) after common bean (Phaseolus vulgaris L.) and pea (Pisum sativum L.). Ascochyta blight, caused by Ascochyta rabiei [Pass] Labr., is a widespread foliar disease that causes extensive crop losses in most regions of the world where the crop is commonly grown. Occurrence and severity of ascochyta blight is weather dependent and environmental conditions favorable to the chickpea crop (>350 mm annual rainfall, 23–25°C) also favor the disease. Ascochyta blight infections may cause 100% yield loss (Nene and Reddy, 1987; Jimenez-Diaz et al., 1993; Acikgoz et al., 1994). Therefore, controlling this disease is essential to ensure stable chickpea production worldwide. Since cultural practices, seed treatment, and foliar application of fungicides for control of ascochyta blight are impractical and uneconomical (Nene and Reddy, 1987), breeding for genetic resistance is a major objective of chickpea improvement programs (Muehlbauer and Singh, 1987).

Sources of resistance to ascochyta blight have been discovered in chickpea germplasm and the inheritance of resistance has been studied by several researchers (Singh et al., 1981; Singh and Reddy, 1993). A single dominant resistance gene was reported in different "desi" (small, angular, and colored seeds) cultivars (Hafiz and Ashraf, 1953; Vir et al., 1975; Eser, 1976). Singh and Reddy (1983) found one recessive gene for blight resistance in ILC-191 and one dominant gene for resistance in ILC-72, ILC-183, ILC-200, and ILC-4935, all of which are "kabuli" (large, ramshead–shaped, and beige-seeded) germplasm lines. Tewari and Pandey (1986) also found two dominant genes in EC-26446, PG-82-1, P-919, P-1252-1, and NEC-2451, and one recessive gene in BRG-8. Kusmenoglu (1990) found two complementary recessive genes for ascochyta blight resistance. Dey and Singh (1993) claimed that two complementary dominant genes controlled resistance and interallelic interactions influenced the simple Mendelian segregation of these genes. There also is evidence that other genes modify the expression of blight resistance (Muehlbauer and Singh, 1987).

Since allelism tests among the reported sources of resistance have not been conducted, the actual number of unique genes for resistance to blight is unclear. In addition, because different screening and disease assessment techniques were used in each study, the same resistance gene could be assigned as a recessive gene in one study and as a dominant gene in another (Ahmad et al., 1952; Hafiz and Ashraf, 1953). The lack of standardized methodology, including control of environmental conditions, restricts comparisons of results from different studies.

Variations in disease reaction across years and locations have been reported for numerous lines (Singh et al., 1981). However, all inheritance studies concerning ascochyta blight resistance in chickpea have been performed using F2 or backcross populations grown for 1 yr at one location. Therefore, a new strategy for determining the inheritance of resistance under field conditions with replication in time and location is needed.

Variable reaction among and within resistant lines has been attributed to the presence of multiple races of the pathogen (Vir and Grewal, 1974; Reddy and Kabbabeh, 1985; Singh and Reddy, 1990). Several sets of disease differential lines were used to classify the pathogen by race (Vir and Grewal, 1974; Gowen, 1982; Porta-Puglia et al., 1986). Several studies generated inconsistent results that have led to the assignment of the same cultivars to the resistant class in one experiment and the susceptible class in another experiment (Jimenez-Diaz et al., 1993). These inconsistencies may have been caused by differences in inoculum concentration, inoculation techniques, environmental conditions, and plant age at inoculation in different studies (Porta-Puglia, 1992). Recent results indicate that different isolates of the pathogen differ in virulance (the amount of disease induced in the host) rather than pathogenicity (Jan and Wiese, 1991; Porta-Puglia et al., 1996). Based on this information, disease reactions for resistant genotypes have been related to quantitative variation under changing environments. For these reasons, the genetic nature of resistance, whether qualitative or quantitative, should be determined (Jimenez-Diaz et al., 1993).

The objective of this study was to determine the number of genes conferring resistance to ascochyta blight in segregating RIL populations derived from crosses of susceptible x resistant chickpea germplasm lines.


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results and discussion
 REFERENCES
 
RILs of chickpea were developed from three crosses (Table 1) using resistant parents, FLIP 84-92C and Dwelley, derived from a common source of resistance, ILC-72 (Fig. 1) . ILC-72 was reported to have a single dominant gene for resistance to ascochyta blight (Singh and Reddy, 1983). Resistant parents FLIP 84-92C(2) and FLIP 84-92C(3) were sister plants from the same germplasm line. The three crosses were advanced by single-seed descent from the F2 to the F5 in the greenhouse from 1995 to 1997. The F5-derived lines of CRIL-3 (chickpea RIL Population 3) and CRIL-7, along with checks including parental lines (Table 2) , were screened in the ascochyta blight nursery at the Washington State University Research Farm, Pullman, WA, in the spring and summer of 1997. Soil series of the farm is a Palouse (fine-silty, mixed mesic Pachic Ultic Haploxeroll). The RILs were further advanced to F7 in the greenhouse and screened in the blight nursery at the same location in the spring and summer of 1998.


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  		Table 1 Crosses used to develop recombinant inbred line (RIL) chickpea populations and the number of RILs in each population

 


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  		Fig. 1 Pedigrees of chickpea parental lines used as sources of ascochyta blight resistance genes

 

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  		Table 2 Mean ascochyta blight disease scores and area under the disease progress curve (AUDPC) of parental and check chickpea lines, and F1 plants

 
All combinations (without reciprocals) of the six parental lines and ILC-72 were made in the greenhouse in 1997. These cross combinations (Table 2) were screened in the blight nursery in the spring and summer of 1998. F1 seed of all combinations were planted in a completely randomized design with 1 to 10 replications depending on availability of hybrid seed.

Planting occurred on 5 May in 1997 and on 30 April in 1998. A susceptible spreader line, `Burpee 5024', was planted every fifth row and along the border of the nursery to promote disease development and spread within the nursery (Reddy et al., 1984). Infected chickpea debris (~50 g per plot) from the previous year was scattered by hand within the plots and disease development was promoted by daily sprinkler irrigation. Disease assessment of RILs, F1 plants, and checks was based on severity of symptoms, which were scored on a 1 to 9 scale, where 1= no visible lesions; 2 = lesions visible by close examination; 3 = a few lesions visible that were easily seen; 4 = many lesions visible, but lesions have not caused irreparable damage to the plants; 5 = large lesions on stem or leaves, some leaf and stem girdling, but the plant is still alive; 6 = many large lesions on stem and leaves, moderate stem and leaf girdling, but the plant probably will survive; 7 = many large lesions on stem and leaves, stem and leaf girdling common, the plant may or may not die but will produce few seeds; 8 = large lesions on stem and leaf common, stem and leaf girdling common, the plant probably will die; and 9 = infection severity such that the plant is dead or dying.

Disease scores of each line were recorded weekly until all susceptible check lines died. Final disease scores were used to classify the RILs as resistant or susceptible. Disease development of individual lines over time was analyzed and compared using AUDPC (Campbell and Madden, 1990). The last three disease scores were used to calculate AUDPC for each line using the formula

where Y is AUDPC, Xi is the blight score of the ith evaluation, Xi+1 is the blight score of the i + 1th evaluation, and (ti+1 - ti) is the number of days between two evaluations.

For all three RIL populations, experiments were planted in an augmented design (Petersen, 1994) with three blocks. Block numbers were calculated from the inequality r >= (10/c - 1) + 1, where r is the block number, and c is the number of check lines. Ten check lines, including parental lines, were replicated as single rows in each block and the remaining rows of the block were assigned to single rows of RILs. Experiments were repeated in 1997 and 1998 using the same experimental design. Analysis of variance of disease scores and AUDPC was performed using PROC GLM of the SAS (SAS Institute, 1989) statistical software package. Disease scores and AUDPC of the RILs were differentiated by Fisher's least significant difference (LSD) (P = 0.05) based on the standard error of mean differences of the 10 check lines and RILs used in each particular CRIL population. The RILs with disease scores and AUDPC less than the resistant parent mean plus the LSD were considered resistant and RILs with disease scores/AUDPC higher than the susceptible parent mean minus the LSD were classified as susceptible (Yildirim et al., 1998). Lines with intermediate scores were classified as intermediate.


   Results and discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
REFERENCES
 
FLIP 84-92C(2) was the most resistant parental line with a disease score of 2.0 from beginning to the end of screening and the smallest AUDPC, which confirmed the resistance (Table 2). PI 359075(1) was highly susceptible and died about the same time as the susceptible spreader Burpee 5024. Dwelley, the resistant parent of the CRIL-6 population had the highest disease score of the resistant parental lines, 3.2, and an AUDPC of 40.83. The susceptibility of Blanco Lechoso, with a disease score of 9.0 and an AUDPC of 123.67, was similar to that of the other susceptible lines. The mean disease scores and AUDPCs of the two FLIP 84-92C purelines were not significantly different from each other.

Of the 21 possible combinations using six parental lines and resistant source line ILC-72, F1 seed was obtained from 13 combinations. F1 plants of all susceptible x susceptible crosses were susceptible and resistant x resistant crosses were resistant (Table 2). F1 plants of FLIP 84-92C(2) x PI 359075(1) (CRIL-3), Dwelley x Blanco Lechoso (CRIL-6), and FLIP 84-92C(3) x C. reticulatum (PI 599072) (CRIL-7) had average disease scores of 5.5, 9.0, and 7.0, respectively, and were significantly different from each other. When PI 359075(1) was the susceptible parent in crosses with resistant parents, the F1 plants had intermediate reactions to blight. The means of these intermediate F1 plants differed depending on which resistant parent was crossed to PI 359075. The F1 of PI 359075(1) with the most resistant parent (FLIP 84-92C(2)) had a disease score of 5.5. However, when PI 359075(1) was crossed with FLIP 84-92C(3), ILC-72, and Dwelley, F1 means were 6.1, 7.4, and 8.0, respectively (Table 2), which differed significantly from each other . Other susceptible x resistant combinations using C. reticulatum and Blanco Lechoso as susceptible parents produced F1 plants that were susceptible to blight. The exception was the cross of FLIP 84-92C(2) x C. reticulatum that had an intermediate reaction (Table 2).

The classification of lines as resistant, susceptible, or intermediate differed slightly when based on disease scores compared with AUDPC values. The correlations between the disease scores and AUDPC were 0.99, 0.98, and for the three CRIL populations, indicating good agreement between the two methods. For simplicity, we used disease scores to determine segregation ratios and to illustrate the distribution of scores from resistant to susceptible lines.

Differences in disease scores and AUDPC among RILs within each population were highly significant (Table 3) . The year effect also was highly significant for CRIL-3 and nearly significant for CRIL-7, which was expected due to the more rapid development of blight in 1998 and the resulting higher disease scores earlier in the season for most of the RILs. There was a highly significant RIL x year interaction for the CRIL-3 and CRIL-7 populations evaluated in 1997 and 1998. Based on the significant RIL x year interaction, we performed a separate classification for each year.


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  		Table 3 Analysis of variance of ascochyta blight disease scores and area under the disease progress curve (AUDPC) in three chickpea recombinant inbred line (RIL) populations

 
In 1997, RILs of CRIL-3 that had intermediate reactions were skewed towards resistance and had a distribution (Fig. 2a) expected of two major genes conferring resistance with several modifiers (Fehr, 1993). In 1998, there was a larger number of lines with intermediate and susceptible reactions and the distribution of intermediates was skewed toward susceptibility (Fig. 2b). However, correlations between disease scores and AUDPC in 1997 and 1998 were 0.86 and 0.87 for CRIL-3, respectively. Segregation among RILs of CRIL-3 fit the expected segregation ratio of 1 resistant:3 susceptible for two recessive complementary genes conferring resistance (Table 4) .



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  		Fig. 2 Frequency distribution of disease scores for reaction to ascochyta blight in the CRIL-3 population. (a) data from 1997 and (b) data from 1998. Resistant and susceptible chickpea parent means are indicated by arrows

 

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  		Table 4 Segregation ratios in three chickpea recombinant inbred line populations based on 2 yr data for reaction to ascochyta blight

 
The CRIL-6 population was screened only in 1998. An LSD of 1.08 was used to compare RIL means with resistant and susceptible parent means. RILs with intermediate scores were included in the susceptible class as was done with the CRIL-3 population. The data fit the segregation ratio of 1 resistant:7 susceptible expected for three recessive complementary genes conferring resistance (Table 4). Most of the intermediate lines had disease scores close to 9 and the frequency distribution of disease scores skewed towards susceptibility (Fig. 3) .



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  		Fig. 3 Frequency distribution of disease scores for reaction to ascochyta blight in the CRIL-6 population in 1998. Resistant and susceptible chickpea parent means are indicated by arrows

 
Despite the significant RIL x year interaction, disease scores were highly correlated between years as were the values for AUDPC in CRIL-7. Segregation of RILs of CRIL-7 showed a good fit to the 1 resistant:7 susceptible ratio expected for three recessive complementary genes (Table 4). The frequency distribution of the lines was also similar to that expected for three major recessive genes (Fig. 4) .



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  		Fig. 4 Frequency distribution of disease scores for reaction to ascochyta blight in the CRIL-7 population. (a) data from 1997 and (b) data from 1998. Resistant and susceptible chickpea parent means are indicated by arrows

 
The mean disease scores of CRIL-3, CRIL-7, and CRIL-6 were 6.03, 6.86, and 8.11, respectively, (Table 5) . The AUDPC values for the three populations followed the same order, with CRIL-3 having the smallest and CRIL-6 the largest AUDPC. One RIL in CRIL-3 and four RILs in CRIL-7 had disease scores that were smaller than the resistant parent, indicating possible transgressive segregation.


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  		Table 5 Mean disease scores, area under the disease progress curve (AUDPC), ranges and standard deviations (SD) for three recombinant inbred line (RIL) populations of chickpea segregating for reaction to ascochyta blight

 
Using AUDPC for individual RILs was not as efficient as using disease scores in our analyses. Although results were slightly different, classification of RILs as resistant or susceptible using the 1 to 9 disease score scale was direct and did not require additional data manipulation. However, using AUDPC values to compare the stability of an individual RIL may be useful in breeding studies because the progression of the disease in individual lines can be noted and monitored.

Resistance to ascochyta blight in ILC-72 was reportedly conferred by a single dominant gene (Singh and Reddy, 1983); however, different degrees of resistance were later recorded for plants grown at other locations. This variation was interpreted as being conferred by additional minor genes (Singh and Reddy, 1989). Based on the segregation of RILs in our study, it appears that three major recessive genes control resistance to ascochyta blight, possibly with several minor modifying genes. Using the same original source of resistance, Kusmenoglu (1990) identified two recessive genes conferring resistance to blight. Since climatic conditions, inoculum density, and plant age affect disease development, different conclusions from various studies might be expected.

Differences in the population means were related to the degree of resistance found in the resistant parent of each population. This could be due to the number of modifier genes in the resistant parent of each population and their transmission to the resistant RILs. Intermediate reactions to blight were shown by 38.6, 14.7, and 27.7% of the RILs in CRIL-3, CRIL-6, and CRIL-7, respectively. Having the highest degree of resistance, FLIP 84-92C might have the largest number of minor genes. A similar mechanism was found for Cu tolerance in Mimulus guttatus Fischer ex DC (Tilstone and Macnair, 1997). They found that a single major gene controlled the presence or absence of tolerance, whereas modifier genes controlled the degree of expression of the tolerance.

The means and ranges of disease scores in the RIL populations closely followed that of the resistant parent. In particular, the two populations where FLIP 84-92C was a parent had the most resistance (Table 5). CRIL-6 had the highest mean disease score, which was consistent with the score for the resistant parent Dwelley, which had the highest disease score of the resistant parents. This could have resulted from the loss of minor genes during the selection process during the development of Dwelley (Fig. 1).

Differences in reaction of the resistant parental lines could be due to the presence of differing numbers of modifier genes in each line. The degree of resistance or susceptibility also depended on which susceptible parent was used in the crosses to develop the RIL populations. Different segregation ratios were observed for CRIL-3 and CRIL-7 populations. Since the resistance source of the three RIL populations was the same (Fig. 1) and two of the resistant parents were different pure lines developed from the same germplasm line, FLIP 84-92C, we expected the RILs to segregate for the same number of major resistance gene(s). In this case, PI 359075(1) must have one of three resistance genes present in FLIP 84-92C, and therefore, only a two-gene segregation ratio was apparent in CRIL-3. Blanco Lechoso and C. reticulatum appear to have susceptible alleles at all three loci, based on the apparent three-gene segregation ratio in CRIL-6 and CRIL-7. Intermediate reaction of F1 plants when PI 359075(1) was used as the susceptible parent also indicated the presence of some minor dominant resistant genes.

We observed variation in the degree of resistance in three RIL populations rather than discrete susceptible and resistant reactions. The intermediate lines with significantly different disease reactions from both susceptible and resistant parents were consistent in both years with slight changes. The normal distributions (Fig. 2, 3, and 4) of RILs in each class (resistant and susceptible) indicated that modifiers with minor effects influenced the expression of resistance in these populations (Fehr, 1993). Without the presence of modifiers, we should have been able to classify RILs into two discrete classes with values close to the parental means plus or minus the LSD. The presumed presence of minor genes may explain the significant RIL x year interaction and the apparent quantitative nature of the inheritance of resistance to blight. Genetic variation caused by minor genes has been reported for several agronomically important traits (Schat and Ten Bookum, 1992; Sourdille et al., 1996; Tilstone and Macnair, 1997; Rebetzke et al., 1998).

Geneticists working with complex traits use many assumptions, such as equality of gene effects and additivity of gene action, to estimate the number of genes controlling particular traits. Quantitative trait loci (QTL) mapping efforts indicate that these assumptions were not always correct (Paterson, 1998). Recent studies on quantitative disease resistance have revealed that the number of loci controlling quantitative resistance are generally less than the number of loci controlling other quantitative traits such as yield or seed weight (Young, 1996). A QTL analysis of blight resistance in chickpea should be conducted.

Epistasis among the QTLs is another concern when studying complex traits. This study did not focus on this subject; however, the genetic background effects observed in three RIL population using the same resistance source may have resulted from interaction of genes in the resistant parent with genes in the susceptible parent's background.Food and Agriculture Organization. 1996

Received for publication October 8, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 




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