Abstract
The amounts and types of epicuticular waxes on onion (Allium cepa) leaves affect the severity of feeding damage by onion thrips (Thrips tabaci), a serious insect pest of onion. Onion plants with light green leaves are referred to as “glossy” and accumulate less epicuticular wax relative to the blue–green (“waxy”) foliage of wild-type onion. The onion cultivar Odourless Greenleaf (OGL) has visually glossy foliage, shows resistance to thrips feeding damage, and has the unique profile of accumulating waxes with 28 or fewer carbons. Plants of glossy OGL were crossed with the glossy inbred B9885 and waxy inbred lines DH2107, DH066619, and B8667. Hybrid progenies from glossy OGL by waxy plants had waxy foliage, indicating recessiveness of the glossy OGL phenotype relative to the waxy phenotype. Hybrids from the cross of glossy OGL with glossy B9885 were also waxy, revealing different genetic bases for the glossy phenotype in these two onions. Hybrid plants were self-pollinated and segregations in F2 families from OGL × waxy crosses fit the expected 3:1 ratio for the single locus at which the homozygous recessive genotype conditions glossy foliage. Segregations in F2 families from crosses of glossy 9885 × glossy OGL fit the 9:7 ratio, supporting two independently segregating loci, where the recessive genotype at either locus conditions the glossy phenotype. Amounts and types of epicuticular waxes on leaves of F2 progenies from crosses of OGL × waxy B8667 and glossy B9885 × OGL were determined using gas chromatography-mass spectrometry. Single-nucleotide polymorphisms were genotyped and genetic maps were constructed. The visually glossy phenotype from OGL and its unique profile of epicuticular waxes were conditioned by one locus on chromosome 6, for which we propose the name glogl. Onion populations such as OGL with unique epicuticular wax profiles will be important germplasms for the development of onion cultivars that suffer less feeding damage from onion thrips compared with waxy onion.
The epicuticular waxes on onion (Allium cepa) foliage are primarily composed of fatty alcohols, alkanes, and a ketone (Damon et al., 2014). The foliage of wild-type onion has a blue–green color, accumulates relatively high amounts of epicuticular waxes, and is referred to as “waxy.” The waxy appearance on leaves of wild-type onion is likely due to large amounts of the ketone 16-hentriacontanone (H16) relative to the other waxes that build as platelets on the leaf surface (Damon et al., 2014). The “glossy” phenotype of onion is characterized by a light green leaf color and low amounts of epicuticular waxes (Damon et al., 2014; Molenaar, 1984). The leaf color and amounts of epicuticular wax on the leaf surface affect feeding damage by onion thrips (Thrips tabaci), which are a serious insect pest of onion worldwide. Onion plants with a light green leaf color and/or different amounts and types of epicuticular waxes support fewer onion thrips per plant and suffer significantly less feeding damage relative to waxy foliage (Boateng et al., 2014; Cramer et al., 2014; Coudriet et al., 1979; Damon et al., 2014; Diaz-Montano et al., 2010; Jones et al., 1934; Munaiz et al., 2020a).
Jones et al. (1944) studied the genetics of glossy plants selected from the onion cultivars Australian Brown and White Persian. For plants selected from ‘Australian Brown’, the glossy phenotype was conditioned by the homozygous recessive genotype at one locus named gl (Jones et al., 1944). For glossy plants from ‘White Persian’, segregations did not fit a single recessive model because too few glossy progenies were observed in families (Jones et al., 1944). Molenaar (1984) completed a genetic study using glossy plants descended from the same selections from ‘White Persian’ identified by Jones et al. (1934) and observed that segregation ratios fit a single recessive model. Munaiz and Havey (2020) mapped the glossy phenotype from ‘White Persian’ to chromosome 8 and named the glwp locus at which the homozygous recessive genotype conditions glossy foliage. The glwp locus interacts with a region on chromosome 1 to increase the numbers of waxy progenies (Munaiz and Havey, 2020), consistent with the observation by Jones et al. (1944) of too few glossy progenies in segregating families.
The onion cultivar Odourless Green Leaf (OGL) has a light green leaf color, is less attractive to thrips relative to waxy onion (Cramer et al., 2014), and has a unique profile of epicuticular waxes (Munaiz et al., 2020b). During this research, we determined the genetic basis of the wax profile and glossy phenotype of OGL, and we showed that it is inherited independently from the glwp locus (Munaiz and Havey, 2020). Germplasms such as OGL with unique wax profiles may be useful for the development of onion cultivars that suffer less damage by onion thrips compared with waxy onion.
Materials and Methods
Seed of OGL was obtained from the U.S. Department of Agriculture (USDA) germplasm collection as PI 289689. Inbred B9885 has glossy foliage (Damon et al., 2014) and inbred B8667 has waxy foliage (Havey and Bohanec, 2007), and both were from the USDA onion-breeding program. Waxy doubled haploids (DH) 2107 and 066619 were described by Hyde et al. (2012). Seeds of these accessions were planted at the Dean Kincaid Farm (Palmyra, WI) and bulbs were produced under normal production conditions. After harvest, bulbs were stored for at least 4 months at 7 °C for vernalization, and planted in greenhouses with 25 °C days and 20 °C nights, and supplemental lighting for 12 to 13 h for OGL or 14 to 15 h for B8667, B9885, DH2107, and DH066619. Individual flowering plants of OGL were paired with plants of the other parents and covered with mesh cages, and houseflies (Musca domestica) were introduced for crossing. Seed was harvested from OGL plants and planted in the greenhouse. Progenies were grown under 12-h days and hybrids were identified by the presence of waxy foliage. At the six-leaf stage, hybrid plants were transferred to a cold room at ≈4 °C with 12 h lighting for at least 4 months. In late April, hybrid plants were transplanted to field plots at the University of Wisconsin Horticulture Research Farm (Arlington, WI) under naturally increasing daylengths. Umbels on individual hybrid plants were covered with mesh cages and blue-bottle flies (Calliphora sp.) were introduced to produce F2 families.
Plants of parents, hybrids, and F2 progenies were grown in a soilless mix (Pro-Mix HP Mycorrhizae; Premier Tech Horticulture, Quakertown, PA) in 11.4-cm pots in a greenhouse with 25 °C days, 20 °C nights, and supplemental lighting for 14 h. Pots were randomly arranged on benches, watered daily, and fertilized with 0.5× Hoagland’s solution (Hoagland and Arnon, 1950) once per week. Eight weeks after the planting of seed, plants had approximately six true leaves and foliage was visually scored as glossy vs. waxy (Fig. 1). Two leaf segments ≈9 cm were collected from the middle region of the fourth or fifth fully expanded leaf from five plants each of OGL, B9885, B8667, and their hybrid progenies, and from individual F2 progenies from the OGL × B8667 and B9885 × OGL families. Each leaf segment was placed in a 16- × 100-mm glass tube (Thermo Fisher Scientific, Waltham, MA) and ≈20 mL of chloroform [high-performance liquid chromatography (HPLC) grade; Sigma-Aldrich, St. Louis, MO] was added to the tube for 1 min; after which, leaf pieces were removed and oven-dried at 80 °C for 5 d, and dry weights were measured. Then 25 μL of docosane (Sigma-Aldrich) dissolved in HPLC-grade chloroform at a stock concentration of 1 mg·mL−1 was added to each tube. The chloroform with docosane was allowed to dry in a fume hood for ≈2 weeks. To prepare for gas chromatography-mass spectrometry (GCMS), dried wax extracts were dissolved in 250 μL chloroform, 300 μL acetronitrile (HPLC grade; Thermo Fisher Scientific), and 105 μL N,O bis(trimethylsilyl)trifluoro-acetamide (HPLC/GC grade; Sigma-Aldrich) and incubated for 30 min at 80 °C. Identification and quantification of waxes involved the GCMS conditions described by Damon et al. (2014) and Khosa et al. (2020).
Phenotypes of waxy B8667 (left), glossy ‘Odourless Greenleaf’ (center), and glossy B9885 (right) onion at the stage of leaf sampling for gas chromatography-mass spectrometry.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 2; 10.21273/JASHS05024-20
DNA were isolated from parents, hybrids, and F2 progenies from the B9885 × OGL and OGL × B8667 crosses using a mini-preparation (Nucleospin 96 Plant II DNA extraction kit; Macherey-Nagel, Bethlehem, PA). DNA concentrations were determined spectrophotometrically (NanoDrop ND-1000; Thermo Fisher Scientific), and quality was assessed by electrophoresis through 1% agarose gels. Two-hundred fifty single-nucleotide polymorphisms (SNPs) evenly spread across the eight chromosomes of onion were genotyped using the KASPar assay (Duangjit et al., 2013; Havey and Ghavami, 2018). Joinmap 5 (Van Ooijen, 2018) was used to assess goodness-of-fit to the expected ratios and for genetic mapping using a logarithm of odds (LOD) of linkage at 6.0.
Individual peak areas from GCMS were adjusted by dividing by the dry weights of leaf pieces. Peak areas from the two samples from each leaf were averaged and used for all analyses. Analyses of variance of the amounts of waxes were calculated using R Studio (R Foundation for Statistical Computing, Vienna, Austria). Visually scored foliar phenotypes were analyzed using the binary model and the amounts of individual and total waxes were analyzed using composite interval mapping (CIM) with the R/qtl package (Broman and Sen, 2009; Broman et al., 2003). Haley Knott regression with 10 marker covariates within a window size of 10 cM was used to determine significant associations (Haley and Knott, 1992) and a 95% significance LOD threshold was used after 1000 permutations. Multiple quantitative trait loci (QTLs) were detected using the stepwiseqtl function dropping one marker at a time with parameters of 0.05 significance level and 1.5-LOD score. Effects of the candidate QTLs were refined using makeqtl, refineqtl, and fitqtl, and the percentages of the phenotypic variation explained by the QTLs were estimated.
Results
Epicuticular waxes on the foliage of OGL.
GCMS analyses of epicuticular waxes revealed that the foliage of OGL accumulated significantly greater amounts of the fatty alcohol 1-hexacosanol (Hex) with 26 carbons (C) and the alkane heptacosane (Heps; C27) and lower amounts of most of the other waxes and total wax relative to waxy B8667 and glossy B9885 (Table 1, Fig. 2). Amounts of Oct1 (C28) were not significantly different for OGL, glossy B9885, waxy B8667, and their hybrids (Table 1). Therefore, the foliage of OGL has the unique phenotype of accumulating C26 and C28 fatty alcohols (Hex and Oct1, respectively) and a C27 alkane (Heps) and significantly lower amounts of waxes with more than 28 carbons compared with waxy onion.
Mean peak areas adjusted for the leaf dry weight of epicuticular waxes detected by gas chromatography-mass spectrometry on onion leaves of ‘Odourless Green Leaf’ (OGL), glossy inbred B9885, waxy inbred B8667, and hybrids from crosses of OGL with B8667 and B9885 with OGL. Means are from the measurements of five plants.
Boxplots for the amounts of (A) fatty alcohol 1-hexacosanol, (B) alkane heptacosane, and (C) ratio (HOH/Total) of the summed amounts of 1-hexacosanol, 1-octacosanol, and heptacosane (HOH) over the sum total of all waxes (Total wax) on foliage of five plants each of glossy ‘Odourless Greenleaf’ (OGL), waxy B8667, glossy B9885 onion, and hybrid progenies. Values on Y-axes in (A) and (B) are peak areas adjusted for leaf dry weights.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 2; 10.21273/JASHS05024-20
Genetic analyses of segregating families from glossy OGL by waxy crosses.
Crosses of OGL plants with waxy B8667, DH2107, and DH066619 produced hybrids with waxy foliage, indicating that the glossy phenotype from OGL is recessive relative to waxy foliage. For 19 of 22 F2 families from the cross of glossy OGL with waxy plants, visual segregations of waxy vs. glossy foliage fit the 3:1 expected ratio (P > 0.05) for a single locus at which the homozygous recessive genotype conditions the glossy phenotype (Table 2). F2 families 2, 3, and 11 (Table 2) had excesses of glossy progenies and fit the expected 3:1 only at P between 0.05 and 0.01. After confirming the homogeneity of errors across families, segregation for waxy vs. glossy foliage summed across families from the crosses of OGL with waxy male parents fit the 3:1 ratio [1049 waxy: 373 glossy (P = 0.284)]. Genetic mapping was completed using 131 SNPs and linkage groups were constructed at an LOD of 6.0. Visual segregations for glossy vs. waxy foliage mapped as a single locus at position 113 cM on chromosome 6 (Supplemental Table 1).
Observed (Obs) segregations and probabilities of goodness-of-fit to expected (Exp) ratios for the numbers of progenies with glossy (Gl) versus waxy (Wx) foliage in F2 families from crosses of glossy ‘Odourless Greenleaf’ (OGL) with glossy (B9885) or waxy (B8667, DH2107, and DH066619) inbred lines of onion.
We calculated the ratio of summed amounts of Hex, Oct1, and Heps (HOH) over total wax (HOH/Total), and OGL was significantly different from B9885, B8667, and hybrids from crosses (Table 1, Fig. 2). A scatterplot of this ratio for F2 progenies from the cross of OGL × waxy B8667 (family 4 in Table 2) showed discrete segregation (Fig. 3) that fit the expected ratio for a single locus at which the homozygous recessive genotype conditioned the glossy OGL phenotype [68 progenies with ratio <0.7: 22 progenies with ratio > 0.7 (P = 0.903)]. Quantitative mapping of the visual phenotype as a binary trait and CIM of the HOH/Total ratio revealed a highly significant QTL at the same position on chromosome 6 at 111 cM, explaining 85% (LOD 37) and 80% (LOD 31.5) of the phenotypic variation, respectively (Table 3). QTL at or near the same position on chromosome 6 were detected and conditioned high amounts of Hex and Heps, and the chromosome region of wild-type waxy B8667 showed significant additive and dominance effects that decreased amounts. Significant QTL conditioning low amounts of the other individual waxes and total wax were detected at or near the same position on chromosome 6, and the chromosome region of B8667 increased amounts (Table 3). Although the amounts of Oct1 were not significantly different between OGL and B8667 (Table 1), CIM revealed a QTL (LOD 3.7) just above the significance threshold (LOD 3.3) on chromosome 5 at 57 cM, explaining 17% of phenotypic variation (Table 3). No QTL were detected for the amounts of the alkane Non in the segregating family from OGL × B8667.
Scatterplots of the ratio of the summed amounts of 1-hexacosanol, 1-octacosanol, and heptacosane (HOH) over the sum total of all waxes (HOH/Total) for F2 progenies from the crosses of glossy OGL with waxy B8667 (A) or glossy B9885 (B) onion. Values were calculated using peak areas adjusted for the leaf dry weights.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 2; 10.21273/JASHS05024-20
Chromosome (Chr) and position (Pos) in centimorgans of the most significant single-nucleotide polymorphism (SNP), SNPs flanking the 1.5 logarithm of odds (LOD) confidence interval, percent variation (Var) explained, observed (Obs) LOD with threshold (Thresh) LOD from permutation analysis, and additive (Add) and dominance (Dom) effects of chromosome region from wild-type waxy B8667 for quantitative trait loci detected by composite interval mapping for the visual (glossy versus waxy) phenotype and amounts of epicuticular waxes on foliage of F2 progenies from the cross of glossy ‘Odourless Greenleaf’ by waxy B8667 onion.
Genetic analyses of segregating families from glossy B9885 × glossy OGL crosses.
Crosses of glossy inbred B9885 × glossy OGL produced hybrids with waxy foliage, indicating that the glossy phenotype from OGL is not conditioned by recessive alleles at the glwp locus on chromosome 8 from B9885 (Munaiz and Havey, 2020). For F2 families from the cross of B9885 × OGL, visual segregations of waxy vs. glossy foliage fit the expected 9:7 ratio for two independently segregating loci where the homozygous recessive genotype at either locus conditions glossy foliage (families 23–25 in Table 2). After confirming homogeneity of errors, segregations summed across F2 families from the cross of B9885 × OGL fit a 9:7 ratio [132 waxy: 102 glossy (P = 0.730)]. This result indicates that the glossy OGL phenotype segregates independently of the glwp locus from glossy B9885 (Munaiz and Havey, 2020).
Genetic mapping was completed using 129 SNPs and DNA from F2 family 24 (Table 2), and linkage groups were constructed at an LOD of 6.0 (Supplemental Table 2). Mapping of glossy vs. waxy foliage as a binary trait revealed two QTL, one on chromosome 6B at 21 cM (LOD = 3.6) and one on chromosome 8 at 26 cM (LOD = 9.0), agreeing with the 9:7 segregation (Tables 2 and 4). The amounts of Hex, Tri, and Heps were significantly different on leaves of glossy OGL and glossy B9885 (Table 1). The CIM of amounts of fatty alcohols Hex and Tri detected a QTL on chromosome 6B at 21 cM (LOD 10.4 and 11.6), explaining 40% and 44% of the phenotypic variation, respectively (Table 4). The chromosome region from OGL increased the amounts of Hex and decreased the amounts of Tri, consistent with the results from the OGL × waxy B8667 family described. Regarding the amounts of the alkane Heps, significant QTL were detected on chromosomes 6B at 21 cM (LOD = 9.7) and 8 at 39 cM (LOD = 8.9), explaining 18% and 13% of the phenotypic variation, respectively (Table 4). Both chromosome regions from OGL had significant additive effects that increased the amounts of Heps (Table 4). The scatterplot of the HOH/Total ratio for F2 progenies from family 24 (Table 2) showed discrete segregation of the phenotype (Fig. 3) that fit a 3:1 ratio [71 progenies < 0.7: 22 progenies > 0.7 (P = 0.765)]. The CIM of the HOH/Total ratio revealed one significant (LOD = 12.1) QTL on chromosome 6B at 21 cM, explaining 45% of the phenotypic variation (Table 4).
Chromosome (Chr) and position (Pos) in centimorgans of the most significant single nucleotide polymorphism (SNP), SNPs flanking the 1.5 logarithm of odds (LOD) confidence interval, percent variation (Var) explained, observed (Obs) LOD with threshold (Thresh) LOD from permutation analysis, and additive (Add) and dominance (Dom) effects of chromosome region from glossy ‘Odourless Greenleaf’ (OGL) for quantitative trait loci detected by composite interval mapping for the visual (glossy versus waxy) phenotype, and amounts of epicuticular waxes on foliage of F2 progenies from the cross of glossy B9885 by glossy OGL onion.
Although OGL and B9885 were not significantly different for amounts of the alkanes Hent and Non, the ketone H16, and total wax, significant QTL were detected on chromosomes 6B and 8. Regarding the amounts of Hent and Non, significant QTL on chromosome 6B at 21 cM (LOD = 9.1 and LOD = 10.3) explained 36% and 24% of the phenotypic variation, respectively, and the chromosome region from OGL decreased the amounts of both alkanes (Table 4). Regarding the amounts of H16, significant QTL were detected on chromosomes 6B at 21 cM (LOD = 10.7) and chromosome 8 at 36 cM (LOD = 13.0), explaining 22% and 32% of the phenotypic variation, respectively (Table 4). The chromosome region on chromosome 8 from OGL increased the amounts of H16, whereas the region on 6B from OGL decreased the amounts of H16. Regarding total wax, a QTL was detected on chromosome 8 at 36 cM (LOD 8.2), explaining 24% of the phenotypic variation (Table 4). The location and effect of QTL controlling the amounts of H16 and total wax are consistent with the glossy phenotype conditioned by the glwp locus (Munaiz and Havey, 2020). No significant QTL were detected for the amounts of Oct1 in the B9885 × OGL family.
Discussion
The foliage of OGL onion accumulates the fatty alcohols Hex (C26) and Oct1 (C28), the alkane Heps (C27), and low amounts of waxes with more than 28 carbons (Table 1). Wax profiles similar to OGL have been reported for other plants conditioned by the cer19 mutant of Arabidopsis thaliana (Rashotte et al., 2001), gl3 mutant of Zea mays (Avato et al., 1987), and gl2 and gl5 mutants of the Brassica oleraceae (Macey and Barber, 1970); all of those do not accumulate significant amounts of waxes with more than 28 carbons. In plants, the synthesis of epicuticular waxes begins with the production of long-chain (>C26) fatty acids that enter the acyl reduction and decarbonylation pathways (Millar et al., 1999; Samuels et al., 2008). In the acyl reduction pathway, fatty acids with even numbers of carbons are used to produce fatty alcohols such as Hex (C26), Oct1 (C28), and Tri (C30). In the decarbonylation pathway, fatty acids lose one carbon, generating waxes with odd numbers of carbons such as the alkanes Heps (C27), Non (C29), and Hent (C31) and the ketone H16 (C31). The accumulation of two fatty alcohols and one alkane on the foliage of OGL indicates that both the acyl reduction and decarbonylation pathways are functioning, and that the availability of the C30 fatty acid (triacontanoic acid) as the substrate for synthesis of waxes with more than 28 carbons may be limiting in OGL leaves.
Results from segregation analyses of the OGL × B8667 and B9885 × OGL families supported a single region on chromosome 6 controlling the glossy foliage and unique wax profile of OGL. SNP i32739_152 mapped within the LOD-1.5 interval for these traits in both the OGL × B8667 and B9885 × OGL families (Tables 3 and 4, Supplemental Tables 1 and 2). Because the same region on chromosome 6 was mapped in two segregating families, we propose the locus name glogl at which the recessive allele from OGL conditions its glossy foliage and unique epicuticular wax profile.
The glossy phenotype from ‘White Persian’ and OGL suffer significantly less feeding damage from onion thrips relative to waxy onion (Cramer et al., 2014; Damon et al., 2014; Jones et al., 1934). However, the glossy phenotype from ‘White Persian’ accumulates significantly lower amounts of total epicuticular waxes compared with waxy onion (Damon et al., 2014; Molenaar, 1984) (Table 1) and shows susceptibility to foliar pathogens (Mohan and Molenaar, 2005). The selection of onion for unique wax profiles, such as high amounts of fatty alcohols and low amounts of H16 (Damon et al., 2014; Munaiz and Havey, 2020), may produce populations that support fewer thrips and suffer less feeding damage from the insect (Munaiz et al., 2020a) but still accumulate adequate amounts of total epicuticular wax for commercial production. Onion populations with unique wax profiles, such as OGL, will also be useful for more basic studies of genes affecting specific profiles of epicuticular waxes.
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Loci, chromosome (Chrom), position (Pos) in centiMorgans, observed (Obs) segregations, and probabilities of goodness-of-fit to the expected (Exp) segregation ratios for the F2 family from the cross of glossy ‘Odourless Greenleaf’ with waxy B8667 onion.
Loci, chromosome (Chrom), position (Pos) in centiMorgans, observed (Obs) segregations, and probabilities of goodness-of-fit to the expected 1:2:1 segregation ratios for the F2 family from the cross of glossy B9885 with glossy ‘Odourless Greenleaf’ onion.