2500

'Si 20O0-

O 1500-

W266 W266ipip W255 x W266ipp

Genotype

Figure 2. Total carotenoid content of carrot rootsfrom genotypes RPRP, rprp, and their hybrid. Bars in-dicate standard errors.

symbol rp to describe the genetic controlof this "reduced pigment" phenotype.

Roots from plants carrying rprp gradu-ally changed from white xylem and phlo-em to a slight yellow tint in the phloemand outer xylem (Figure 1). Mature roots(120 days after planting) from plants car-rying rprp were similar in size and shapeto orange roots of W266. Carotenoid pig-mentation of these mature roots was de-tectable (Figure 2), indicating the pres-ence of relatively small amounts of pig-ment in rprp as compared to RPRP plants.Mature roots had a whitish-yellowish ap-pearance and contained 141 jtg carotene/gdry weight. By contrast, orange pigmentedroots of W266 contained nearly 1,800 (igcarotene/g dry weight. Carotene contentincreases sharply during early stages ofroot growth and levels off during the grow-ing season and during storage (Werner1940). Pigmentation of the rprp genotypefollowed this pattern, however, develop-ment of carotenoids was minimal com-pared to standard orange roots.

The first several leaves of plants carry-ing rprp were white or speckled with white(Figure lb), indicating an effect of rp onleaf chlorophyll content. This whiteningwas not evident beyond the sixth leaf, sug-gesting the effect of rp is developmentallyregulated. Indeed, mature plants carryingrprp cannot be differentiated from plantscarrying RPRP by the color of their foliage.Reduction in carotenoid content is char-acteristic of many chlorophyll mutants be-cause the absence of carotenoid pigmentsrenders chlorophyll susceptible to pho-tooxidation (Aronoff 1966). Curiously, theeffect of rp continues to inhibit carotenoidsynthesis but not chlorophyll synthesisduring growth and development.

The finding of recessive alleles condi-

tioning nonpigmentation in carrot roots iscontrary to results reported by Buishandand Gabelman (1978), Imam and Gabel-man (1968), Kust (1970), and Laferriereand Gabelman (1968), who all determinedthat lack of pigmentation is controlled bydominant alleles. Kust (1970) described anepistatic relationship between the allelesY Y,, and Y2 with two pigment enhancingalleles, 10 and 0. He hypothesized that thenumber of 10 and 0 alleles had to be great-er than the number of Y, Y,, and Y2 allelesfor the presence of orange root color. Hefurther suggested that the recessive geno-type yyy,y,y2y2ioiooo should be white sinceit did not have the dominant pigment en-hancing alleles; however, no verification ofthis hypothesis was ever provided.

Lack of agreement regarding recessive-ness of nonpigmentation with the resultsreported by numerous workers suggeststhe lack of pigmentation in W266rp is per-haps due to a lesion in a carotenoid bio-synthesis gene that has not yet been stud-ied. Since mature roots from rprp plantsexhibit small amounts of beta carotene, rpdoes not completely block carotenoid syn-thesis. This character may prove useful indissecting the complex inheritance of ca-rotenoids in carrot.

From the Department of Horticulture, University ofWisconsin-Madison, 1575 Linden Drive, Madison, Wl.

The Journal of Heredity 1996:87(5)

References

Aronoff S, 1966. The chlorophylls—an introductory sur-vey. In: The chlorophylls (Vernon LP and Seely GR,eds). New York: Academic Press; 3-20.

Banga 0, 1964. Origin and distribution of the westerncultivated carrot. Genet Agraria 17:357-370.

Buishand JG and Gabelman WH, 1979. Investigations onthe inheritance of color and carotenoid content inphloem and xylem of carrot roots (Daucus carota L).Euphytica 28:611-632.

Imam MK and Gabelman WH, 1968. Inheritance ol anumber of phenotypes in Daucus carota L. (PhD disser-tation). Madison, Wisconsin: University of Wisconsin.

Kust AF, 1970. Inheritance and differential formation ofcolor and associated pigments in xylem and phloem ofcarrot, Daucus carota L. (PhD dissertation). Madison,Wisconsin: University of Wisconsin.

Laferriere L and Gabelman WH, 1968. Inheritance ofcolor, total carotenoids, alphaorotene, and beta-car-otene in carrots, Daucus carota L. Proc Am Soc Hort Sci93:408-418.

Simon PW and Wolff XY, 1987. Carotenes in typical anddark orange carrots. J Agric Food Chem 35:1017-1022.

Werner HO, 1940. Dry matter, sugar, and carotene con-tent of morphological portions of carrots through thegrowing and storage season. Proc Am Soc Hort Sci 38:267-272.

Received August 1, 1995Accepted December 31, 1995

Corresponding Editor: Kendall R- Lamkey

Inheritance of KernelResistance to Fusariumgraminearum in MaizeC. Chungu, D. E. Mather, L. M.Reid, and R. I. Hamilton

Inheritance of maize (Zea mays L) kernelresistance to ear rot caused by Fusariumgraminearum Schwabe was investigatedin generations derived from a cross be-tween resistant (CO325) and susceptible(CO265) maize inbred parents. Parents,F,, F2, and backcross generations wereevaluated in two locations in eastern Can-ada in 1993 and 1994. Plants were inoc-ulated with a macroconidial suspensionusing a kernel-stab method 15 days aftersilk emergence. Disease severity was as-sessed at harvest using a seven-class rat-ing scale. Significant differences were ob-served among the generation means in allenvironments. In general, the F, did notdiffer significantly from the resistant parentexcept at one location in 1993. The fre-quency distribution of the F2 and back-cross generations showed continuousvariation. Generation means analysis in-dicated that resistance to F. graminearumwas under genetic control with both sim-ple (additive and dominance) and digenic(dominance x dominance) effects contrib-uting to the total genetic variation amongthe generation means. Weighted leastsquare regression indicated that morethan 68% of the genetic variation could beexplained by additive effects. Estimates ofthe number of effective factors affectingkernel resistance ranged from 4.6 to 13.7.

Fusarium graminearum Schwabe (sexualstate: Gibberella zeae Schwein) may entermaize (Zea mays L.) ears via the silk orthrough wounds made by insects or birds.According to Koehler (1942), entry via thesilk and/or silk channel is the most com-mon mode of entrance of many pathogens.Plants may require different resistancemechanisms to defend themselves againstdifferent modes of fungal entry. Reid et al.(1992a,c) have presented evidence for re-sistance in the silk tissue that acts by pre-venting the fungus from growing rapidlydown the silk to the kernels. However, ge-notypes possessing this resistance mech-anism may not have any means of inhib-iting the spread of the fungus from kernelto kernel should the fungus bypass thesilk or succeed in overcoming the silk re-sistance. Resistance mechanisms in the

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kernel tissue might be useful as alterna-tives or complements to silk resistance.

There have been several reports on theinheritance of resistance to F. graminear-um and other Fusarium ear-rotting patho-gens, all based on studies in which artifi-cial inoculation techniques were used.Boling and Grogan (1965) evaluated sixgenerations derived from two inbred linesfor gene effects involved in the resistanceto F. moniliforme Sheld. They inoculatedplants 10 days after silking by shooting aspore-covered BB pellet into the ear about5 cm below the tip. They reported that in-heritance of resistance to F. moniliformewas due to additive, dominance, and epi-static gene effects and estimated that it in-volved two gene pairs. Odiemah and Man-ninger (1982) assessed the inheritance ofresistance to F. graminearum in six gener-ations using two inoculation methods;that study involved injection of about 2 mlof spore suspension between the silks andplacement of a colonized toothpick be-tween the silks. They concluded that ad-ditive, dominance, and epistatic effectswere important. Chiang et al. (1987) useddiallel analysis to investigate resistance toF. graminearum. Plants were inoculated byplacing a colonized toothpick at the tip ofthe ear within the base of the silks. Resultsof that study showed that additive geneeffects were important in resistance to earrot. Reid et al. (1992b) also used diallelanalysis to study resistance to F. grami-nearum infection. They inoculated plantsby injecting 2 ml of spore suspension intothe silk channel. They reported that inher-itance of silk resistance seemed to involvedominance. In another study, Reid et al.(1994) examined resistance to infectionvia the silk in six generations of crossesbetween a highly resistant inbred line andthree susceptible inbred lines. There ap-peared to be one major dominant gene af-fecting resistance, but its expression wassensitive to environmental conditions.

In most of the studies cited above, in-oculum was either applied to the silks orwas introduced to the kernels by methodsthat wounded both the kernel and the un-derlying cob tissue. Wounding of the cobtissue could allow the pathogen to enterthe kernels from the cob tissue ratherthan simply spreading from kernel to ker-nel. To assess differences in kernel resis-tance, it may be best to introduce inocu-lum to the kernels without wounding thecob tissue. Chungu et al. (1996) and Reidand Hamilton (in press) have describedthe use of a kernel-stab inoculation tech-nique that introduces a macroconidial sus-

pension to developing kernels withoutwounding the cob tissue. The use of aspore suspension may be preferable to theintroduction of mycelial tissue (as in thetoothpick method). The use of mycelial in-oculum can lead to intense disease symp-toms even in resistant lines, making it dif-ficult to discriminate effectively betweenlines (Ullstrup 1970). The purpose of thecurrent study was to use the kernel-stabinoculation method to examine the inher-itance of kernel resistance to F. graminear-um ear rot in a cross between susceptibleand resistant maize inbreds.

Materials and Methods

We chose maize inbreds CO325 (PI) andCO265 (P2) as the resistant and suscepti-ble parents for this study, based on theirresponse to inoculation with a macrocon-idia suspension of F. graminearum with thesilk-channel injection (Reid et al. 1992c)and kernel-stab method [Chungu et al.(1996)]. From the F, generation, we de-rived the F2 generation by selfing, and twobackcross generations (BCP1 and BCP2)by crossing to the two parental inbreds.The PI, P2, F,, F2, BCP1, and BCP2 gener-ations of CO325 x CO265 were evaluatedin a randomized complete block designwith four blocks at two locations (Ste-Anne-de-Bellevue, Quebec, Canada, andOttawa, Ontario, Canada) in 1993 and1994. Each plot of the PI, P2, and F, gen-erations consisted of seven rows of 14plants. Each plot of the F2, BCP1, and BCP2generations consisted of 10 rows of 14plants. The inter- and intrarow spacingwere 0.75 m and 0.20 m, respectively.

We produced inoculum using modifiedBilay's liquid medium consisting of 2 gKH2PO<, 2 g KNO3, 1 g MgSO4, 1 g KCI, 1 gdextrose, 0.2 g MnSO4, FeCl3, 2 g ZnSO< in1 L of water. A 1 cm2 plug of F. graminear-um culture maintained on potato dextroseagar was suspended in 250 ml of auto-claved liquid medium in a 500 ml Erlen-meyer flask. Before inoculation we filteredthe inoculum through two layers of cheesecloth and diluted it to a spore count of 5x 105 macroconidia/ml. Fifteen days aftersilk emergence, primary ears of the center12 ears of each of the five (PI, P2, and F,)or eight (F2) BCP1, and BCP2) inner rowsof each plot were inoculated using a ker-nel-stab technique. In this inoculationtechnique [Chungu et al. (1996); Reid andHamilton (in press)] a probe consisting offour nails (1.5 cm) fixed to a cylindricalwooden handle is dipped into the inocu-lum and then used to stab through the

Table 1. Mean ear rot symptom severity ratings"of generations from a cross between maizeinbreds CO325 and CO265 inoculated withFusarium graminearum at two sites in 2 years

Ste-Anne-de-Bellevue Ottawa

Generation

CO325 (PI)BCP1F,F2

BCP2CO265 (P2)

1993

3.43c3.59c3.48c4.74b5.41a5.29a

1994

2.99e3.22d3.15de3.67c4.10b5.10a

1993

2.93e3.28d3.14d3.79c4.46b4.71a

1994

4.38b4.39b4.16b4.42b5.38a5.63a

Means followed by the same letter within the columnsare not significantly different.

• Based on a scale of 1-7, where 1 = no symptoms pres-ent, 2 = 1-3%, 3 = 4-10%, 4 = 11-25%, 5 = 26-50%, 6= 51-75%, and 7 = 76-100% of the ear infected.

husk to wound three to four kernels in themiddle of the ear. We harvested the inoc-ulated ears in mid-October and assessedthe severity of their ear rot symptoms us-ing a seven-class rating scale where 1 =no symptoms present, 2 = 1-3%, 3 = 4-10%, 4 = 11-25%, 5 = 26-50%, 6 = 51-75%,and 7 = 76-100% of the ear infected. Weconsidered plants with severity ratings of3 or lower to be resistant, those with rat-ings of 4 to be moderately resistant, andthose with ratings of 5 or above to be sus-ceptible.

Data Analyses

A separate ANOVA for disease severitywas calculated for each environment. Thegeneration means were computed andcompared using the Duncan's multiplerange test (Steel and Torrie 1980). Within-plot variances were estimated and pooledacross the four replicates. Each genera-tion mean was weighted by the reciprocalof the variance of the mean for that gen-eration and the method of Mather andJinks (1982) was used to estimate additive,dominance, and epistatic genetic effects.Parameters not significantly different fromzero were eliminated from the model andgoodness-of-fit of each model was testedby a weighted chi square. Tests of signifi-cance for the gene effects were obtainedby dividing estimates by their correspond-ing standard errors and subsequentlycomparing them with the two-tailed Stu-dent's t test at 0.01 and 0.05 probabilitylevels.

The additive genetic variance (VJ wasestimated using the following formula: VA

= 2(VP2) - (VBC, + V ^ where V,,, V^,,and VBC2 represent variances of F2 and ofbackcrosses to PI and P2, respectively.This method assumes that the nonherita-

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80

40

080

40

05

CDCD

"5CDO)S"cCD

0Q_

080

40

080

40

080

40

080

40

0

CO325

1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7

Disease Severity Rating (scale 1-7)

n=332SEO015

n=357SE=0.014

n=393SE=0.011

1 2 3 4 5 6 7

Figure 1. Percent frequency distribution of disease severity of six generations at Ste-Anne-de-Bellevue in 1993 (A) and 1994 (B), and at Ottawa in 1993 (C) and 1994 (D); n = numberof diseased ears in the experiment and SE = standard error.

ble components of variation are compa-rable for the F2 and backcross generations.Narrow-sense heritability was computedas follows: h2 = VJVn. The number of ef-fective factors (K) controlling kernel resis-tance was estimated using the equationRT/8VA (Falconer 1989), where RT is thedifference between parents.

Results and Discussion

All of the inoculated ears showed at leastsome ear rot symptoms. The kernel inoc-ulation technique used in this study incit-ed sufficient infection to differentiate be-tween resistant and susceptible genotypesat both locations in both years.

Due to heterogeneity of error variances,we performed separate ANOVAs for eachenvironment. We observed significant dif-ferences (P < .05) in disease severityamong the six generation means in eachenvironment (Table 1). Means for the F,generation were similar to those for theresistant parent (CO325). Disease severity

values were higher in 1993 than in 1994 atSte-Anne-de Bellevue, but the reverse wastrue at Ottawa. These differences in symp-tom severity between years may havebeen due to differences in available mois-ture for fungal development during Augustand September. At Ste-Anne-de-Bellevue,precipitation was higher in September1993 than in September 1994. At Ottawa,it rained more frequently during the 2weeks after inoculation in 1994 than it didin 1993.

The F2 and backcross generationsshowed continuous variation (Figure 1).The distributions of the backcrosses wereskewed toward their recurrent parents.From these distributions, it appears thatseveral loci control the inheritance of ker-nel resistance to the pathogen.

The regression analysis indicated thatadditive gene effects were predominant,explaining more than 68% of the total ge-netic variation among the generations (Ta-ble 2). Odiemah and Manninger (1982),who used diallel analysis, also found sig-

nificant additive effects for resistance toFusarium graminearum ear rot, but theseeffects were relatively small compared tothe dominance gene effects. In our study,the magnitudes of the additive gene ef-fects in comparison with the mean effectswere higher than those of dominance anddominance X dominance genetic effects,suggesting that additive gene effects areimportant in the inheritance of kernel re-sistance in the cross studied.

Dominance gene effects for resistancewere negative and significant at Ste-Anne-de-Bellevue in 1994, but not in the otherthree environments (Table 2). However, inthose three environments there weresmall but significant dominance X domi-nance interactions. Perhaps the locus orloci that caused the dominance effects inone environment interacted with otherloci (or with each other) in the other threeenvironments. There could also be oppos-ing dominance effects at different loci;generation means analysis can fail to de-tect dominance in such cases (Boling and

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Table 2. Estimates of additive, dominance, and dominance x dominance genetic effects on Fusariumgraminearum ear rot symptom severity, and the relative contributions of these effects to the genotypesum of squares (% SS) in generations from a maize cross CO325 x CO265, at two sites in 2 years

Geneticparam-eter

m»dhi1

i

Ste-Anne-de-Bellevue

1993

Estimate"

4 . 7 3 "- 1 . 3 3 "

- 1 . 1 7 "

SSSS

98.8

1.2

1994

Estimate

4 . 7 3 "- 1 . 0 0 "-0.90*

% SS

68.531.5

Ottawa

1993

Estimate

4 . 0 2 "- 1 . 0 5 "

-0.87*

%SS

95.9

4.1

1994

Estimate

5.01**-0.77**

-0.86**

56SS

83.9

16.1

*,** Significant at the 0.05 and 0.01 probability levels, respectively.

• Based on weighted least square estimates of the parameters fitted in the genetic model.

* m = mean, d = additive, h = dominance, i = additive x additive, 1 = additive x dominance, and j = dominancex dominance.

Table 3. Estimates of additive genetic variance (VJ, F, variance, narrow-sense heritability (h2), andtheir standard errors, and number of effective factors (K) for kernel resistance to Fusariumgraminearum ear rot in generations derived from a maize cross CO325 x CO265 in experiments grownat two sites in 2 years

„ Ste-Anne-de-BellevueGener-ation 1993

Ottawa

1994 1993 1994

h2

K

0.075 ± 0.02900.104 ± 0.01900.720 ± 0.42195.8

0.041 ± 0.02160.067 ± 0.01400.610 ± 0.4843

13.6

0.029 ± 0.01870.043 ± 0.01300.670 ± 0.2153

13.7

0.042 ± 0.02330.075 ± 0.01300.560 ± 0.23024.6

Grogan 1965; Hallauer and Miranda 1988).Certainly it appears that the dominancecomponent of the genetic variation in thiscross interacts with environmental fac-tors; this is similar to what Reid et al.(1994) reported for a dominant gene con-ditioning resistance to F. graminearum in-fection via the silk. The negative signs as-sociated with the dominance effects sug-gest that dominance was in the directionof greater resistance.

Because we collected data on an indi-vidual plant basis, it was possible to ex-amine variances as well as means. Thevariances obtained from the segregatinggenerations permitted estimation of theheritable component of variation and thenumber of effective factors controlling ker-nel resistance. More than 56% of the totalvariation was attributable to additive vari-ation (Table 3), corresponding to the re-sults of the generation means analysis.The estimates for number of genes affect-ing kernel resistance were 5.8 and 13.6 atSte-Anne-de-Bellevue, and 13.7 and 4.6 atOttawa, in 1993 and 1994, respectively.These estimates support the conclusion ofChiang et al. (1987) that three or moregenes could be involved in controlling re-sistance to ear rot.

The kernel-stab inoculation method wasconsistent at inciting infection in all fourenvironments, a prerequisite for effectiveselection. Our results indicate that addi-

tive, dominance, and dominance X domi-nance gene effects are important in the in-heritance of kernel resistance to F. grami-nearum, with a predominance of additivegene effects. It may be possible to identifyplants with intermediate resistance fromcrosses derived between resistant andsusceptible parents. Since only two inbredparents were used, caution should be ex-ercised when making inferences on resultsobtained in this work. The limitations) in-volved in using two fixed parents could beaddressed by carrying out a more elabo-rate study that utilizes several inbred par-ents. Nevertheless, the experiments de-scribed here provide some evidence thatinheritance of kernel resistance could bedue to several genes.

From McGill University, Macdonald Campus, Plant Sci-ence Department, 21111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada (Chungu and Math-er) and the Plant Research Centre, Agriculture andAgri-Food Canada, Ottawa, Ontario K1A 0C6, Canada(Reid and Hamilton). PRC contribution #1616. Thiswork was supported by Pioneer Hi-Bred (Canada), Ag-riculture and Agri-Food Canada, the Natural Sciencesand Engineering Research Council of Canada, and theGovernment of Zambia. We thank N. Brown and Y. Chenfor their valuable technical assistance.

The Journal of Heredity 1996:87(5)

References

Boling MB and Grogan CO, 1965. Gene action affectinghost resistance to Fusarium ear rot of maize. Crop Sci5:305-307.

Chiang MS, Hudon M, Devaux A, and Ogilvie I, 1987.Inheritance of resistance to Gibberella zeae ear rot inmaize. Phytoprotection 68:29-33.

Chungu C, Mather DE, Reid LM, and Hamilton RI, 1996.Response of maize inbred lines resistance to Fusariumgraminearum infection via silk, kernel and cob tissue.Plant Disease 80:81-84.

Falconer DS, 1989. Introduction to quantitative genet-ics. New York: Longman Scientific.

Hallauer AR and Miranda JB, 1988. Quantitative genet-ics in maize breeding. Ames, Iowa: Iowa State Univer-sity Press.

Koehler B, 1942. Natural mode of entrance of fungi intocorn ears and some symptoms that indicate infection.J Agric Res 64:421^*42.

Mather K and Jinks JL, 1982. Biometrical genetics: thestudy of continuous variation. Cambridge: CambridgeUniversity Press.

Odiemah M and Manninger I, 1982. Inheritance of resis-tance to Fusarium ear rot in maize. Acta PhytopathAcad Sci Hung 17:91-99.

Reid LM, Bolton AT, Hamilton RI, Woldermariam T, andMather DE, 1992a. Effect of silk age on resistance ofmaize to Fusanum graminearum. Can J Plant Pathol 14:293-298.

Reid LM and Hamilton RI, in press. Effect of inoculationposition, timing, macroconidial concentration and irri-gation on resistance of maize to Fusarium graminearuminfection through kernels. Can J Plant Pathol.

Reid LM, Mather DE, Bolton AT, and Hamilton RI, 1994.Evidence for a gene for silk resistance to Fusarium gra-minearum Schw. ear rot of maize. J Hered 85:118-121.

Reid LM, Mather DE, Hamilton RI, and Bolton AT, 1992b.Diallel analysis of resistance in maize to Fusarium gra-minearum infection via the silk. Can J Plant Sci 72:915-923.

Reid LM, Mather DE, Hamilton RI, and Bolton AT, 1992c.Genotypic differences in the resistance of maize silk toFusarium graminearum. Can J Plant Pathol 14:211-214.

Steel RGD and Torrie JH, 1980. Principles and proce-dures of statistics. New York: McGraw-Hill.

Ullstrup AJ, 1970. Methods of inoculating corn earswith Gibberella zeae and Diplodia maydis. Plant Dis Rep54:658-662.

Received August 2, 1995Accepted January 19, 1996

Corresponding Editor: Prem P. Jauhar

Mutator-lnduced CytoplasmicMutants in Barley: GeneticEvidence of Activation of aPutative ChloroplastTransposon

A. R. Prina

The four mutants described here were vi-sually selected among the selfed progeny ofa chloroplast mutator (cpm/cpm) genotype.Due to their mode of inheritance they weredesignated as cytoplasmic lines (CLs). Oneof them, CL3 was a homogeneous viridis(light-green) type, while the other three pre-sented diverse positional patterns of varie-gation and also had different expression de-pending on the stage of growth. They

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