identification of in vivo-induced haploid seeds in maize based on...

Identification of in vivo-induced haploid seeds in maize based on oil content V. Mirdita, W. Schipprack, and A.E. Melchinger Abstract In vivo haploid induction has become a routine tool for rapid line development in maize. However, distinguishing haploid (H) from diploid crossing (C) seeds is problematic for many germplasm due to poor expression or suppression of the currently used R1-nj embryo marker. A new approach for sorting H and C seeds based on their oil content (OC) was examined. Ten source germplasm, including single crosses, synthetics and landraces, were pollinated by high-oil (HO) inducer UH600 with OC=10.8%. The average difference (1.79%) between the mean OC of C and H seeds was more than twice the standard deviation (SD) within each fraction. Thus, sorting H and C seeds based on OC smaller or greater than an a priori chosen threshold t was generally more reliable than based on the R1-nj embryo marker. Another ten source germplasm were pollinated with normal-oil inducer UH400 with OC=3.0%. Since the difference (0.65%) between OC of C and H seeds was approximately of the same magnitude as the SD, both fractions overlapped too much for reliable sorting. The discrimination of H and C seeds based on their OC looks very promising, even for heterogeneous source materials such as landraces, provided a HO inducer and a stringent threshold t are used. In combination with high-throughput platforms for automated sorting of single seeds for OC, this opens new avenues for extending the application and increasing the efficiency of the double haploid technology in maize.

Identification of in vivo-induced haploid seeds in maize based on oil

content

V. Mirdita, W. Schipprack, and A.E. Melchinger Institute of Plant Breeding, Seed Science and Population Genetics

University of Hohenheim, Stuttgart, Germany

e-mail: [email protected] Phone: 49 711 - 459 - 22334

2 50th Annual Illinois Corn Breeders School © VM 03.03.2014

Outline

● Introduction

● State-of-the-art of DH production

● Current status of marker systems

● New identification system based on oil content

● New inducers for haploid seed identification

● Summary and conclusions


● Acceleration of line development ● Early evaluation of potential hybrid cultivars ● Maximum additive variance from the beginning ● No masking effects due to residual

heterozygosity ● Reduced costs of maintainance breeding ● Good per se performance of lines ● Early protection of outstanding lines by PVP ● Simplified logistics

Main advantages of DH technique


State-of-the-art of DH production

F1 seeds

haploids

1 2

3

4

4

inducer

source germplasm

DH line 1

DH line 2


Requirements of an effective marker system

easy, cheap and early detection

reliable, unambiguous unbiased selection

small FDR and FNR

independent of environment

stable expression in different genetic

background

high throughput, automatable


Marker currently used: R1-nj

x

outcross or selfing

H (aploid) seed

C (rossing) seed

Embryo: purple Colorless

embryo

Aleurone: purple

Aleurone: purple


Pros (+): Easy Cons (-): Labor-intensive Cheap No automation Early detection Error-prone

Marker currently used: R1-nj


Limitations of 1-nj system

has purple aleurone color

harbors dominant anthocyanin inhibitor genes suppressing R1-nj expression

Accuracy and speed of H seed identification depends on trained staff

Haploid seeds identification is hampered if source population:

no clear marker = ?


FDR = FP

FP TP +

Misclassification in selecting H/C seeds

FN

FNR = FN TP +

Type I error = FDR

Type II error = FNR

C H

True positive

TP

False negative

FN

True negative

TN

False positive

FP

H

Test result of

True

con

ditio

n

C


C H

H

Test result of

C

TP

FN

TN

FP “Gol

d“ s

tand

ard

FDR

FNR

True H 128 = 8.90 %

True C 1310 = 91.10 %

Ligueless

Numerical example for marker

C


red root coloration

GMO traits e.g.GFP

quality traits

oil content

oil composition

protein

Alternative marker systems


x

H seed colorless

root purple

root C seed

Principle of haploid identification based on red root colour


Bruker mq 20 the minispec Nuclear Magnetic Resonance (NMR) Analyzer

Principle of haploid identification based on oil content

Mixture of H and C seeds

H-seeds (lower oil content)

C-seeds (higher oil content)

Choice of threshhold for oil

content

Oil measurement of single seeds


Suggestion of Rotarenco et al. (2007)

Would a HO inducer solve the problem?

Freq

uenc

y (%

)

Discriminate H and C seeds based on OC

Oil content (%)


Foundations of HO-inducer system

Melchinger et al. 2013 Oil content (%)

µC seeds HO-Inducer

2 3 4 5 6 7 8 1

µH seeds

Den

sity

10.8

C seed

H seed HIR = +


“Gol

d“ s

tand

ard

Example: HO- inducer system

FDR = 11

11 76 + = 12.6%

8 FNR =

8 76 + = 9.52%

Threshold t = 4.40% OC CH

TP FN

TN FP

H

Test result

C

True H

True C C


Distribution of OC in H/C seeds

= 12.6%

= 9.52 %

t

Oil content (%)

Freq

uenc

y

8 FNR =

8 76 +

Example: 919 seeds from cross (S072 x P213) x UH601

FDR = 11

11 76 +


Foundation of HO-inducer system

Important factors FDR FNR

Hapl. induction rate (HIR) ∆OC = µC - µH σC, σH Threshold t

↓

↓

↓

↓ ↓

↓

↑

↑ ↓

↑

↑

↑


HIR: well-known property of HO-Inducer (I) little variation among source germplasm (SG) ∆OC: µC - µH µC = (µI - µSG) and µH = µSG

and if SD = P1 x P2 then µSG = (µP1 + µP2)

µC - µH = (µI - µSG)

σC, σH: use prior information from other similar germplasm

Choose t based on theory and desired FDR and FNR

assuming additive inheritance of OC

21

21

21

Foundation of HO-inducer system


Choice of t in HO-system

Software available for CDF player: www.wolfram.com/cdf-player


“Proof of concept“ - Results 37,454 seeds from 10 source germplasm x UH600 Classification based on R1-nj Classification based on OC

Melchinger et al. 2014, in press.

“Gold standard“:

visual scoring of

plants in field

false “positives“


OC R1-nj Misclassification of seeds:

vs

60

40

0

20

80

100 FDR (%) FNR (%)


Identification of H seeds based on OC

C seeds H seeds

NO Inducer UH400

Oil content (%)

HO Inducer UH600


“Proof of concept“ successfully completed

2 HO-inducers available: UH600, UH601

UHOH develops further HO-inducers

UHOH has high-throughput system for H

seed identification based on OC

Summary and conclusions


HO inducer lines

Haploid induction rate (HIR): 10.2 ± 1.3% 6.6 ± 2.7%

Oil content: 10.8 ± 0.7% 11.7 ± 0.4%

Marker system: OC of seeds: + + R1-nj marker: + + B1 marker: + Pollen production: + +

UH601 UH600

UH600 UH601


References

https://plant-breeding.uni-hohenheim.de

Melchinger et al. 2013, Sc. Rep. 3

Melchinger et al. 2014, Crop Sci. in press

Rotarenco et al. 2007, MNL 81:11


Acknowledgements

Project members:

A. E. Melchinger V. Mirdita

Technical team: F. Mauch R. Volkhausen

T. Schmid N. Friedl

Internal funds from University of Hohenheim

F. Mauch

Financial support:

identification of in vivo-induced haploid seeds in maize based on...

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