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

40

60

80

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