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Understanding a Rapid Cycling Winter Wheat Background From ‘Goodstreak × Apogee’ Using the KASP Assay
Rungravee Boontung1, Ahmed Sallam1,2, Liuling Yan3 and P. Stephen Baenziger1
1Department of Agronomy and Horticulture, University of Nebraska-Lincoln2Department of Genetics, Faculty of Agriculture, Assiut University, Egypt
3Department of Plant and Soil Sciences, Oklahoma State University
Introduction
Background : Backcrossing is slowed by long generation times due to
vernalization. Expedited backcrossing can be achieved by introducing
rapid cycling alleles into a “winter” wheat background.
Objective : To study the genetics of rapid cycling and the effects of
single-locus and multi-locus genes on flowering date and plant height.
Approach : Tall winter wheat cultivar Goodstreak was crossed with
super dwarf and early flowering spring wheat Apogee. The F5 from
single seed descent and BC1F1 were planted and measured for
flowering date and plant height. DNA was extracted from leaf tissue at
seedling stage. Plant genotypes were analyzed by KASP genotyping and compared with phenotype data.
Materials and Methods
Plant material
• A spring wheat cultivar Apogee, used as a donor parent carrying
genes for early flowering and semi-dwarf (Vrn-A1, Ppd-D1, Rht-B1, Rht-D1) was crossed to Goodstreak (vrn-A1, ppd-D1, rht-B1, rht-D1)
to create rapid cycling winter wheat background lines. Single seed
descent method was performed until the F5 generation. Seeds from the
two earliest flowering plants from the F2 and F3 were selected and
planted and crossed back to Goodstreak to generate the BC1F1.
• 170 individuals of F5 Goodstreak × Apogee and 104 individuals of
BC1F1 of Goodstreak × Apogee/Goodstreak were planted without
vernalization in greenhouse (16 hr photoperiod, average night-time
temperature was 18o C and average daytime temperature was 24o C).
Phenotyping
• The number of days from planting to flowering and plant height were
measured.
Genotyping
• Leaf tissue from seedlings was extracted for DNA using BioSprint 96
protocols.
• Six KASP markers : Vrn-A1, Vrn-B1, Vrn-D1, Ppd-D1, Rht-B1 and
Rht-D1 were used for SNP genotyping. The information of KASP
markers can be downloaded at
http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/kasp_downlo
ad.php.
• SNP genotyping was performed using LGC Genomics KASP system
fluorescent assays. The assay information is available at
www.lgcgroup.com. Genotyping data from fluorescence was plotted
using KlusterCaller software (LGC Genomics, Hoddeson, UK).
• The single-locus and multi-locus at Vrn-A1, Ppd-D1, Rht-B1 and Rht-
D1 loci effect on flowering date and plant height were calculated
using the GLM procedure in SAS 9.4 (SAS Institute, Inc., Cary, NC)
Conclusions
• To develop rapid cycling lines, we recommend using markers to
identify the genetic pool of early lines followed by phenotyping to
find a smaller subset of earliest lines with additional genes.
• Vrn-A1 marker had significant effect on flowering date, but Ppd-D1
marker did not have a significant effect on flowering date in the F5
population.
• Vrn-A1 and Ppd-D1 had a significant effect on flowering date in the
BC1F1 population.
• Both Rht-B1 and Rht-D1 had significant effects on plant height in
the F5 and BC1F1 population.
• 37 multi-locus genotypes in the F5 were significantly different in
flowering date and plant height. Homozygous multi-locus for spring
type GG,CC,TT,TT was associated with early flowering, which
flowered 66.5 days earlier than Goodstreak and this multi-locus was
also associated with shortest plants in the F5 population.
• Rapid cycling lines can be created where the spring wheat alleles are
present in an otherwise winter wheat background.
Acknowledgement
• Caixia Liu, Department of Agronomy and Horticulture, University of Nebraska-
Lincoln assisted with transferring the KASP technology to our lab.
References
• Grogan, S.M., G. Brown-Guedira, S.D. Haley, G.S. McMaster, S.D. Reid, J. Smith,
and P.F. Byrne. 2016. Allelic variation in developmental genes and effects on winter
wheat heading date in the US great plains. PloS One 11:e0152852.
• Rasheed, A., W. Wen, F. Gao, S. Zhai, H. Jin, J. Liu, Q. Guo, Y. Zhang, S.
Dreisigacker, and X. Xia. 2016. Development and validation of KASP assays for
genes underpinning key economic traits in bread wheat. Theor. Appl. Genet.
129:1843-1860.
• Semagn, K., R. Babu, S. Hearne, and M. Olsen. 2014. Single nucleotide
polymorphism genotyping using kompetitive allele specific PCR (KASP): Overview
of the technology and its application in crop improvement. Mol. Breed. 33:1-14.
Figure 1. Genotypic cluster plots of Vrn-A1, Ppd-D1, Rht-B1 and Rht-D1
in the F5 of Goodstreak × Apogee (A) and in the BC1F1 of Goodstreak ×
Apogee/Goodstreak (B). Data points represent the fluorescence result of each DNA sample.
Table 2. ANOVA table for three-genotypes (two homozygous and one
heterozygous) examining the effect of markers on flowering date and plant height in the F5 population.
Figure 2. Means of flowering date (days) and plant height (cm) from eight
multi-locus genotypes that were related with earliest flowering and eight
multi-locus genotypes that were related with shortest plants in the F5 (A)
and all eight multi-locus genotypes in the BC1F1 (B) compared with multi-locus genotype of Apogee and multi-locus genotype of Goodstreak.
Results
Markers EfficiencyAllele
TypeSpring Winter
Vrn-A1 Useful GG AA
Vernalization genesVrn-B1 Not useful CC GG
Vrn-D1 Not useful CC GG
Ppd-D1 Useful CC TT Photoperiod genes
Rht-B1 Useful TT CCHeight genes
Rht-D1 Useful TT GG
Table 1. Six KASP markers were used in this experiment. However, the
results from Vrn-B1 and Vrn-D1 did not give a good genotyping cluster plot when using KlusterCaller software, hence were not used.
0
0.5
1
0 0.5 1
Y
X
Vrn-A1: F5
Winter (A:A)
Spring (G:G)
Heterozygous (G:A)
Apogee (G:G)
Goodstreak (A:A)
NTC
0
0.5
1
0 0.5 1
Y
X
Ppd-D1: F5
Winter (T:T)
Spring (C:C)
Heterozygous (C:T)
Apogee (C:C)
Goodstreak (T:T)
NTC
0
0.5
1
0 0.5 1
Y
X
Rht-B1: F5
Tall (C:C)
Dwarf (T:T)
Heterozygous (T:C)
Apogee (T:T)
Goodstreak (C:C)
NTC
0
0.5
1
0 0.5 1
Y
X
Rht-D1: F5
Tall (G:G)
Dwarf (T:T)
Heterozygous (T:G)
Apogee (T:T)
Goodstreak (G:G)
NTC
A
0
0.5
1
0 0.5 1
Y
X
Vrn-A1: BC1F1
Winter (A:A)
Heterozygous (G:A)
Apogee (G:G)
Goodstreak (A:A)
NTC
0
0.5
1
0 0.5 1
Y
X
Ppd-D1 : BC1F1
Winter (T:T)
Heterozygous (C:T)
Apogee (C:C)
Goodstreak (T:T)
NTC
0
0.5
1
0 0.5 1
Y
X
Rht-B1: BC1F1
Tall (C:C)
Heterozygous (T:C)
Apogee (T:T)
Goodstreak (C:C)
NTC
0
0.5
1
0 0.5 1
Y
X
Rht-D1: BC1F1
Tall (G:G)
Heterozygous (T:G)
Apogee (T:T)
Goodstreak (G:G)
NTC
B
Marker Genotype df
Days to Flowering Plant Height
Mean
squareR2 Mean
squareR2
Vrn-A1 GG vs GA vs AA 2 7755.41** 0.30 1208.34* 0.05
Ppd-D1 CC vs CT vs TT 2 140.49 0.01 941.57 0.03
Rht-B1 TT vs TC vs CC 2 640.68 0.02 8486.46** 0.27
Rht-D1 TT vs TG vs GG 2 1077.60* 0.04 8812.93** 0.30
Table 3. ANOVA table for two-genotypes (one homozygous and one
heterozygous) examining the effect of markers on flowering date and plant height in the BC1F1 population.
Table 4. ANOVA table for the effect of the parental genotypes (Vrn-A1,
Ppd-D1, Rht-B1, and Rht-D1 loci) in the progeny on flowering date and
plant height in the F5 and BC1F1 population. 37 parental genotypes were
found in the F5 population and 8 heterozygous vs. Goodstreak genotypes (recurrent parent) were found in the BC1F1 population.
PopulationParental
genotypesdf
Days to Flowering Plant Height
Mean
squareR2 Mean
squareR2
F5 37 36 593.80** 0.40 1171.03** 0.71
BC1F1 8 7 2888.64** 0.98 617.37** 0.56
** significant at the 0.01 probability level* significant at the 0.05 probability level
Marker Genotype df
Days to Flowering Plant Height
Mean
squareR2 Mean
squareR2
Vrn-A1 AA vs GA 1 11342.09** 0.55 85.25 0.01
Ppd-D1 TT vs CT 1 2221.26** 0.11 40.64 0.01
Rht-B1 CC vs TC 1 356.40 0.02 1123.92** 0.15
Rht-D1 GG vs TG 1 54.58 0.01 547.52** 0.14
** significant at the 0.01 probability level
** significant at the 0.01 probability level
0
20
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100
120
Da
ys
to F
low
erin
g
A
0
20
40
60
80
100
120
Hei
gh
t
0
20
40
60
80
100
120
140
Da
ys
to F
low
erin
g
B
0
20
40
60
80
100
120
Hei
gh
t
GG = Spring
AA = Winter
CC = Spring
TT = Winter
TT = Dwarf
CC = Tall
TT = Dwarf
GG = Tall
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