Euphytica 111: 61–65, 2000. 61 © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

Quantitative genetic analysis of erect glandular trichome density in diploid alfalfa

J. Gonzalez-Garc´ ´ıa1,I.M.Ray2,∗, J.A. Henning3 & L.W. Murray2 1 1 Facultad de Ciencias Agr´ıcolas y Forestales, Universidad Aut´onoma de Chihuahua. Km 2 /2 Carretera Delicias- Rosales, Cd. Delicias, Chih., C.P. 33000, M´exico; 2Dep. of Agronomy and Horticulture and University Statistics Center, respectively, New Mexico State University, Las Cruces, New Mexico 88003-0003, U.S.A.; 3USDA-ARS, Corvallis, Oregon 97331, U.S.A., (∗author for correspondence)

Received 3 August 1998; accepted 6 June 1999

Key words: alfalfa, genetics, heritability, trichomes

Summary The presence of glandular trichomes may protect alfalfa (Medicago sativa L.) against certain stem-, leaf-, and fruit-eating insect pests. In order to determine appropriate breeding methods to select for this trait, this study char- acterized quantitative genetic parameters of erect glandular trichome density in ‘KS94GH6’ diploid (2n=2x=16) alfalfa. Eight female and five male plants were crossed in a Design II mating to produce 40 full-sib families. Glandular trichome density was determined on these families under replicated greenhouse conditions in Las Cruces and Los Lunas, New Mexico, U.S.A. The effect of males was significant (p ≤ 0.10) across the two environments. Variation among females was not significant within either location or across locations. The dominance genetic 2 2 variance (σ D = 0.210) was greater than the additive genetic variance (σ A = 0.111). The average degree of domin- ance exceeded a value of ‘1’ indicating that erect glandular trichome density in KS94GH6 may be influenced by digenic epistasis, and/or repulsion phase linkage disequilibrium. The large contribution of non-additive effects was 2 reflected by a low narrow-sense heritability estimate (hn = 0.25). The results indicate that further improvements in erect glandular trichome density in KS94GH6 alfalfa will require replicated progeny testing. Approaches to introgress this trait into cultivated tetraploid alfalfa are discussed.

Introduction erect glandular trichomes in several annual and per- ennial wild species of Medicago has been shown to An important factor of maximizing alfalfa (Medicago protect against certain stem-, leaf-, and fruit-eating sativa L.) forage yield and quality involves controlling insect pests (Small, 1986; Sorensen et al., 1986, insect pests (Manglitz & Ratcliffe, 1988; Marten et 1994). Upon contact with an insect body the gland-tips al., 1988) including the pea aphid (Acyrthosiphon rupture and the trichome exudate rapidly polymer- pisum Harris), blue alfalfa aphid (Acyrthosiphon kon- izes resulting in entrapment/immobilization of insects doi Shinji), spotted alfalfa aphid (Therioaphis macu- (Kreitner & Sorensen, 1979; Hasidoko et al., 1992; lata Buckton), and the alfalfa weevil (Hypera postica Hesk et al., 1992; Yu et al., 1992). The presence of Gyllenhai). Chemical control of these pests is not certain volatile and non-volatile compounds may also always cost-effective and the number of pesticide op- work together to deter insects from feeding in alfalfa tions available in alfalfa has diminished. Early harvest, (Ferguson et al., 1982). Core (1993) hypothesized that as a cultural practice to control the alfalfa weevil, is volatile compounds in the leaves and stems of wild al- also an effective management tool but reduces forage falfa species may work in conjunction with glandular yield (Keith et al., 1982). An alternative pest man- trichomes to produce antixenosis. agement option is to utilize host plant mechanisms to Most currently available glandular-haired alfalfa increase resistance to insect pests. The presence of germplasms are not agronomically desirable and 62 demonstrate wide variation among plants for the dis- lar trichome density and distribution (Sorensen et al., tribution and density of glandular hairs. Introgression 1986). KS94GH6 possesses erect glandular hairs on of this trait into a cultivated background can be done peduncles, pedicels, and seed pods and is resistant to more efficiently if genetic parameters influencing erect the pea and blue alfalfa aphid and the alfalfa weevil glandular trichome density are known. Evidence in al- (Sorensen et al., 1986). Eight female and five male falfa and other crops suggests that glandular trichome parents were randomly selected from KS94GH6 and density is quantitatively-inherited (Kitch et al., 1985; werecrossedbyhandinaDesignIImating(Com- Ågren & Schemske, 1992). Ågren & Schemske (1992) stock & Robinson, 1948) using vacuum emasculation. reported a realized heritability of 0.38 for trichome Seed representing the resulting 40 full-sib families was number on the edge of the first leaf using divergent se- planted in greenhouses at Las Cruces and Los Lunas, lection in a rapid-cycling population of Brassica rapa. New Mexico, U.S.A. Full-sib families were replicated The broad-sense heritability for trichome density was in four blocks in a randomized complete block design 2 high (hb = 0.86) in kidney bean (Phaseolus vulgaris at both locations. Each full-sib family was represented L.), however, the narrow-sense heritability estimate in each block by four plants. Greenhouse temperat- 2  ◦ was very low (hn = 0.10) reflecting a large magnitude ures were maintained at 24 5 C in both locations. of nonadditive genetic effects (Park et al., 1994). Plants were grown under natural light supplemented Vallejo et al., (1994a, 1994b) reported that broad- with 18 h fluorescent lights at Las Cruces and 18 h sense heritabilities of foliar glandular trichomes and sodium vapor lights at Los Lunas. Plants were fertil- polyphenol oxidase (PPO) activity ranged from 0.48 ized weekly with 100 ml of 10 mM N, 5 mM P, and to 0.77 in a population obtained from an interspecific 4mM K. cross of potato (Solanum spp.). In reciprocal backcross Four month-old plants were evaluated for erect populations of Solanum, narrow-sense heritability es- glandular trichome density along a 1 mm transect of timates for trichome density and PPO activity were the stem under 30× magnification according to Kitch 2 very low (hn = 0.04 to 0.24), suggesting little ad- et al. (1985). Data for each plant were collected on the ditive genetic variance for these traits. In tetraploid third fully elongated internode from the shoot apex for alfalfa, Kitch et al., (1985) partitioned the phenotypic each of three stems that possessed 10 internodes. Erect variance for glandular trichome density into additive glandular trichome density was expressed on a full-sib (29%), non-additive (16%), general (between-plant) family-mean basis by averaging the stem measure- environmental (29%), and special (within-plant) en- ments over the four plants representing each family vironmental (26%) variance components. They also within each block. Males (M), females (F), crosses reported a narrow-sense heritability estimate of 0.55 (C), and replicates (R) were considered as random ef- for glandular trichome density. fects while locations (L) were fixed effects. Analysis Research describing the genetic mechanisms that of variance within locations was conducted as a ran- control erect glandular trichome density in wild dip- domized complete block design with treatments in an loid species of alfalfa is limited and may be useful 8 female × 5 male factorial. Analysis of variance over for designing breeding strategies to introgress this trait locations was conducted as a randomized complete into cultivated tetraploid alfalfa. The objectives of this block design with blocks nested within locations, and study were to determine the contribution of additive treatments in an 8 female × 5 male factorial. Mean and non-additive genetic variances to the total geno- squares of the F × R(L), M × R(L), and F × M × R(L) typic variance of glandular trichome density in diploid interactions were not different and were subsequently alfalfa, and to estimate the narrow-sense heritability of pooled as an error term for the analysis over locations. this trait. Differences between locations were not detected in the analysis of variance. Therefore, narrow-sense herit- ability was determined on male and female half-sib 2 Materials and methods families using additive variation (σ A) estimates from the male and female components of variance over loc- The alfalfa germplasm evaluated in this study, ations. Variance components were obtained from the ‘KS94GH6’, was derived from a population of dip- expected mean squares (Hallauer & Miranda, 1988). loid (2n=2x=16) alfalfa [Medicago sativa var. viscosa The narrow-sense heritability estimate based on males (Rchb.)] which had been subjected to six cycles of was calculated as phenotypic recurrent selection for increased glandu- 63

Table 1. Mean squares from analysis of vari- Table 2. Genetic parameters and their standard errors for erect ance for erect glandular trichome density in glandular trichome density in KS94GH6 diploid alfalfa KS94GH6 diploid alfalfa over two environ- ments Genetic Male Female Weighted parameter estimate estimate average Source of variation df Mean square 2    σ A 0.111 0.095 0.001 0.051 0.041 0.061 Location (L) 1 22.489 ¯ ∗∗ d >1 >1 Replication/(L) 6 6.205 2    ∗∗ hn 0.250 0.207 0.002 0.146 0.106 0.156 Crosses (C) 39 1.119 2 Females (F) 7 0.799 σ A = Additive genetic variance. ∗ Males (M) 4 2.562 d¯ = Average degree of dominance. ∗ 2 F × M 28 0.992 hn = Narrow-sense heritability. C × L 39 0.514 F × L 7 0.369 × M L 4 0.361 not reported because the effect of locations and of the × × ∗ F M L 28 0.573 crosses × location, male × location, and female × Error 234 0.396 location interactions were not significant (Table 1). C.V. (%) 19.5 The lack of differences between locations may be due ∗ Significant at p = 0.10. to evaluation of trichome density under greenhouse ∗∗ Significant at p = 0.01. conditions which reduces climatic variation. When averaged over both locations the 40 full-sib families − 2 = possessed a mean of 3.21 glandular trichomes mm 2 hn 2 2 + 2 + 2 + 2 + 2 with a range of 1.54. The range in trichome density 4σm/(4σm 4σ 4σ /l 4σ /l σe /lr) mf ml mfl and the significant mean square for crosses suggests genotypic variation for this trait in KS94GH6. Vari- 2 2 2 2 2 with σ m, σ mf, σ ml, σ mfl,andσe representing the ation for trichome density among males was signi- variance components due to males, male × female, ficant over locations (p ≤ 0.10), however, variation male × location, and male × female × location, and among females was not detected. The contrasting res- the experimental error variance component, respect- ults between males and females may have resulted ively. Narrow-sense heritability was estimated in a from the small sample size used in our study, and wide similar fashion using the female sources of variation. variation for glandular trichome density and distribu- Narrow sense heritability was also calculated using tion among KS94GH6 plants, as reported by Sorensen a weighted average of the male and female variance et al. (1986). 2 components. The dominance genetic variance (σ D) Estimates of additive genetic variance, the average was determined as degree of dominance, and narrow-sense heritability were reported for males, females, and the weighted average of females and males to provide a com- σ 2 = 4σ 2 D mf plete genetic description of this trait (Table 2). The 2 magnitude of the dominance genetic variance (σ D = ¯  The average degree of dominance based on males (dm) 0.210 0.022) compared to the additive genetic vari- ¯ and females (df) was computed as ance estimates, and the average degree of dominance for males and females (d¯> 1) indicate a large contribu- √ √ tion of non-additive genetic effects for erect glandular ¯ = 2 2 ¯ = 2 2 dm (2σmf/σm) and df (2σmf/σf ) trichome density. The results suggest that digenic epistasis may be present in KS94GH6 which would result in an upward bias of d.¯ Loci controlling glandu- lar trichome density may also be in repulsion phase Results and discussion linkage disequilibrium because such linkages should 2 2 underestimate σ A and overestimate σ D resulting in an Variation for glandular trichome density among the overestimate of d¯ (Hallauer & Miranda, 1988). The 40 full-sib families was significant within each loca- importance of nonadditive effects in this population tion (data not shown). Analyses within locations were could also reflect that six cycles of selection for in- 64 creased trichome density and distribution could have KS94GH6 possessed glandular trichomes (Sorensen et caused allele frequencies to deviate from 0.5, as well al., 1986). Following six cycles of recurrent pheno- as favor linkage disequilibrium, resulting in biased typic selection for increased trichome distribution and genetic parameter estimates. frequency, all Cycle 6 plants (i.e., KS94GH6) pos- The importance of non-additive effects for erect sessed glandular trichomes. This suggests that some glandular trichome density was also reflected by loci controlling the presence of this trait may be fixed low narrow-sense heritability estimates, which ranged in KS94GH6. If major loci controlling this trait are 2 2 from hn = 0 for females, to hn =0.25formales fixed in KS94GH6 then early generation selection re- (Table 2). The heritability of this trait in diploid alfalfa sponse for this trait in CADL may be greater than was similar to the narrow-sense heritabilities reported indicated by our data. After practicing selection for 2 for glandular trichome density in Phaseolus sp. (hn = this trait in CADL, glandular trichomes could eventu- 2 0.10 to 0.17; Park et al., 1994), Brassica rapa (hn = ally be transferred to the tetraploid level via 2n gam- 0.38; Ågren & Schemske, 1992), and Solanum sp. etes, triploid bridge crosses, or colchicine doubling of 2 (hn = 0.04 to 0.24; Vallejo et al., 1994a, 1994b). The CADL plants. Our results indicate that erect glandular heritability of erect glandular trichomes in KS94GH6 trichome density is strongly influenced by nonadditive was less than that reported in an interspecific tetraploid effects in KS94GH6. Optimization of glandular trich- 2 population of Medicago (hn = 0.55), where the es- ome density and distribution, for effective pest res- 2 timate for hn was based on the regression of half-sib istance in CADL or cultivated tetraploid populations, progeny on their parents (Kitch et al., 1985). Differ- may require replicated progeny testing. Reciprocal re- 2 ences between our hn estimates and those reported current selection could be used, if deemed necessary, by Kitch et al. (1985) most likely resulted from the at either the diploid or tetraploid level to exploit both two populations being genetically distinct. Heritability the additive and nonadditive effects controlling this of this trait in tetraploid alfalfa, however, may have trait. also been overestimated due to the potential to trans- mit dominance effects to tetraploid half-sibs (Wricke & Weber, 1986). The diploid narrow-sense heritabil- Acknowledgement 2 ity estimate reported for males (hn = 0.25) may also overestimate the true value because variation among Research was supported by the New Mexico Agricul- females was not detected. The lack of variation among tural Experiment Station. females also prevented the determination of maternal effects on this trait. Given that all heritability estimates were low, the data indicate that evaluation of erect glandular trichome density in KS94GH6 alfalfa re- References quires replicated progeny testing in order to identify Ågren, J. & D.W. Schemske, 1992. Artificial selection on trichome parents that will be useful for future improvements in number in Brassica rapa. Theor Appl Genet 83: 673–678. this trait. Bingham, E.T. & T.J. McCoy, 1979. 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