Hereditas 126: 255-259 (1997)

Ploidy manipulations and genetic markers as tools for analysis of quantitative trait variation in progeny derived from triploid plantains RODOMIRO ORTIZ, KATHELYNE CRAENEN and DIRK VUYLSTEKE Plantain and Banana Improvement Program, International Institute of Tropical Agriculture, High RainfuEl Station, Onne, Rivers State, Nigeria

Ortiz, R., Craenen, K. and Vuylsteke, D. 1997. Ploidy manipulations and genetic markers as tools for analysis of quantitative trait variation in progeny derived from triploid plantains. - Hereditas 126 255-259. Lund, Sweden. ISSN 0018-0661. Received January 30, 1997. Accepted June 4, 1997.

Plantains (Musa spp. AAB group) are starchy bananas widely grown in Africa and tropical America by small landholders. Genetic analysis of the plantain genome is difficult due to its triploidy and high sterility. Ploidy manipulations (scaling up and down the number of chromosomes) and interspecific plantain-banana hybridization opened the path for the genetic amelioration of the crop and for the investigation of its genome for further manipulations. This report shows the associated effects of ploidy and of the major loci controlling vegetative fruit parthenocarpy and resistance to black sigatoka fungal leaf spot on growth and yield characteristics of plantain-banana euploid hybrids. The number of copies of the black sigatoka resistance allele (bs,) and of the fruit parthenocarpy gene ( P , ) in addition to their intralocus interaction and ploidy level were all found to significantly affect bunch and fruit characteristics in euploid plantain-banana hybrids. Epistasis significantly affected fruit weight and size in one cross but not in another. Significant multiple regression models combining ploidy and genetic markers explained 15 % to 85 YO of quantitative trait variation (QTV). The amount of QTV accounted by ploidy and genetic markers varied according to the characteristic and cross in which the markers were examined.

Rodomiro Ortiz, Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, 40 Thor- valdsensvej, DK 1871 Frederiksberg C, Denmark

The phenotypic expression of quantitative characters is often controlled by many minor genes (or polyge- nes). Each polygene affects slightly the observed quantitative trait variation (QTV) (FALCONER and MACKAY 1996). However, major genes that are asso- ciated with specific pronounced qualitative pheno- typic expression and are inherited in a Mendelian fashion, may also produce significant variation in certain quantitative traits (SAX 1923; THODAY 1961; MACKAY 1995). The effect of major genes on QTV can be explained by linkage or pleiotropism and may act in an additive or non-additive manner. The non- additive gene interaction can be either within the locus (dominance or intralocus interaction) or be- tween loci (epistasis or interlocus interaction) (FAL-

The independent effect of ploidy and genetic mark- ers in quantitative trait variation has been investi- gated recently in Musa (ORTIZ and VUYLSTEKE 1995a; VANDENHOUT et al. 1995; CRAENEN and ORTIZ 1996). However, knowledge of combined effects of specific types of gene action in the plantain genome is lacking. Hence, we investigated the joint ploidy and genetic effects of marker loci on segregat- ing euploid plantain-banana hybrids in crosses be- tween triploid French plantains and a wild homozygous diploid banana.

CONER and MACKAY 1996).

MATERIALS AND METHODS

Euploid hybrids were obtained from interspecific in- terploidy crosses between triploid plantains and a diploid wild banana (VUYLSTEKE et al. 1993a). Hy- brid seeds were in vitro germinated (VUYLSTEKE et al. 1990). Both plantain parents have a duplex and simplex genotype for fruit parthenocarpy (ORTIZ and VUYLSTEKE 1995a) and black sigatoka resistance (ORTIZ and VUY LSTEKE 1994a), respectively; whereas the wild banana has a recessive genotype for both loci (SIMMONDS 1952; ORTIZ and VUYLSTEKE 1994a).

Specific genotypes (Table 1) were assigned to re- spective loci in diploid and tetraploids as explained earlier (ORTIZ and VUYLSTEKE 1994a, 1995a; CRAE- NEN and ORTIZ 1996). The tetraploid hybrids with parthenocarpic fruits were duplex ( P , P , p , p , ) for the fruit parthenocarpy locus ( P , ), while their full-sib diploids were either heterozygous ( P , p , ) hybrids with parthenocarpic fruits or homozygous recessive ( p I p I ) with nonparthenocarpic fruits. Genotypes were not assigned to triploid hybrids. The genotype of the hybrids for the black sigatoka resistance locus (bs, ) was determined by their host response to black siga- toka. Genotyping of the bs, locus was confirmed by selfing or sibmating a sampling of euploid hybrids (ORTIZ and VUYLSTEKE 1994a). As expected, putative

256 R. Ortiz et al. Hereditas 126 (1997)

Table 1. Genetic effects in marker loci of segregating euploid plantain-banana hybrids Marker Ploidy Phenotype Intralocus interaction

Additivity Dominance'/Net effect'

Fruit parthenocarpy locus' Diploid non-parthenocarpic 0 0

parthenocarpic 1 1 Tetraploid non-part henocarpic 0 0

parthenocarpic 2 1

locus* Diploid susceptible 1 0 less susceptible 2 2 resistant 2 2

Tetraploid susceptible 2 0 less susceptible 2 0 resistant 4 4

Black sigatoka resistance

recessive or nulliplex genotypes for the bs, locus did not show segregation after selfing or sibmating among themselves. However, the offspring of suscep- tible hybrids displayed all the phenotypic variation expected for heterozygous genotypes.

Types of gene action within each locus followed conventional definitions of quantitative genetics (FALCONER and MACKAY 1996) and have been ex- plained for Musa hybrids elsewhere (ORTIZ 1995a). The effect of allele substitution was defined in terms of changing the genotypic value by substituting the dominant allele for its recessive counterpart, which depends on the allele frequency in respective locus. The dominance in the fruit parthenocarpy locus (SIM- MONDS 1952) and the net effect of the black sigatoka resistance locus (CRAENEN and ORTIZ 1997) results from the interaction of alleles at the same locus. Genetically, this intralocus interaction represents the difference between the value of the whole genotype and the sum of the value of its genic parts, i.e., a statistical interaction effect (DOOLITTLE 1987).

Ploidy was initially determined by phenotypic as- sessment, and later confirmed by stomata examina- tion and chromosome countings (VANDENHOUT et al. 1995). There were 13 tetraploids, 2 triploids, and 16 diploids from the cross Obino 1'Ewai x Calcutta 4 (N = 31) and 6 tetraploids, 2 triploids and 44 diploids from the cross Bobby Tannap x Calcutta 4 (N = 52).

Quantitative traits were recorded in early evalua- tion trials (plant and ratoon crops) using the opti- mum single-row plots of 5 plants per genotype (ORTIZ and VUYLSTEKE 1995b) at Onne (southeast- ern Nigeria), which is in a secondary center of plan- tain diversity (VUYLSTEKE et al. 1993b). Site characterization and cultural practices for on-station

trials have been described elsewhere (VUYLSTEKE et al. 1993a; ORTIZ 1995b).

One-way analyses of variance were carried out for specific gene action at each individual locus (ED- WARDS et al. 1987) and two-way analyses of variance were performed for each epistatic interaction (STU- BER et al. 1987). The effect of ploidy (independent variable) on quantitative trait variation (dependent variable) was determined by linear regression analy- ses (SOKAL and ROHLF 1981). Multiple regression models included all significant sources of variation, unless there was co-linearity between the independent variables. The coefficient of determination (R2) ex- plained the percentage of total variation due to the model, while the F-test indicated the statistical signifi- cance of the genetic model (SOKAL and ROHLF 1981). Residuals from the multiple regression models for each trait within each cross were tested by Durbin- Watson statistics (SOKAL and ROHLF 1981) to deter- mine the reliability of the data sets and the potential utilization of the regression model on marker assisted selection. Broad-sense heritability (H), based on plot means, was previously calculated from the ratios of variance components (i.e., genetic variance/pheno- typic variance) (ORTIZ and VUYLSTEKE 1995a).

RESULTS AND DISCUSSION

The analysis of variance, based on two segregating markers (Table 2), revealed that the effect of allele substitution (additive gene action) at both black siga- toka resistance (bs, ) and fruit parthenocarpy ( P , ) loci significantly affected ( P < 0.001) both bunch weight and fruit size. Likewise, dominance at both loci sig- nificantly affected ( P < 0.05 or P < 0.001) fruit length and circumference, while dominance at the P , locus

Hereditas 126 (1997) Variation in progeny derived from triploid plantains 257

Table 2. Analysis of variance for quantitative traits bused on two segregating markers (bs,, black sigatoka resistance; PI, fruit parthenocarpy) and ploidy in the euploid hybrids derived from crossing triploid female fertile plantains with diploid male fertile bananas

Source of variation Significance of F-test for respective quantitative trait

DF PH HTSh BW H F AFW FL FC DFF

Bobby Tannap x Calcutta 4 Effect of allele substitution in P , locus (A,) in bs, locus (A2)

Intra-locus interactions Dominance in P, locus (D,) Net effect in bs, locus (D2)

Inter-locus interactions (epistasis) A, A2 A' D2 A2 x D, D, x D2 Ploidy R2 ("A,) regression model F-test regression model Analysis of residuals

Obino 1'Ewai x Calcutta 4 Effea of allele substitution in P , locus (A, ) in hs, locus (A2) Intra-locus interactions Dominance in P , locus (D,) Net effect in bs, locus (D2)

Inter-locus interactions (epistasis) A, x A2 A, x D2 A2 x D, D, x D2 Ploidy R2 ("A)) regression model F-test regression model Analysis of residuals

Broad-sense heritability (YO)

2 2

1 2

1 1 1 1

2

2 2

1 2

1 1 1 1 2

NS NS

NS NS

NS NS NS NS

NS

NS -

-

*** *

NS *

* * * NS

56

NS

70

***

**

NS NS

NS NS

NS NS NS NS NS

NS -

-

NS NS

NS NS

NS NS NS NS

NS

NS

56

-

-

*** ***

** **

NS NS NS NS

39

NS

***

***

*** ***

NS ***

NS NS NS NS

81

NS

88

***

***

* NS

* NS

NS NS NS NS NS 15

I *

** NS

*** NS

NS NS NS NS NS 34

NS

75

**

** NS

* *

NS NS NS NS

19

NS

*

*

NS NS

* NS

NS NS NS NS

NS 17

NS

82

*

*** ***

** ***

NS NS NS NS

54

NS

***

***

*** ***

NS ***

NS NS NS NS

83

NS

94

***

***

*** ***

*** **

NS NS NS NS

40

I

*

***

*** ***

* ***

* * * NS

76

I

94

***

***

*** ***

*** ***

NS NS NS NS

53

NS

***

***

*** ***

* ***

** ** ** NS

85

NS

94

***

***

NS NS

NS NS

NS NS NS NS

NS

NS -

-

NS NS

NS NS

NS NS NS NS

NS

NS -

-

14

DF = degrees of freedom, PH = plant height at flowering (cm), HTSh = height of tallest sucker at harvest, BW = bunch weight (kg plant-'), H =number of hands (nodals clusters of fruits) bunch-', F = total number of fruit bunch-', AFW = average fruit weight (g), FL = fruit length, FC = fruit circumference, DFF = days for fruit filling. NS, *, **, and *** indicate non-significant or significant variation at 5%1, 1%, and 0.1%) levels. I = inconclusive test of residuals. Broad-sense heritability values as reported by ORTIZ and VUYLSTEKE (1 995a)

significantly affected (P < 0.05) the number of hands and fruits in both crosses. Intralocus interactions of the bs, locus affected both bunch weight and fruit traits.

There was a significant epistatic interaction (P < 0.05 or P < 0.01) between the additive and non-addi- tive effects (except dominant x dominant) of both bs, and P, loci for plant height, fruit length and circum- ference (Table 2). However, these effects were only observed in Obino 1'Ewai x Calcutta 4, while no sig- nificant interactions were observed in Bobby Tan-

nap x Calcutta 4. Additive effects of both loci plus intralocus interaction in bs, and epistasis significantly affected plant height in Obino 1'Ewai x Calcutta 4. However, plant height was not affected by the segre- gation of either marker in progeny of Bobby Tan- nap x Calcutta 4. This supports the observation that a dwarfism locus segregates in progeny from Bobby Tannap x Calcutta 4 (ORTIZ and VUYLSTEKE 1995~). Similarly, the lack of significant effects of both ge- netic markers and ploidy on height of tallest sucker supports the theory that apical dominance in Musa is

258 R. Ortiz et al. Hereditas 126 (1997)

Table 3. Best multiple regression models, based on ploidy ( X ) and genetic effects in marker loci (bs,, PI), which explain quantitative trait variation in bunch fruit traits of the segregating offspring from Obino I’Ewai x Calcutta 4

Trait Multiple regression model Standard error

2.4 kg plant-’ Number of hands (H) 1.2 hands Number of fruits (F) F=55.3 D,+44 31.3 fruits Av. fruit weight (AFW) Fruit length (FL)

Fruit circumference (FC)

Bunch weight (BW) BW (kg) = 4.4 X + 3.5 A, + 4.2 D, - 5.5 A, + 6.3 H = 3.61 D, + 3.1

AFW(g) = 58.0 X + 33.2 A, + 49.4 Dl - 7.3 A, - 65.5 FL (cm) = 4.2 - 10.2 A, - 7.0 D, + 6.5 A2 - 0.01 (A, x D,) + 9.3 (A, x D1) 2.9 - 8.0 A, - 4.7 D, + 4.4 A, + 0.4 (A, x A,) - 0.2 (A, x D2)

22.4 g

2.4 cm

1.2 cm + 6.2 (A, x D,)

X = ploidy level, A, = effect of allele substitution in P, locus, A, = effect of allele substitution in bs, locus, D, = effect of dominance (i.e., intra-locus interaction) in P, locus, D, = net effect of intra-locus interaction in bs, locus

controlled by only one single recessive gene with variable expressivity, incomplete penetrance, and ge- netic specificity (ORTIZ and VUYLSTEKE 1994b).

Significant differences between the three ploidy levels (P < 0.001) were observed for bunch weight and fruit size in both families but only for plant height in Obino 1’Ewai x Calcutta 4 and only for number of fruits in Bobby Tannap x Calcutta 4. There was no significant increase or decrease at dif- ferent ploidy levels for days to fruit filling, number of hands in both crosses and for plant height in Bobby Tannap x Calcutta 4.

Most of the genetic variation for plant height, bunch weight and fruit size (H2) was accounted for by the gene action of the markers and ploidy level in progeny from Obino 1’Ewai x Calcutta 4. The level of genetic variation accounted for by the defined parameters was measured by the coefficient of determination (R2 in Table 2) of the multiple regression model. In contrast, only a small portion of the total variation for bunch weight and fruit size was explained by these same genetic parameters in progeny from Bobby Tannap x Calcutta 4. This suggests that additional loci may be influencing these traits in progeny from Bobby Tannap x Cal- cutta 4.

Genetic parameters having significant effects in progeny from Obino 1’Ewai x Calcutta 4 were in- cluded in the best multiple regression model (Table 3). Certain genetic parameters, although significant in the analysis of variance, were omitted in the model because they provided redundant informa- tion, i.e., when included the model had a singular matrix. In cases of redundancy only the parameters with the highest partial coefficient of regression were considered.

The regression models (based on ploidy and genetic effects in the markers bs, and P , loci) explained up to 85 YO of the genetic quantitative trait variation in

bunch and fruit traits expressed by progeny from Obino 1’Ewai x Calcutta 4 (Table 3). The coefficients of regression suggest that the additive and non-addi- tive effects in the P, locus as well as ploidy account for a significant portion of the variation in fruit weight. However, intralocus interaction in the P, locus accounted for most of the variation in number of hands and fruits per bunch. To the authors’ knowledge this could be the first report of dissecting quantitative traits in Musa with the aid of conven- tional genetic markers.

The Durbin-Watson analyses of residuals (Table 2) were non-significant or inconclusive in the quantita- tive traits measured. This suggests that marker-as- sisted selection based on both genetic markers may result in significant gains in bunch weight and fruit weight of plantain-banana hybrids. The accumulation of favourable alleles (either dominant P, ) or recessive (bs, ) genes) through recurrent selection should result in a population showing heavy bunches with thick and weighty fruits. This is likely to be especially effective in polyploid progeny from Obino 1’Ewai x Calcutta 4.

Irrespective of the precision of our genetic models, our analysis shows that it was possible to dissect quantitative trait variation in progeny derived from a triploid organism like plantain. Moreover, the re- ported effects of the genetic markers were expected since ploidy, fruit parthenocarpy, and host-response to black sigatoka significantly influence the pheno- type for bunch and fruit traits in Musa (SIMMONDS 1995; ORTIZ and VUYLSTEKE 1995a; CRAENEN and ORTIZ 1996). The recent development of DNA mark- ers in Musa (KAEMMER et al. 1992; FAURE et al. 1993; HOWELL et al. 1994; JARRET et al. 1994) may enhance our capability for dissecting quantitative trait variation and advanced genetic manipulations for the betterment of the plantain and banana genomes.

Hereditas 126 (1997) Variation in progeny derived from triploid plantains 259

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