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Identification of QTL for Popping Characteristics Using a BC2F2 Population and Comparison with Its F2:3 Population in Popcorn
Agricultural Sciences in China
February 2009
2009, 8(2): 137-143
Identification of QTL for Popping Characteristics Using a BC2F2 Population and Comparison with Its F2:3 Population in Popcorn LI Yu-ling, DONG Yong-bin, NIU Su-zhen and CUI Dang-qun Agronomy College, Henan Agricultural University, Zhengzhou 450002, P.R.China
Abstract Normal corn germplasm can be used to improve popcorn (Zea mays L.) through 1-2 backcrosses with popcorn as recurrent parents. Popping characteristics of derived lines from popcorn × dent or flint corn crosses play a determinant role in popcorn breeding. Advanced backcross QTL methods can effectively combine QTL identification and plant breeding. 220 selected BC2F2 families developed from a cross between Dan 232, a dent corn inbred line, and N04, an elite popcorn inbred line, were evaluated for three popping characteristics, popping volume, flake size, and popping rate, under two environmental conditions. Using composite interval mapping, a total of 10 significant QTLs were detected, and of these, 2 to 4 QTLs were identified for each trait. Six QTLs had favorable alleles contributed by Dan 232. Comparison with the 15 QTLs detected in the F2:3 families showed that 3 QTLs were the same in both populations. The QTLs should be redetected in generations developed through severe selection. Improved N04 and near isogenic lines could be developed from this BC2F2 population through selfing or another 1 to 2 backcrosses with N04. Key words: Zea mays L., popcorn, BC2F2 population, quantitative trait loci, popping characteristics
INTRODUCTION Popping characteristics of lines derived from popcorn × dent or flint corn crosses play a determinant role in popcorn breeding. Thus, normal corn can be used to improve popcorn germplasm. The achievement of this objective requires one to two backcrosses with popcorn as the recurrent parent (Dofing et al. 1991; Li et al. 2002; Ziegler and Ashman 1994). Progeny or lines from such crosses have been obtained by successive selfing and selection for important target traits in each population (Ashman 1991; Li and Lu 2000; Li et al. 2005; Robbins and Ashman 1984). Popping characteristics exhibit quantitative inheritance patterns with a continuous distribution of phenotypic values in segregation populations (Ziegler and Received 24 May, 2008
Ashman 1994). QTLs for popping characteristics have been detected with early generations BC1 (Lu et al. 2003) and F 2:3 (Babu et al. 2006; Li et al. 2006a,b, 2008) derived from crosses between popcorn and dent or flint corn inbred lines. Our F2:3 population has also been used to reveal the genetic relationship between popping expansion volume and two yield components (Li et al. 2008). However, a simulation has shown that the efficiency of marker-assisted selection (MAS) declines in later generations, owing in part to the recombination between markers and QTLs (Gimelfarb and Lande 1994; Edwards and Page 1994). Comparative QTL mapping for grain yield, yield components, and plant traits in the F2:3 and F6:7 generations in normal corn further suggests that some of the QTLs detected during early generations of maximum linkage disequilibria are owing to multiple, linked genes that are separated via recombi-
Accepted 16 July, 2008
Correspondence LI Yu-ling, Ph D, E-mail: [email protected]
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nation (Austin and Lee 1996a,b). Backcrossed populations have the reduction in background genetic variation that is necessary for a more precise estimation of QTL effects (Butruile et al. 1999). Favorable QTL alleles are identified through advanced backcross populations and improved lines with genomes nearly identical to those of the recurrent parent are obtained. Therefore, near isogenic lines (NILs) containing the QTL of interest can be rapidly developed (Bernacci et al. 1998; Tanksley and Nelson 1996; Xie et al. 2006) and put to use in breeding, further genetic research, or production. This strategy has been successfully applied in tomato (Bernacchi et al. 1998; Fulton et al. 1997; Tanksley et al. 1996), rice (Moncada et al. 2001; Septiningsih et al. 2003; Xiao et al. 1998), normal corn (Ho et al. 2002), wheat (Huang et al. 2003), and barley (Pillen et al. 2003). In this study, three popping characteristics were investigated in a population of 220 BC2F2 families derived from a cross between a dent corn inbred (Dan 232) and an elite popcorn inbred (N04). Our objective was to locate and characterize genetic factors associated with trait variation. Early unselected (F2:3) and later selected (BC2F2) populations developed from the same parents were evaluated in the same environment. Therefore, the study design also included a comparison between QTL detection in the BC2F2 population with results from a previous study using F2:3 families from the same parents evaluated under the same environmental conditions (Li et al. 2007).
MATERIALS AND METHODS Population development The two mapping populations of maize were developed from a cross of the dent corn inbred line Dan 232 and the popcorn inbred line N04. The F2:3 population has been described by Li et al. (2007). For the BC 2F 2 population, the F1 plants were backcrossed to N04 as the recurrent parent to develop 235 BC1F1 plants. All BC1F1 plants were backcrossed a second time to N04 as the recurrent parent to develop BC2F1 seeds and were selfed simultaneously. The 72 best BC2F1 families were selected based on grain weight per plant (> N04), 100grain weight (> N04), and popping expansion volume
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( N04) of selfed ears of BC1F1 plants, and grown in the field to produce BC2F2 seeds. Subsequently, 220 of 812 BC2F2 families were selected with the same criterion used to select the best BC2F1.
Field trials and trait evaluation The 220 BC2F2 families, F1, and both parents were evaluated in a completely random design of one-row plots with two replications under similar two environments as the F2:3 population, spring and summer, at the same location in 2004 (Li et al. 2007). The rows were 4 m long with 0.67 m spacing between rows. Plots were planted by hand at a density of 60 000 plants ha-1. Standard cultivation management practices were used at each environment. The test of three popping characteristics for the F2:3 population was conducted as described by Li et al. (2007) at Zhengzhou National Maize Improvement Sub-Center of China, Henan Agricultural University, China. Briefly, the naturally dried ears of each plot with the optimum moisture for popping (13.5 ± 0.5)% were shelled manually and bulked for popping expansion tests. A sample of 100 kernels from each plot was individually popped with a BZ-99 popping machine (Shanghai Duoli Food Machine Building Company, Shanghai, China). After popping, the popped volume was measured in the graduated 1l glass cylinder, while the unpopped kernel volume was measured in a 100 mL graduated measuring cylinder. Popping volume (PV) refers to the popped volume per 100 kernels. Flake size (FS) was calculated as the total popped volume divided by the number of popped kernels. Popping rate (PR) was calculated based on the number of unpopped kernels in 100 kernels after popping. Trait measurements averaged over the two replicate experiments were used as the preliminary data in the analyses.
SSR analysis and map construction Twenty seeds per BC2F2 family, F1, and the two parents were cultivated in a climatic chamber, and the leaf tissues (< 2 weeks old) were collected and bulked for each entry. All leaf samples were stored at -80°C until DNA was extracted using a CTAB procedure (Saghai
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Identification of QTL for Popping Characteristics Using a BC2F 2 Population and Comparison with Its F 2:3 Population in
Maroof et al. 1984). SSR analysis was conducted as reported by Senior and Heun (1993). The 193 markers that clearly showed co-dominant segregation in the F2:3 population (Li et al. 2007) were chosen to genotype the 220 BC2F2 families. Twentythree pairs of SSR markers that showed serious segregation distortion were excluded from the analysis. The linkage map was constructed with Joinmap 3.0 b (van Ooijen and Voorrips 2001) using 170 pairs of SSR markers, according to the genetic linkage map constructed using the 259 F2 population derived from the same cross (Li et al. 2007). This linkage map covered 10 maize chromosomes with a total length of 1575.1 cM and an average interval of 9.3 cM.
Statistical analysis Broad sense heritability and confidence intervals were calculated as described by Li et al. (2007). QTL analysis was carried out on linkage maps consisting of 170 SSR markers. Since the variance components for the interactions between family and environment for the three traits were all not significant in the combined variance analysis, the phenotypic data was comprised of untransformed PV, FS, and PR from pooled data across environments. Composite interval mapping (CIM) was used to map QTLs and to estimate their effects on the three popping characteristics (Zeng 1994) with QTL Cartographer ver. 2.5 (Wang et al. 2006).
RESULTS Performance of three popping characteristics in BC2F2 families and comparison with the F2:3 population All traits differed greatly among the BC2F2 families, with
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PV and FS showing a pattern of continuous distribution and PR showing a skewed distribution toward the recurrent parent type (Table 1). The means of PR and FS for BC2F2 families approached the recurrent parent N04, while the mean PV was significantly higher than N04. This result indicated that most of the families had popping characteristics similar to those of N04, and also dent corn inbred Dan 232 may have favorable alleles of QTL for the three traits, especially for PV and FS. Compared with the F2:3 population, the performance of PV and PR was significantly higher in the BC2F2 population, especially at the lowest limit. However, the performance of FS was higher in the F2:3 population owing to the considerably higher FS of Dan 232. The ranges and broad sense heritability of all traits were significantly reduced.
QTL detection for each trait and comparison with the F2:3 population A total of 10 QTLs were found to be significantly associated with three popping characteristics in the BC2F2 families. The QTLs were located on chromosomes 3, 5, 7, and 8, with 1 to 2 per chromosome (Table 2). The QTLs detected in the F2:3 families have been described by Li et al. (2007). Four QTLs for PV were located on chromosomes 3, 5, 7, and 8. The contributions to the phenotypic variations of a single QTL varied between 5.6 and 8.0%, with qBPV7-1 recording the highest contribution. The total contributions were 27.9%. The positive alleles of three QTLs on chromosomes 5, 7, and 8 were contributed by Dan 232, the dent corn parent. In the F2:3 population, six QTL were detected on chromosomes 1 (three), 6, 7, and 8. Only the QTL on chromosome 7 was found in the same chromosome interval, bnlg339umc1112, in both populations, with positive alleles all contributed by Dan 232.
Table 1 Popping characteristics in P1 (N04), P2 (Dan232), and BC2F2 families evaluated over two seasons and comparison with F2:3 families Traits1)
N04
Dan 232
PV (mL) FS (mL) PR (%)
173.8 1.9 90.3
50.0 5.1 3.3
Range
Mean± SD
100.0-241.3 190.0 ± 23.7 1.2-2.4 1.9 ± 0.2 65.6-98.6 92.2 ± 4.6
BC2F2 families CV (%) Skewness 12.9 11.5 4.9
-0.4 -0.2 -2.3
Kurtosis
hB2 (CI)2)
Range
F2:3 families Mean ± SD
0.4 -0.17 8.0
0.44 (0.26-0.57) 0.41 (0.22-0.55) 0.44 (0.26-0.57)
53.8-283.1 1.4-3.3 12.0-93.4
155.3 ± 46.1 2.3 ± 0.4 60.5 ± 16.1
hB2 (CI) 0.82 (0.75-0.87) 0.62 (0.51-0.70) 0.85 (0.79-0.89)
PV, popping volume; FS, flake size; PR, popping rate. hB2, broad sense heritability; CI, confidence interval. All experiments were performed in duplicate. 1) 2)
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Four QTLs on chromosomes 3, 5, 7, and 8 were also detected for FS. The contributions to phenotypic variations for a single QTL varied between 5.3 and 9.3%, with qBFS7-1 recording the highest contribution. The total contribution to phenotypic variations was 28.8%. The positive alleles of three QTLs on chromosomes 5, 7, and 8 were contributed by Dan 232. Only the positive allele of qBFS3-1 was contributed by the popcorn parent N04. In the F2:3 population, four QTLs for FS were detected on chromosomes 1, 2, 5, and 7. Only the QTL on chromosome 7 was located in the near chromosome intervals bnlg339-umc1112 and bnlg1070-bnlg339 in the two populations, respectively, with positive alleles all contributed by the dent corn parent. Two QTLs affecting PR were identified. Both were located on chromosome 1. The contributions to phenotypic variations for a single QTL were 8.1 and 11.0%, with qBPR1-2 the highest. The total contribution was 19.1%, with all positive alleles contributed by the popcorn parent. Only one QTL was also detected in the same chromosome interval, bnlg1643-umc1885, in the F2:3 population.
DISCUSSION Comparison of QTLs detected in the BC2F2 and F2:3 populations for three popping characteristics For the same three popping characteristics, 10 and 15 QTLs were detected in the BC2F2 and F2:3 populations, respectively. Only three QTLs (accounting for 30%) observed in the BC2F2 population were located in the same or near chromosome intervals as in the F 2:3 population. Seven QTLs were specific to the BC2F2 progeny and twelve were only detected in the F 2:3 population. Inconsistent detection of QTLs can be caused by several factors, such as population types (parents and progeny types), sampling variation, genetic heterogeneity of the phenotype, environments, and molecular markers (Austin and Lee 1996a, b). Through a simulation study, Moreno-Gonzalez (1993) showed that different generations have different efficiencies for the estimation of marker-associated QTL effects by multiple regression. Beavis et al. (1994) suggested that
Table 2 Putative QTL associated with three popping characteristics in the BC2F2 families and comparison with F2:3 generations from the cross of Dan 232 × N04 Trait Popping volume (PV)
Population BC2F2
F2:3 3)
Flake size (FS)
BC2F2
F2:3
Popping rate (PR)
BC2F2 F2:3
QTL qBPV3-1 qBPV5-1 qBPV7-1 qBPV8-1 qPV1-1 qPV1-2 qPV1-3 qPV6-1 qPV7-1 qPV8-1 qBFS3-1 qBFS5-1 qBFS7-1 qBFS8-1 qFS1-1 qFS2-1 qFS5-1 qFS7-1 qBPR1-1 qBPR1-2 qPR1-1 qPR1-2 qPR1-3 qPR6-1 qPR8-1
Marker interval
Chro./bin 1)
LOD
Additive effect
R2 (%)2)
umc2275-umc1273 umc1389-phi109188 bnlg339-umc1112 umc1069-phi233376 umc1269-umc1948 umc2083-umc1281 bnlg1643-umc1885 phi299852-umc2165 bnlg1070-bnlg339 umc2147-bnlg2082 umc2275-unc1273 umc1389-phi109188 bnlg339-umc1112 umc1069-phi233376 phi427913-bnlg1429 umc2214-phi101049 bnlg1346-bnlg2305 bnlg339-umc1112 bnlg1811-umc2227 bnlg1643-umc1885 umc1269-umc1948 umc2083-umc1281 bnlg1643-umc1885 phi299852-umc2165 umc1360-bnlg1863
3.07-3.08 5.03 7.03 8.08-8.09 1.01 1.05-1.06 1.08-1.11 6.07 7.03 8.03 3.07-3.08 5.03 7.03 8.08-8.09 1.01 2.1 5.07 7.03 1.04 1.08-1.10 1.01 1.05-1.06 1.08-1.11 6.07 8.02-8.03
4.1 3.5 4.4 3.5 9.8 5.0 4.6 7.6 3.9 6.7 3.2 3.2 4.5 3.3 10.8 4.3 4.8 4.7 4.2 3.3 4.4 6.4 4.5 5.5 3.8
15.1 -14.2 -14.7 -13.2 22.1 10.5 7.2 22.7 -17.1 8.9 0.1 -0.1 -0.2 -0.1 0.2 0.1 -0.1 -0.2 3.1 3.4 5.0 6.5 4.0 6.6 3.6
7.1 7.2 8.0 5.6 13.0 7.5 9.6 10.5 5.2 8.2 7.0 5.3 9.3 7.2 15.6 5.3 6.6 7.0 8.1 11.0 6.2 10.2 9.7 8.2 4.9
Bin locations of the nearest marker. R2, the percent of phenotypic variance explained by each QTL. 3) QTL detected in the F2:3 families have been described by Li et al. (2007). 1) 2)
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Identification of QTL for Popping Characteristics Using a BC2F 2 Population and Comparison with Its F 2:3 Population in
genetic background was one explanation for differences in QTL detection among two F3 backcrossed lines (Stuber et al. 1992), F4 lines, and topcrossed populations from the same B73 × Mo17 cross. Genetic mapping with hybrid progeny across three testers and two generations (F2:3 and F6:7) for grain yield and grain moisture showed that individual tester QTL effects were not consistent in rank or detection across generations (Austin et al. 2000). In our study, since both the F2:3 and BC2F2 populations were evaluated under the same environments, and the same polymorphic markers were used to analyze the genotypes of both populations, the inconsistency in QTL detection may mainly be attributed to the backcross and selection while the BC2F 2 population was under development; hence, the great difference in genetic background between the two populations. During the development of advanced backcross populations, some non-recurrent introgressions may be selected against (Butruille et al. 1999; Septiningsih et al. 2003). Based on Mendelian expectation with no selection, only 25% of the plants in each BC2F2 family will contain the donor parent allele for a given introgression. This “dilution effect” by non-carrier individuals can mask small effects of non-recurrent parent QTL alleles present at low frequencies (Ho et al. 2002). In the BC2F2 population developed in this study, rigorous selection was conducted in the BC1 and BC2 generations, resulting in large changes in population structure. The allele frequency and genotypic ratio were 83% N04 to 15% Dan 232 and 68% N04/N04 homozygotes to 30% N04/Dan 232 heterozygotes in the BC 2F 2 population, respectively (Niu 2006). In contrast, an allele frequency of 50% N04 to 50% Dan 232 and genotypic ratios of 25% N04/N04 homozygotes to 51% N04/Dan 232 heterozygotes to 24% Dan 232/Dan 232 homozygotes occurred in the F2:3 population (Li et al. 2006b). The unequal allele frequencies may cause a reduction in power for detecting QTLs. This may be one of the reasons why considerably fewer QTLs were detected in the BC2F2 population. Different trait heritability and QTLs with different degrees of dominant effects in the F2:3 population may be the other two reasons for QTL inconsistency across the two populations. Butruille et al. (1999) considered that traits with high heritability allowed for the detection of a large number of QTLs. Austin and Lee (1996a,
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b) experienced more consistent detection of F2:3 QTLs additive to dominant gene action versus those with overdominance in the F6:7 generation. In our present study, the heritability for three popping characteristics was significantly reduced in the BC2F2 population owing to smaller trait variations (Table 1). Of the fifteen QTLs detected in the F2:3 population, fourteen (93.3%) QTLs expressed different degrees of dominant effects (Li et al. 2007). Therefore, QTLs should be redetected in generations developed through severe selection. Great caution should be taken to use QTLs detected in early generations in segregation in further MAS, fine mapping, or near-isogenic line development. Only QTLs with perfect stability across populations and environments will be good candidate QTLs for MAS. Comparing the QTLs detected by Lu et al. (2003) and Babu et al. (2006) using different BC1 and F2:3 populations under different environments, the QTLs at two chromosome regions may be taken into first consideration. At bin location 5.03 for qBPV5-1 and qBFS5-1 in this study, a QTL for flake volume was detected by Babu et al. (2006). Lu et al. (2003) also identified a QTL for popping expansion volume on 5S. Similarly, at bin location 3.07-3.08 for qBPV3-1 and qBFS3-1, a QTL for percent unpopped kernels was found by Babu et al. (2006) at 3.07. Most QTLs for popping characteristics may be clustered around specific regions on chromosomes 1, 3, and 5. However, a QTL at bin location 7.03 for PV and FS in this study, and a QTL for flake volume and percent unpopped kernels at bin location 9.04 and for flake volume at bin location 10.04 detected by Babu et al. (2006) may be germplasm specific. The discovery of different QTLs for different popping characteristics in this study and by Babu et al. (2006) for different germplasms provides ample scope for an effective pyramiding approach using MAS.
Utility of BC2F2 families in popcorn breeding Previous studies have shown that normal maize germplasm can be effectively used in popcorn breeding through 1 to 2 backcrosses with popcorn germplasm as the recurrent parent (Dofing et al. 1991; Ziegler and Ashman 1994; Li and Lu 2000; Li et al. 2002). We used an AB (advanced backcross)-QTL method to com-
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bine QTL analysis with plant breeding (Bernacchi et al. 1998; Fulton et al. 1997; Huang et al. 2003; Moncada et al. 2001; Septiningsih et al. 2003; Tanksley et al. 1996; Xiao et al. 1998; Xie et al. 2006). Also, the AB population development process is consistent with the way by which normal maize germplasm is utilized in popcorn breeding. Since the 220 BC2F2 families were maintained by selection in BC1 and BC2 according to grain weight per plant (> N04), 100-grain weight (> N04), and popping expansion volume ( N04), some favorable alleles for these phenotypes must have improved simultaneously. In fact, of the 220 BC2F 2 families, the grain weight per plant of 210 families (95%) and 100-grain weight of 208 families (95%) were higher than N04, and among these, the popping expansion volume of 35 families (16%) was nearly the same or higher than N04 (Niu 2006). Therefore, these 35 families could be used to replace N04 in popcorn breeding only after 1 to 2 generations of self-pollination. Meanwhile, QTL-NILs for grain weight per plant and/ or 100-grain weight could be developed through selfing or another 1 to 2 backcrosses with N04.
Acknowledgements This work was funded by the Natural Science Foundation of Henan Province of China (0511032900).
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February 2009
2009, 8(2): 137-143
Identification of QTL for Popping Characteristics Using a BC2F2 Population and Comparison with Its F2:3 Population in Popcorn LI Yu-ling, DONG Yong-bin, NIU Su-zhen and CUI Dang-qun Agronomy College, Henan Agricultural University, Zhengzhou 450002, P.R.China
Abstract Normal corn germplasm can be used to improve popcorn (Zea mays L.) through 1-2 backcrosses with popcorn as recurrent parents. Popping characteristics of derived lines from popcorn × dent or flint corn crosses play a determinant role in popcorn breeding. Advanced backcross QTL methods can effectively combine QTL identification and plant breeding. 220 selected BC2F2 families developed from a cross between Dan 232, a dent corn inbred line, and N04, an elite popcorn inbred line, were evaluated for three popping characteristics, popping volume, flake size, and popping rate, under two environmental conditions. Using composite interval mapping, a total of 10 significant QTLs were detected, and of these, 2 to 4 QTLs were identified for each trait. Six QTLs had favorable alleles contributed by Dan 232. Comparison with the 15 QTLs detected in the F2:3 families showed that 3 QTLs were the same in both populations. The QTLs should be redetected in generations developed through severe selection. Improved N04 and near isogenic lines could be developed from this BC2F2 population through selfing or another 1 to 2 backcrosses with N04. Key words: Zea mays L., popcorn, BC2F2 population, quantitative trait loci, popping characteristics
INTRODUCTION Popping characteristics of lines derived from popcorn × dent or flint corn crosses play a determinant role in popcorn breeding. Thus, normal corn can be used to improve popcorn germplasm. The achievement of this objective requires one to two backcrosses with popcorn as the recurrent parent (Dofing et al. 1991; Li et al. 2002; Ziegler and Ashman 1994). Progeny or lines from such crosses have been obtained by successive selfing and selection for important target traits in each population (Ashman 1991; Li and Lu 2000; Li et al. 2005; Robbins and Ashman 1984). Popping characteristics exhibit quantitative inheritance patterns with a continuous distribution of phenotypic values in segregation populations (Ziegler and Received 24 May, 2008
Ashman 1994). QTLs for popping characteristics have been detected with early generations BC1 (Lu et al. 2003) and F 2:3 (Babu et al. 2006; Li et al. 2006a,b, 2008) derived from crosses between popcorn and dent or flint corn inbred lines. Our F2:3 population has also been used to reveal the genetic relationship between popping expansion volume and two yield components (Li et al. 2008). However, a simulation has shown that the efficiency of marker-assisted selection (MAS) declines in later generations, owing in part to the recombination between markers and QTLs (Gimelfarb and Lande 1994; Edwards and Page 1994). Comparative QTL mapping for grain yield, yield components, and plant traits in the F2:3 and F6:7 generations in normal corn further suggests that some of the QTLs detected during early generations of maximum linkage disequilibria are owing to multiple, linked genes that are separated via recombi-
Accepted 16 July, 2008
Correspondence LI Yu-ling, Ph D, E-mail: [email protected]
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nation (Austin and Lee 1996a,b). Backcrossed populations have the reduction in background genetic variation that is necessary for a more precise estimation of QTL effects (Butruile et al. 1999). Favorable QTL alleles are identified through advanced backcross populations and improved lines with genomes nearly identical to those of the recurrent parent are obtained. Therefore, near isogenic lines (NILs) containing the QTL of interest can be rapidly developed (Bernacci et al. 1998; Tanksley and Nelson 1996; Xie et al. 2006) and put to use in breeding, further genetic research, or production. This strategy has been successfully applied in tomato (Bernacchi et al. 1998; Fulton et al. 1997; Tanksley et al. 1996), rice (Moncada et al. 2001; Septiningsih et al. 2003; Xiao et al. 1998), normal corn (Ho et al. 2002), wheat (Huang et al. 2003), and barley (Pillen et al. 2003). In this study, three popping characteristics were investigated in a population of 220 BC2F2 families derived from a cross between a dent corn inbred (Dan 232) and an elite popcorn inbred (N04). Our objective was to locate and characterize genetic factors associated with trait variation. Early unselected (F2:3) and later selected (BC2F2) populations developed from the same parents were evaluated in the same environment. Therefore, the study design also included a comparison between QTL detection in the BC2F2 population with results from a previous study using F2:3 families from the same parents evaluated under the same environmental conditions (Li et al. 2007).
MATERIALS AND METHODS Population development The two mapping populations of maize were developed from a cross of the dent corn inbred line Dan 232 and the popcorn inbred line N04. The F2:3 population has been described by Li et al. (2007). For the BC 2F 2 population, the F1 plants were backcrossed to N04 as the recurrent parent to develop 235 BC1F1 plants. All BC1F1 plants were backcrossed a second time to N04 as the recurrent parent to develop BC2F1 seeds and were selfed simultaneously. The 72 best BC2F1 families were selected based on grain weight per plant (> N04), 100grain weight (> N04), and popping expansion volume
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( N04) of selfed ears of BC1F1 plants, and grown in the field to produce BC2F2 seeds. Subsequently, 220 of 812 BC2F2 families were selected with the same criterion used to select the best BC2F1.
Field trials and trait evaluation The 220 BC2F2 families, F1, and both parents were evaluated in a completely random design of one-row plots with two replications under similar two environments as the F2:3 population, spring and summer, at the same location in 2004 (Li et al. 2007). The rows were 4 m long with 0.67 m spacing between rows. Plots were planted by hand at a density of 60 000 plants ha-1. Standard cultivation management practices were used at each environment. The test of three popping characteristics for the F2:3 population was conducted as described by Li et al. (2007) at Zhengzhou National Maize Improvement Sub-Center of China, Henan Agricultural University, China. Briefly, the naturally dried ears of each plot with the optimum moisture for popping (13.5 ± 0.5)% were shelled manually and bulked for popping expansion tests. A sample of 100 kernels from each plot was individually popped with a BZ-99 popping machine (Shanghai Duoli Food Machine Building Company, Shanghai, China). After popping, the popped volume was measured in the graduated 1l glass cylinder, while the unpopped kernel volume was measured in a 100 mL graduated measuring cylinder. Popping volume (PV) refers to the popped volume per 100 kernels. Flake size (FS) was calculated as the total popped volume divided by the number of popped kernels. Popping rate (PR) was calculated based on the number of unpopped kernels in 100 kernels after popping. Trait measurements averaged over the two replicate experiments were used as the preliminary data in the analyses.
SSR analysis and map construction Twenty seeds per BC2F2 family, F1, and the two parents were cultivated in a climatic chamber, and the leaf tissues (< 2 weeks old) were collected and bulked for each entry. All leaf samples were stored at -80°C until DNA was extracted using a CTAB procedure (Saghai
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Identification of QTL for Popping Characteristics Using a BC2F 2 Population and Comparison with Its F 2:3 Population in
Maroof et al. 1984). SSR analysis was conducted as reported by Senior and Heun (1993). The 193 markers that clearly showed co-dominant segregation in the F2:3 population (Li et al. 2007) were chosen to genotype the 220 BC2F2 families. Twentythree pairs of SSR markers that showed serious segregation distortion were excluded from the analysis. The linkage map was constructed with Joinmap 3.0 b (van Ooijen and Voorrips 2001) using 170 pairs of SSR markers, according to the genetic linkage map constructed using the 259 F2 population derived from the same cross (Li et al. 2007). This linkage map covered 10 maize chromosomes with a total length of 1575.1 cM and an average interval of 9.3 cM.
Statistical analysis Broad sense heritability and confidence intervals were calculated as described by Li et al. (2007). QTL analysis was carried out on linkage maps consisting of 170 SSR markers. Since the variance components for the interactions between family and environment for the three traits were all not significant in the combined variance analysis, the phenotypic data was comprised of untransformed PV, FS, and PR from pooled data across environments. Composite interval mapping (CIM) was used to map QTLs and to estimate their effects on the three popping characteristics (Zeng 1994) with QTL Cartographer ver. 2.5 (Wang et al. 2006).
RESULTS Performance of three popping characteristics in BC2F2 families and comparison with the F2:3 population All traits differed greatly among the BC2F2 families, with
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PV and FS showing a pattern of continuous distribution and PR showing a skewed distribution toward the recurrent parent type (Table 1). The means of PR and FS for BC2F2 families approached the recurrent parent N04, while the mean PV was significantly higher than N04. This result indicated that most of the families had popping characteristics similar to those of N04, and also dent corn inbred Dan 232 may have favorable alleles of QTL for the three traits, especially for PV and FS. Compared with the F2:3 population, the performance of PV and PR was significantly higher in the BC2F2 population, especially at the lowest limit. However, the performance of FS was higher in the F2:3 population owing to the considerably higher FS of Dan 232. The ranges and broad sense heritability of all traits were significantly reduced.
QTL detection for each trait and comparison with the F2:3 population A total of 10 QTLs were found to be significantly associated with three popping characteristics in the BC2F2 families. The QTLs were located on chromosomes 3, 5, 7, and 8, with 1 to 2 per chromosome (Table 2). The QTLs detected in the F2:3 families have been described by Li et al. (2007). Four QTLs for PV were located on chromosomes 3, 5, 7, and 8. The contributions to the phenotypic variations of a single QTL varied between 5.6 and 8.0%, with qBPV7-1 recording the highest contribution. The total contributions were 27.9%. The positive alleles of three QTLs on chromosomes 5, 7, and 8 were contributed by Dan 232, the dent corn parent. In the F2:3 population, six QTL were detected on chromosomes 1 (three), 6, 7, and 8. Only the QTL on chromosome 7 was found in the same chromosome interval, bnlg339umc1112, in both populations, with positive alleles all contributed by Dan 232.
Table 1 Popping characteristics in P1 (N04), P2 (Dan232), and BC2F2 families evaluated over two seasons and comparison with F2:3 families Traits1)
N04
Dan 232
PV (mL) FS (mL) PR (%)
173.8 1.9 90.3
50.0 5.1 3.3
Range
Mean± SD
100.0-241.3 190.0 ± 23.7 1.2-2.4 1.9 ± 0.2 65.6-98.6 92.2 ± 4.6
BC2F2 families CV (%) Skewness 12.9 11.5 4.9
-0.4 -0.2 -2.3
Kurtosis
hB2 (CI)2)
Range
F2:3 families Mean ± SD
0.4 -0.17 8.0
0.44 (0.26-0.57) 0.41 (0.22-0.55) 0.44 (0.26-0.57)
53.8-283.1 1.4-3.3 12.0-93.4
155.3 ± 46.1 2.3 ± 0.4 60.5 ± 16.1
hB2 (CI) 0.82 (0.75-0.87) 0.62 (0.51-0.70) 0.85 (0.79-0.89)
PV, popping volume; FS, flake size; PR, popping rate. hB2, broad sense heritability; CI, confidence interval. All experiments were performed in duplicate. 1) 2)
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Four QTLs on chromosomes 3, 5, 7, and 8 were also detected for FS. The contributions to phenotypic variations for a single QTL varied between 5.3 and 9.3%, with qBFS7-1 recording the highest contribution. The total contribution to phenotypic variations was 28.8%. The positive alleles of three QTLs on chromosomes 5, 7, and 8 were contributed by Dan 232. Only the positive allele of qBFS3-1 was contributed by the popcorn parent N04. In the F2:3 population, four QTLs for FS were detected on chromosomes 1, 2, 5, and 7. Only the QTL on chromosome 7 was located in the near chromosome intervals bnlg339-umc1112 and bnlg1070-bnlg339 in the two populations, respectively, with positive alleles all contributed by the dent corn parent. Two QTLs affecting PR were identified. Both were located on chromosome 1. The contributions to phenotypic variations for a single QTL were 8.1 and 11.0%, with qBPR1-2 the highest. The total contribution was 19.1%, with all positive alleles contributed by the popcorn parent. Only one QTL was also detected in the same chromosome interval, bnlg1643-umc1885, in the F2:3 population.
DISCUSSION Comparison of QTLs detected in the BC2F2 and F2:3 populations for three popping characteristics For the same three popping characteristics, 10 and 15 QTLs were detected in the BC2F2 and F2:3 populations, respectively. Only three QTLs (accounting for 30%) observed in the BC2F2 population were located in the same or near chromosome intervals as in the F 2:3 population. Seven QTLs were specific to the BC2F2 progeny and twelve were only detected in the F 2:3 population. Inconsistent detection of QTLs can be caused by several factors, such as population types (parents and progeny types), sampling variation, genetic heterogeneity of the phenotype, environments, and molecular markers (Austin and Lee 1996a, b). Through a simulation study, Moreno-Gonzalez (1993) showed that different generations have different efficiencies for the estimation of marker-associated QTL effects by multiple regression. Beavis et al. (1994) suggested that
Table 2 Putative QTL associated with three popping characteristics in the BC2F2 families and comparison with F2:3 generations from the cross of Dan 232 × N04 Trait Popping volume (PV)
Population BC2F2
F2:3 3)
Flake size (FS)
BC2F2
F2:3
Popping rate (PR)
BC2F2 F2:3
QTL qBPV3-1 qBPV5-1 qBPV7-1 qBPV8-1 qPV1-1 qPV1-2 qPV1-3 qPV6-1 qPV7-1 qPV8-1 qBFS3-1 qBFS5-1 qBFS7-1 qBFS8-1 qFS1-1 qFS2-1 qFS5-1 qFS7-1 qBPR1-1 qBPR1-2 qPR1-1 qPR1-2 qPR1-3 qPR6-1 qPR8-1
Marker interval
Chro./bin 1)
LOD
Additive effect
R2 (%)2)
umc2275-umc1273 umc1389-phi109188 bnlg339-umc1112 umc1069-phi233376 umc1269-umc1948 umc2083-umc1281 bnlg1643-umc1885 phi299852-umc2165 bnlg1070-bnlg339 umc2147-bnlg2082 umc2275-unc1273 umc1389-phi109188 bnlg339-umc1112 umc1069-phi233376 phi427913-bnlg1429 umc2214-phi101049 bnlg1346-bnlg2305 bnlg339-umc1112 bnlg1811-umc2227 bnlg1643-umc1885 umc1269-umc1948 umc2083-umc1281 bnlg1643-umc1885 phi299852-umc2165 umc1360-bnlg1863
3.07-3.08 5.03 7.03 8.08-8.09 1.01 1.05-1.06 1.08-1.11 6.07 7.03 8.03 3.07-3.08 5.03 7.03 8.08-8.09 1.01 2.1 5.07 7.03 1.04 1.08-1.10 1.01 1.05-1.06 1.08-1.11 6.07 8.02-8.03
4.1 3.5 4.4 3.5 9.8 5.0 4.6 7.6 3.9 6.7 3.2 3.2 4.5 3.3 10.8 4.3 4.8 4.7 4.2 3.3 4.4 6.4 4.5 5.5 3.8
15.1 -14.2 -14.7 -13.2 22.1 10.5 7.2 22.7 -17.1 8.9 0.1 -0.1 -0.2 -0.1 0.2 0.1 -0.1 -0.2 3.1 3.4 5.0 6.5 4.0 6.6 3.6
7.1 7.2 8.0 5.6 13.0 7.5 9.6 10.5 5.2 8.2 7.0 5.3 9.3 7.2 15.6 5.3 6.6 7.0 8.1 11.0 6.2 10.2 9.7 8.2 4.9
Bin locations of the nearest marker. R2, the percent of phenotypic variance explained by each QTL. 3) QTL detected in the F2:3 families have been described by Li et al. (2007). 1) 2)
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Identification of QTL for Popping Characteristics Using a BC2F 2 Population and Comparison with Its F 2:3 Population in
genetic background was one explanation for differences in QTL detection among two F3 backcrossed lines (Stuber et al. 1992), F4 lines, and topcrossed populations from the same B73 × Mo17 cross. Genetic mapping with hybrid progeny across three testers and two generations (F2:3 and F6:7) for grain yield and grain moisture showed that individual tester QTL effects were not consistent in rank or detection across generations (Austin et al. 2000). In our study, since both the F2:3 and BC2F2 populations were evaluated under the same environments, and the same polymorphic markers were used to analyze the genotypes of both populations, the inconsistency in QTL detection may mainly be attributed to the backcross and selection while the BC2F 2 population was under development; hence, the great difference in genetic background between the two populations. During the development of advanced backcross populations, some non-recurrent introgressions may be selected against (Butruille et al. 1999; Septiningsih et al. 2003). Based on Mendelian expectation with no selection, only 25% of the plants in each BC2F2 family will contain the donor parent allele for a given introgression. This “dilution effect” by non-carrier individuals can mask small effects of non-recurrent parent QTL alleles present at low frequencies (Ho et al. 2002). In the BC2F2 population developed in this study, rigorous selection was conducted in the BC1 and BC2 generations, resulting in large changes in population structure. The allele frequency and genotypic ratio were 83% N04 to 15% Dan 232 and 68% N04/N04 homozygotes to 30% N04/Dan 232 heterozygotes in the BC 2F 2 population, respectively (Niu 2006). In contrast, an allele frequency of 50% N04 to 50% Dan 232 and genotypic ratios of 25% N04/N04 homozygotes to 51% N04/Dan 232 heterozygotes to 24% Dan 232/Dan 232 homozygotes occurred in the F2:3 population (Li et al. 2006b). The unequal allele frequencies may cause a reduction in power for detecting QTLs. This may be one of the reasons why considerably fewer QTLs were detected in the BC2F2 population. Different trait heritability and QTLs with different degrees of dominant effects in the F2:3 population may be the other two reasons for QTL inconsistency across the two populations. Butruille et al. (1999) considered that traits with high heritability allowed for the detection of a large number of QTLs. Austin and Lee (1996a,
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b) experienced more consistent detection of F2:3 QTLs additive to dominant gene action versus those with overdominance in the F6:7 generation. In our present study, the heritability for three popping characteristics was significantly reduced in the BC2F2 population owing to smaller trait variations (Table 1). Of the fifteen QTLs detected in the F2:3 population, fourteen (93.3%) QTLs expressed different degrees of dominant effects (Li et al. 2007). Therefore, QTLs should be redetected in generations developed through severe selection. Great caution should be taken to use QTLs detected in early generations in segregation in further MAS, fine mapping, or near-isogenic line development. Only QTLs with perfect stability across populations and environments will be good candidate QTLs for MAS. Comparing the QTLs detected by Lu et al. (2003) and Babu et al. (2006) using different BC1 and F2:3 populations under different environments, the QTLs at two chromosome regions may be taken into first consideration. At bin location 5.03 for qBPV5-1 and qBFS5-1 in this study, a QTL for flake volume was detected by Babu et al. (2006). Lu et al. (2003) also identified a QTL for popping expansion volume on 5S. Similarly, at bin location 3.07-3.08 for qBPV3-1 and qBFS3-1, a QTL for percent unpopped kernels was found by Babu et al. (2006) at 3.07. Most QTLs for popping characteristics may be clustered around specific regions on chromosomes 1, 3, and 5. However, a QTL at bin location 7.03 for PV and FS in this study, and a QTL for flake volume and percent unpopped kernels at bin location 9.04 and for flake volume at bin location 10.04 detected by Babu et al. (2006) may be germplasm specific. The discovery of different QTLs for different popping characteristics in this study and by Babu et al. (2006) for different germplasms provides ample scope for an effective pyramiding approach using MAS.
Utility of BC2F2 families in popcorn breeding Previous studies have shown that normal maize germplasm can be effectively used in popcorn breeding through 1 to 2 backcrosses with popcorn germplasm as the recurrent parent (Dofing et al. 1991; Ziegler and Ashman 1994; Li and Lu 2000; Li et al. 2002). We used an AB (advanced backcross)-QTL method to com-
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bine QTL analysis with plant breeding (Bernacchi et al. 1998; Fulton et al. 1997; Huang et al. 2003; Moncada et al. 2001; Septiningsih et al. 2003; Tanksley et al. 1996; Xiao et al. 1998; Xie et al. 2006). Also, the AB population development process is consistent with the way by which normal maize germplasm is utilized in popcorn breeding. Since the 220 BC2F2 families were maintained by selection in BC1 and BC2 according to grain weight per plant (> N04), 100-grain weight (> N04), and popping expansion volume ( N04), some favorable alleles for these phenotypes must have improved simultaneously. In fact, of the 220 BC2F 2 families, the grain weight per plant of 210 families (95%) and 100-grain weight of 208 families (95%) were higher than N04, and among these, the popping expansion volume of 35 families (16%) was nearly the same or higher than N04 (Niu 2006). Therefore, these 35 families could be used to replace N04 in popcorn breeding only after 1 to 2 generations of self-pollination. Meanwhile, QTL-NILs for grain weight per plant and/ or 100-grain weight could be developed through selfing or another 1 to 2 backcrosses with N04.
Acknowledgements This work was funded by the Natural Science Foundation of Henan Province of China (0511032900).
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