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a Center for Applied Genetic Technologies, 111 Riverbend Road, The University of Georgia, Athens, GA, 30602, USA
b Advanta Seeds, Balcarce Research Station, Ruta 226, KM 60.3 (7620), Balcarce PCIA DE BS. AS., ARGENTINA
c Department of Experimental Statistics, Clemson University, Clemson, SC 29634
* Corresponding author (sjknapp{at}uga.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: INDEL, insertion-deletion • LG, linkage group • QTL, quantitative trait locus • RIL, recombinant inbred line • SSR, simple sequence repeat
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unshellable, small-seeded, high-oil (oilseed) cultivars were developed between 1920 and 1955 by direct selection for increased seed oil concentration and indirect selection for small seeds and thin pericarps (hulls) in open-pollinated populations (Putt, 1940, 1997; Heiser, 1951, 1977; Pustovoit, 1964). Cultivars producing 380 g kg–1 of seed oil may have been developed as early as 1915; however, the seed oil concentrations of cultivars commonly grown between 1900 and 1915 reportedly ranged from 200 to 300 g kg–1 (Putt, 1997). Selection in open-pollinated populations increased seed oil concentrations to 430 g kg–1 by 1935 and 490 g kg–1 by 1955 (Pustovoit, 1964; Heiser, 1977; Seiler, 1985, 1994; Putt, 1997; Seiler and Brothers, 1999), thereby greatly increasing the economic importance of sunflower as an oilseed and further differentiating the confectionery and oilseed market classes (Pustovoit, 1964; Putt, 1997).
Seeds of confectionery and oilseed cultivars are distinguished by differences in shellability, hull color, seed weight and morphology, and kernel-to-pericarp weight ratio, in addition to seed oil concentration (Beard, 1981; Seiler, 1997). The seeds of confectionery cultivars are typically gray or white, black or brown striped, and easily dehulled or shelled, whereas the seed of oilseed cultivars are typically black (dark purple) and difficult to dehull. Seeds of confectionery cultivars are larger and have lower kernel-to-pericarp weight ratios than seeds of oilseed cultivars. Hull color differences are produced by pigments in the epidermal, hypodermal, and phytomelanin layers (Johnson and Beard, 1977; Leon et al., 1996; Miller and Fick, 1997). The epidermis is either unpigmented, solid brown or black, or black- or brown-striped, the hypodermis is either anthocyanin pigmented (black) or unpigmented, and phytomelanin is either present (black) or absent (Johnson and Beard, 1977; Leon et al., 1996; Miller and Fick, 1997). Several hull pigment loci have been identified through phenotypic analyses of mutations (reviewed by Miller and Fick, 1997). The allelism of many of the mutations is not known, and only Hyp, a hypodermis pigment locus, has been genetically mapped (Leon et al., 1996).
The first high-oil cultivars were apparently developed by introgressing allelic diversity from wild populations into open-pollinated, low-oil cultivars (Pustovoit, 1964; Heiser et al., 1969; Semelczi-Kovacs, 1975; Putt, 1997). Selection within domesticated, low-oil populations per se cannot be completely ruled out—land race selec tion between 1880 and 1915 could have played a role in advancing sunflower as an oilseed (Putt, 1997). Significant genetic variability for seed oil concentration is present in wild sunflower germplasm, e.g., seed oil concentrations ranged from 191 to 355 g kg–1 among 340 wild H. annuus populations screened by the USDA (http://www.ars-grin.gov). The upper end of the range was even greater among 93 wild populations of H. anomalus S. F. Blake, H. petiolaris Nutt., H. debilis Nutt., and other taxa—minimum, mean, and maximum seed oil concentrations were 262, 346, and 457 g kg–1, respectively.
Once hybrid seed production systems were developed (Leclercq, 1969; Kinman, 1970), the focus in sunflower breeding rapidly shifted to the development of high-oil inbred lines from an assortment of open-pollinated, high-oil populations and cultivars, the progenitors of most of the early high-oil inbred lines on which the hybrid seed industry was initially built (Korell et al., 1992; Cheres and Knapp, 1998). While present-day hybrids commonly produce 440 to 490 g kg–1 of seed oil, CM 612 (PI 546351), CM 630 (PI 566828), and other inbred lines producing 500 to 530 g kg–1 of seed oil have been described (Dedio and Rashid, 1991, 1994; http://www.ars-grin.gov). Our goal was to identify phenotypic and quantitative trait loci (QTL) underlying genetic variability for pericarp pigments, seed oil concentration, and other seed traits in a recombinant inbred line (RIL) mapping population (Tang et al., 2002) developed from a hybrid between large-seeded, low-oil (RHA280) and small-seeded, high-oil (RHA801) inbred lines developed in the early period of single-cross hybrid breeding in sunflower (Fick et al., 1974a; Roath et al., 1981).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The RILs were field grown at Corvallis, OR, in the summers of 2000 and 2001 in 6.1-m-long rows spaced 0.9 m apart. Two replications of the RILs and parent inbred lines were planted in randomized complete blocks in May of both years. Fifty-six kilograms per hectare of 20N-10P-15K- 7S fertilizer was broadcast preplant and incorporated during the primary tillage operation. The seed bed was prepared by disking and harrowing, and seeds were planted 2.54 cm deep. The within-row seeding rate was 40 seeds per 6.1 m. Plants were hand-thinned to produce a within row spacing of one plant per 0.3 m 2 wk postemergence. Capture [bifenthrin = (2-methyl-3-phenyl-phenyl)methyl 3-(2-chloro-3,3,3-trifluoro-prop-1-enyl)-2,2-dimethyl-cyclopropane-1-carboxylate, 0.11 kg ai ha–1] and Hoelon {diclofop-methyl = methyl (RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propionate, 2.35 L ha–1} were applied postplant, preemergence mixture for broadleaf and grass weed control and were activated by a minimum of 1.27 to 2.54 cm of water applied through sprinkler irrigation, rainfall, or both. Subsequent to thinning, 100.8 kg per ha of N was applied by banding urea (46N-0-0). The crop was sprinkler irrigated at 2-wk intervals throughout the first 12 wk of the growing season; 1.28 to 2.54 cm of water was applied at each interval. Capitula were harvested in late September of both years, dried in a forced-air gas seed drier for 48 to 72 h at 42 to 44°C, threshed, and cleaned.
The RILs were phenotyped at the onset of flowering for presence and absence of apical branching (20 plants per RIL per replication were phenotyped). Four to 12 physiologically mature seeds per RIL were visually phenotyped for hypodermal and phytomelanin pigments in the pericarp by dissecting through successive pericarp cell layers (epidermis, hypodermis, and phytomelanin) as described by Leon et al. (1996). Capitula were harvested from 10 individuals per RIL per replication. Primary capitula were harvested from unbranched RILs, whereas primary and two secondary capitula were harvested from branched RILs. Kernel weight (kwt), pericarp weight (pwt), and kernel-to-pericarp weight ratio (kpr = kwt/pwt) were measured on 10 seeds per RIL per replication produced in 2000. The 10 seeds per RIL were manually dehulled with a scalpel and separated into kernel and hull fractions. Five-gram samples of seeds were randomly drawn from each replication and pooled into a single 10-g sample for seed oil concentration (soc) analyses by nuclear magnetic resonance (Leon et al., 1995). We measured seed length (sl), width (sw), and depth (sd) on 10 randomly sampled seeds per RIL per replication and 100-seed weight (swt) on 100 randomly sampled seeds per RIL per replication.
We constructed a genetic linkage map for the QTL analysis using three phenotypic loci (B, P, and Hyp) and 203 simple sequence repeat (SSR) or insertion-deletion (INDEL) marker loci selected from the original 577-locus RHA280 x RHA801 genetic linkage map described by Tang et al. (2002) and Yu et al. (2003). The original map was constructed by genotyping 94 RILs; hence, 203 SSR or INDEL markers were genotyped on 79 additional RILs for the present study. The terminal-most or near terminal-most SSR or INDEL marker loci from each linkage group were selected for genotyping in addition to SSR or INDEL marker loci spaced as evenly as possible throughout each linkage group. The genotyping methods and statistical analyses used to construct the map have been described elsewhere (Tang et al., 2002).
Statistical Analyses
The phenotypic distributions for seed traits were tested for normality by the Kolmogorov- Smirnov (D) statistic. D was estimated by PROC UNIVARIATE of the Statistical Analysis System (SAS) (http://www.sas.com). Progeny-mean (RIL-mean) heritabilities (h2) and additive genetic correlations (rG) were estimated for RILs among years and replications for 100-seed weight and seed length, width, and depth, among years for seed oil concentration, and among replications for kernel and pericarp weight and kernel-to-pericarp weight ratio. RILs, years, and replications were identified as random effects for variance and covariance component analyses. Variance components were estimated by the REML method of SAS PROC VARCOMP and covariance components were estimated by the REML method of SAS PROC MIXED as described by Holland et al. (2001). RIL-mean heritabilities (h2) and genetic correlations (rG) were estimated as described by Falconer and MacKay (1996) and Bernardo (2002).
Composite interval mapping (CIM), as implemented in QTL-Cartographer (Zeng, 1993, 1994; Zeng et al., 1999; Basten et al., 2001), was used to scan for additive intralocus QTL effects. CIM analyses were performed on least square means for RILs estimated by SAS PROC MIXED, where RIL effects were fixed and replication, year, and RIL x year interaction effects were random. Tests of significance of RIL, replication, year, and RIL x year effects were performed with Type III F statistics estimated by SAS PROC GLM. Rank correlations between least square means for RILs between years were estimated by SAS PROC CORR. CIM analyses were performed on RIL least square means across years (RIL x year effects were nonsignificant for every trait).
CIM analyses were performed with a 5-cM window width and significant cofactors identified by QTL-Cartographer; 10 significant cofactors were identified and selected for CIM analyses of soc and five significant cofactors were identified and selected for CIM analyses of other seed traits. B, P, and Hyp were used as cofactors for every trait. Likelihood-odds (LOD) thresholds for declaring statistical significance (the presence of QTL) were calculated by permutation testing with 1000 permutations (Churchill and Doerge, 1994). Permutation thresholds ranged from 2.9 to 3.2 for the eight traits. One-LOD support intervals were calculated as described by Conneally et al. (1985) and Lynch and Walsh (1998). The additive effects (a) and phenotypic coefficients of determination (R2P) for individual QTL were estimated by CIM.
Multilocus QTL analyses were performed with seven genetic marker loci as independent variables in mixed linear models (Littel et al., 1991, 1996). The loci selected as independent variables—B, P, Hyp, and four SSR marker loci (ORS371, ORS188, ORS1068, and ORS484)—were tightly linked to QTL identified by CIM for multiple seed traits. Statistical analyses were performed by SAS PROC MIXED, where genotype (G) effects were fixed and RIL nested in genotype (RIL:G), year (Y), replication (R), and genotype x year (G x Y) effects were random. Variance components were estimated by the REML method, Type III sums of squares and F statistics were estimated for genotype effects, least square means were estimated for genotypes, and additive (a), additive x additive (a x a), and additive x additive x additive (a x a x a) effects were estimated by ESTIMATE statements.
Statistical analyses were performed on homozygotes only because heterozygotes were rare and produced severely unbalanced data and missing cells. We performed an incomplete 27 factorial analysis by estimating first-, second-, and third-order effects (a, a x a, and a x a x a, respectively) among the seven genetic marker loci. RIL:G and G x Y variance components were nonsignificant and were subsequently pooled with other random residual effects to maximize error degrees of freedom (dfe); dfe were 215 for seed oil concentration, 540 for 100-seed weight and seed length, width, and depth, and 237 for kernel and pericarp weight and kernel-to-pericarp weight ratio. Genetic coefficients of determination were estimated for a effects only (R2G = SSG/SSRIL) and a, a x a, and a x a x a effects combined (R2G* = SSG*/SSRIL), where SSG is the sum of squares for a effects, SSG* is the sum of squares for a, a x a, and a x a x a effects, SSRIL is the sum of squares among RILs, and SSG, SSG*, and SSRIL were estimated by SAS PROC GLM (Littel et al., 1996).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 = 0.01, p = 0.91) for F6:7 RILs (85.2 b b: 2.7 B b: 85.2 B B). The B locus mapped to LG 10 (Fig. 1). The observed segregation ratio for Hyp, 83 opaque white (Hyp Hyp): 2 segregating (Hyp hyp): 88 transparent (hyp hyp), was not significantly different from the expected segregation ratio (2 = 0.41; p = 0.65). The Hyp locus mapped to LG 16 (Fig. 1). The observed segregation ratio for P, 71 non- pigmented (p p): 1 segregating (P p): 101 black (P P), was significantly different from the expected segregation ratio (2 = 6.9, p = 0.008) because of an excess of P P RILs. The segregation ratios for SSR marker loci tightly linked to P had similarly distorted segregation ratios. The P locus mapped to LG 17 (Fig. 1).
Heritabilities and Genetic Correlations
The phenotypic distributions were normal for soc, sl, sw, and sd and approximately normal but slightly positively skewed for swt, kwt, pwt, and kpr (the phenotypic distributions for soc x kpr and soc x swt are shown in Fig. 3 for branched and unbranched RILs). Significant genetic variability was observed among RILs for every trait. Year effects were significant for every trait, whereas RIL x year interaction effects were nonsignificant for every trait measured in both years; kwt, pwt, and kpr were only measured 1 yr. The rank correlations for RIL least square means between years were significant, high, and positive for the five traits measured in both years (0.85 for soc, 0.88 for swt, 0.92 for sl, 0.90 for sw, and 0.85 for sd). RIL-mean heritabilities were exceptionally high and ranged from 0.92 to 0.98 (Table 2).
Strong genetic correlations were observed among the eight seed traits (Table 3 and Fig. 3). Oil concentration was negatively genetically correlated with pericarp weight (–0.75) and 100-seed weight (–0.58) and positively genetically correlated with kernel-to-pericarp weight ratio (0.85). Kernel and pericarp weight were positively genetically correlated with seed length, width, and depth (0.76–0.88) and 100-seed weight (0.94).
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QTL Identified by Composite Interval Mapping
Using CIM, we identified 40 QTL for eight seed traits in 14 marker intervals on 10 linkage groups (Fig. 1 and Table 4). Three-fourths of the QTL were clustered on linkage groups (LG) 5 (five QTL), 10 (eight QTL), 16 (six QTL), and 17 (10 QTL). QTL on seven linkage groups affected multiple traits (Fig. 1). The QTL identified on LG 10, 16, and 17 were centered on or near B, Hyp, and P, respectively, whereas QTL identified on LG 1, 4, 5, and 9 were centered on or near four SSR marker loci (ORS371, ORS1068, ORS484, and ORS188, respectively). Single-trait QTL were identified for sl on LG 8, sd on LG 12, and swt on LG 14 (Fig. 1). Four to six QTL were identified for each trait and collectively explained 52.3 to 77.5% of the phenotypic variability per trait (individual QTL explained 3.0 to 52.5% of the phenotypic variability per trait) (Table 4).
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Six QTL were identified on LG 1, 4, 9, 10, 16, and 17 for seed oil concentration (Fig. 1 and Table 4). The QTL individually explained 3.1 to 22.5% and collectively explained 55.7% of the phenotypic variability for soc. QTL for soc on LG 10, 16, and 17 were centered on the B, Hyp, and P loci, respectively, and QTL alleles for increased oil were transmitted by the oilseed parent (RHA801). B-, Hyp-, and P-linked QTL (soc10.1, soc16.1, and soc17.1) explained 22.5, 10.5, and 3.1% of the phenotypic variability, respectively. R2P for the third largest QTL (soc9.1) was 9.1%.
Five QTL were identified on LG 5, 9, 10, 14, and 17 for 100-seed weight (Fig. 1 and Table 4). The QTL collectively explained 73.4% of the phenotypic variability. B- and P-linked QTL (swt10.1 and swt17.1) explained 52.5 and 7.4% of the phenotypic variability for swt, respectively. The minor QTL swt9.1 and swt14.1 explained 3.2 and 3.6% of the phenotypic variability, respectively. The RHA280 allele from four of the five QTL increased 100-seed weight, whereas the RHA280 allele for one of the five QTL (swt5.1) decreased 100-seed weight. The swt5.1 QTL had the third largest effect.
Four QTL were identified for kwt on three linkage groups (LG 5, 10, and 16) and collectively explained 57.3% of the phenotypic variability (Fig. 1 and Table 4). Two linked QTL were identified on LG 16 (kwt16.1 and kwt16.2). Favorable alleles for the B-linked QTL (kwt10.1) were transmitted by the unbranched, low-oil parent (RHA280), whereas favorable alleles for the other three QTL (kwt5.1, kwt16.1 and kwt16.2) were transmitted by the branched, high-oil parent (RHA801). Similarly, five QTL were identified for pwt on four linkage groups (LG 4, 5, 10, and 17) and collectively explained 77.5% of the phenotypic variability. Favorable alleles were transmitted by the low-oil parent for one of the five QTL (pwt5.1) (Table 4). Two linked QTL were identified for pwt on LG 17 (pwt17.1 and pwt17.2). Coincident QTL were identified for swt, kwt, and pwt in DNA marker intervals on linkage groups 5 (kwt5.1, pwt5.1, and swt5.1), 10, (kwt10.1, pwt10.1, and swt10.1), and 17 (pwt17.2 and swt17.1) (Fig. 1). By contrast, kwt and pwt QTL were not identified in two DNA marker intervals where small-effect swt QTL were found (swt9.1 and swt14.1) (Table 4).
Five QTL were identified for kernel-to-pericarp weight ratio on LG 1, 4, 10, 16, and 17 and collectively explained 52.3% of the phenotypic variability. RHA801 alleles for every QTL increased kpr (Fig. 1 and Table 4). The five kpr QTL were coincident with five of six SOC QTL and, as noted earlier, the effects of the B-, Hyp-, and P-linked QTL (kpr10.1, kpr16.1, and kpr17.1) were virtually equal and individually explained 13.5 to 15.2% of the phenotypic variance for kpr.
Five, 4, and 6 QTL were identified for sl, sw, and sd, respectively, and collectively explained 75.9, 73.9, and 73.1% of the phenotypic variance, respectively (Fig. 1 and Table 4). QTL for sl, sw, and sd were clustered on LG 5, 10, 16, and 17 and overlapped SOC, swt, kwt, pwt, and kpr QTL. QTL for sl, sw, and sd on LG 5 and 16 had positive additive effects (alleles from the high- oil parent increased seed length, width, and depth), as was observed for overlapping swt, kwt, and pwt QTL on LG 5 and 16 where alleles from the high-oil parent increased 100-seed, kernel, and pericarp weight. The three B-linked QTL for sl, sw, or sd (sl10.1, sw10.1, and sd10.1) explained 41.7, 44.0, and 43.6% of the phenotypic variability and the five P-linked QTL for sl, sw, or sd (sl17.1, sl17.2, sw17.1, sw17.2, and sd17.1) explained 10.5, 9.2, 13.7, 12.7, and 13.4% of the phenotypic variability.
Multilocus QTL Analyses
Multilocus QTL analyses were performed with phenotypic and DNA marker loci (B, P, Hyp, ORS371, ORS188, ORS1068, and ORS484) identified by CIM to be tightly linked to QTL for multiple seed traits (Fig. 1). The array of marker genotypes observed among the RILs was insufficient for performing a complete 27 factorial analysis but sufficient for performing an incomplete 27 factorial analysis with first-, second-, and third-order (a, a x a, and a x a x a, respectively) effects among B, P, Hyp, ORS371, ORS188, ORS1068, and ORS484 (Fig. 4
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)–every a, a x a, and a x a x a effect was estimable, 89 out of 128 possible homozygous genotypes were observed among 153 RILs, 20 RILs were heterozygous for one or more loci, and less than half of the genotypes were replicated (were observed in two or more RILs). Using a significance threshold of p 
0.05, 39 out of 56 a, 70 out of 168 a x a, and 119 out of 280 a x a x a effects were significant among the eight seed traits (Fig. 4–6). Using a more stringent significance threshold (p 0.0001), 24 a, 11 a x a, and 36 a x a x a effects were significant. R2G* ranged from 0.63 to 0.82; hence, two-thirds to fourth-fifths of the phenotypic variability among RILs was associated with QTL (Table 2). B-, P-, and Hyp-linked QTL alone were associated with more than half of the phenotypic variability among RILs–R2G* ranged from 0.50 to 0.63 for the eight seed traits in complete 23 factorial analyses performed with only B, P, and Hyp as the independent variables.
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–6). While positive and negative epistatic interactions were observed for every trait, most were negative (intragenic and intergenic interaction effects had opposite signs) and crossover in nature. The sign differences we observed between intragenic (a) and intergenic (a x a and a x a x a) effects (Fig. 4–6) and between sums of significant (p < 0.05) intragenic and intergenic effects (Table 5) illustrate the predominantly negative or antagonistic (Holland, 2001) nature of epistasis among QTL for seed traits in RHA280 x RHA801. If the intragenic effect of a QTL was positive (increased the mean), most of the intergenic effects between the QTL and other QTL were negative (decreased the mean). Sums of significant intergenic QTL effects were greater than sums of significant intragenic QTL effects for six of the eight traits (swt, sl, sw, sd, kwt, and pwt) (Table 5). Conversely, sums of significant intergenic QTL effects were less than sums of significant intragenic QTL effects for seed oil concentration and kernel-to-pericarp weight ratio. The intergenic to intragenic effect ratio was lowest for seed oil concentration (0.03) and greatest for kernel weight (7.07) and seed length (13.85) (Table 5).
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–6). Hyp- and ORS371-linked QTL for seed weight, length, width, and depth affected other traits through predominantly negative epistatic interactions (Fig. 4–6). ORS484 was selected for multilocus QTL analyses because of tight linkage to QTL for 100-seed weight, seed length and depth, and kernel and pericarp weight (Fig. 1 and Table 4). Contrary to other QTL, ORS484 alleles transmitted by the high-oil parent increased 100-seed weight, seed length, width, and depth, and pericarp weight (Fig. 1 and 4–6). The additive effect of ORS484 on seed oil concentration was nonsignificant because of the canceling effect of the crossover additive x additive interaction effect; however, ORS484 affected seed oil concentration through one positive (ORS371 x ORS188 x ORS484) and two negative (B x ORS484 and ORS371 x ORS484) epistatic interactions (Fig. 4). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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–6). Selection for increased seed oil concentration seems to have operated on multiple QTL affecting several genetically correlated seed traits. Thus far, 54 QTL have been identified for eight seed traits on 14 linkage groups (20 DNA marker intervals) in RHA280 x RHA801 (Fig. 1 and 4–6) and an F2 population developed from an oilseed x wild (HA89 x ANN1238) hybrid (Burke et al., 2002). The number of loci producing genetic variability for seed traits obviously could be less than 54 because of pleiotropy. The genomic regions flanking B, P, and Hyp apparently harbor multiple linked or pleiotropic QTL and seem to be hotbeds of allelic diversity for seed and other traits in sunflower. Of the seven QTL clusters identified in RHA280 x RHA801, two on the lower end of LG 17 and one on LG 10 overlapped the 100-seed weight, length, and width QTL identified by Burke et al. (2002) in HA89 x ANN1238 (Fig. 1 and Table 4). Conversely, none of the swt, sl, and sw QTL on LG 2, 3, 5, 6, 8, 9, 12, and 13 identified by Burke et al. (2002) overlapped QTL identified in RHA280 x RHA801 (Fig. 1). The P locus sits in the middle of the LG 17 QTL cluster, whereas the B locus sits in the middle of the LG 10 QTL cluster (Fig. 1). No branching QTL were identified by Burke et al. (2002) on LG 10. The 1.0-LOD support interval for a 100-seed weight QTL on LG 17 in HA89 x ANN1238 overlapped 1.0-LOD support intervals for a 100-seed weight QTL (swt17.1) and several other seed trait QTL (SOC17.1, kpr17.1, sl17.2, sw17.2, and sd17.1) on LG 17 in RHA280 x RHA801 (Fig. 1). Hence, the 23.6-cM ORS561-ORS811 interval on LG 17 harbors several QTL affecting multiple correlated and uncorrelated domestication and post- domestication traits (e.g., seed oil concentration, seed shattering, and self-pollination). Between the RHA280 x RHA801 and HA89 x ANN1238 analyses, 23 QTL have been identified for 17 traits in the ORS561-ORS811 interval on LG 17.
The segregation of branching and hull pigment loci in RHA280 x RHA801 facilitated genetic mapping of B, P, and Hyp and the assignment of B, P, and Hyp locations using the public linkage group nomenclature (Tang et al., 2002; Yu et al., 2003). B and Hyp were previously mapped by means of proprietary RFLP markers and independent linkage group nomenclatures, each differing from the public linkage group nomenclature (Leon et al., 1996; Mestries et al., 1998; Gentzbittel et al., 1999). Hull color differences distinguishing "striped" confectionery from "black" oilseed cultivars and RHA280 from RHA801 were caused by P and Hyp mutations (Table 1; Fig. 1 and 2). The allelism of P and Hyp to previously identified hull pigment mutations (Miller and Fick, 1997) is not known. P could be the phytomelanin pigment locus described by Mosjidis (1982). Hyp is the hypodermal pigment locus described by Leon et al. (1996).
The analysis of hull pigment loci in RHA280 x RHA801 was stimulated by the discovery of large-effect QTL for seed oil concentration linked to Hyp in ZENB8 x HA89 (Leon et al., 1996, 2003), and led to the discovery of multiple large-effect QTL, linked not only to Hyp, but to P (Table 4 and Fig. 1 and 4–6). P- and Hyp-linked QTL epistatically interacted with each other, with B-linked QTL, and with other QTL in RHA280 x RHA801 (Fig. 4 –6). By contrast, Hyp- linked QTL did not epistatically interact with other QTL in ZENB8 x HA89 (Leon et al., 1995, 2003). Moreover, epistatically interacting QTL for seed oil concentration have not been identified in other genetic analyses in sunflower (Leon et al., 1995, 2003; Mestries et al., 1998; Mokrani et al., 2002; Bert et al., 2003).
B, P, and Hyp either directly (pleiotropically) produce or are tightly linked to QTL producing genetic variability for seed traits in sunflower. B has long been known to profoundly affect several capitula and seed traits (Ross, 1939; Fick et al., 1974b; Dedio, 1980), and B-linked QTL have been shown to affect seed oil concentration and 100-seed weight in two high-oil x high-oil mapping populations, GH x PAC (Mestries et al., 1998) and XRQ x PSC8 (Bert et al., 2003). The B-linked seed oil QTL identified in GH x PAC and XRQ x PSC8 were similar in magnitude but opposite in sign—branching increased seed oil concentration in GH x PAC and decreased seed oil concentration in XRQ x PSC8. Mestries et al. (1998) concluded that B pleiotropically affected seed oil concentration, whereas Bert et al. (2003) concluded that seed oil concentration was affected by QTL tightly linked to B. Neither possibility can be ruled out; however, the direction of the effect of B in XRQ x PSC8 (Bert et al., 2003) runs counter to the effects of B in RHA280 x RHA801 (Fig. 3) and other genetic backgrounds (Ross, 1939; Fick et al., 1974b; Dedio, 1980; Mestries et al., 1998).
Gametic-phase linkage disequilibrium between B and linked QTL cannot be ruled out, particularly since seed weight and length QTL were identified on LG 10 in HA89 x ANN1238, a population where B was not segregating (Burke et al., 2002). We aligned LG 10 from HA89 x ANN1238 and RHA280 x RHA801 using common SSR marker loci (Tang et al., 2002; Yu et al., 2003) to ascertain whether QTL for swt and sl in the former were tightly linked to the B locus in the latter (QTL for seed oil concentration were not mapped in HA89 x ANN1238). The ORS815- ORS684 interval flanks B in RHA280 x RHA801 (Yu et al., 2003); hence, B seems to be in the middle of the 23-cM ORS878-ORS613 interval in HA89 x ANN1238, proximal to the swt and sl QTL identified by Burke et al. (2002). The latter were deduced to overlap B-linked QTL for seed traits identified in RHA280 x RHA801 (Fig. 1). The lack of branching QTL and presence of seed trait QTL in the ORS1088-ORS613 interval in HA89 x ANN1238 and large effects of B-linked QTL on seed oil concentration in opposite directions in different genetic backgrounds suggests QTL identified in the DNA marker interval flanking the B locus could have been caused by the pleiotropic effects of B and linkage disequilibrium between B and QTL (Mestries et al., 1998; Burke et al., 2002; Bert et al., 2003; Fig. 1).
The utility of exotic germplasm for enhancing the performance of elite cultivars for quantitative traits normally cannot be assessed through phenotypic analyses alone, although special phenotypic methods have been developed for identifying exotic populations and inbred lines carrying novel favorable alleles for enhancing the parents of elite single-cross hybrids (Dudley, 1984, 1987, 1988; Bernardo, 1990; Metz, 1994). QTL analyses, by contrast, are designed to identify favorable alleles in exotic and elite germplasm sources (Tanksley and Nelson, 1996; Tanksley et al., 1996; Lynch and Walsh, 1998, p. 477–489; Barton and Keightley, 2002; Doerge, 2002). One of our goals was to assess whether or not favorable alleles for seed oil concentration were present in the low-oil inbred line RHA280, an exotic, albeit narrow, source of genetic diversity for breeding high-oil sunflower. Favorable alleles were present in nearly equal numbers in the parents of three high-oil x high-oil crosses (Mestries et al., 1998; Mokrani et al., 2002; Bert et al., 2003). By contrast, Leon et al. (2003) found no favorable alleles for increasing seed oil concentration in the low-oil parent (ZENB8) of a low-oil x high-oil cross (ZENB8 x HA89), and we found no favorable alleles for increasing seed oil concentration in the low-oil parent (RHA280) of our low-oil x high-oil cross (Fig. 1 and 4–6 and Table 4). RHA280 and other confectionery inbred lines in the public germplasm collection (http://www.ars-grin.gov) originated from a narrow genetic base (Cheres and Knapp, 1998) and carry many of the alleles found in elite oilseed inbred lines (Tang and Knapp, 2003); hence, favorable alleles for increasing seed oil concentration and enhancing the performance of elite oilseed cultivars could be scarce in confectionery but should be common in wild sunflower germplasm (Burke et al., 2002; Gandhi et al., 2005).
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ACKNOWLEDGMENTS |
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Received for publication January 4, 2005.
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