Future line: Standardization of SNPs to

various crops for commercial lot testing.

DOYLE, J. J AND DOYLE, J.L, 1990, Isolation of

plant DNA from fresh tissue. Focus,12: 13-15.

MAO AND DENG, X.L, 1992, Hybrid Nee seed production

techniques in three Hybrid Rice Seed line method of

hybrid Nee. Production Technology- Training Manual,

published by Directorate of Rice Research, Hyderabad,

47-48.

Genetic purity refers to the percentage of contamination by seeds or

genetic material of other varieties or species and is thus, a quality

assurance check. It has been reported that 1% reduction in genetic purity

reduces seed yield by 100kg/ha (Mao et al., 1992).

Genetic purity of seeds can be assured by different methods starting from

conventional Grow Out Test (GOT) to advanced techniques like protein,

isozyme and DNA markers i.e., RFLP, RAPD, SSR and SNP.

In this study we collate the conventional GOT with molecular marker

testing using SSRs and with GBS via SNPs identified by dd-RAD

sequencing.

Use of microsatellite markers and SNPs for genetic purity

testing of maize (Zea mays L.) F1 hybrid Satya Srii, V.,1 Nethra, N1., Lohithashwa, H.C2., Ramanappa, T.M1. and Devaraju, P.J3

1AICRP on Seed Technology Research, 2 Department of Genetics and Plant Breeding and

3Department of Seed Science and Technology, University of Agricultural Sciences, GKVK, Bengaluru-560065, India

Introduction

Material and methods

Hybrids used for the study – MAH-14-5 (CAL-1443 × CML-451)

Hema (NAI-137 × MAI-105)

Field GOT analysis:

SSR marker analysis

GBS method for SNP analysis

Lot testing by using identified SSRs and SNPs

SNP ANALYSIS

Results

MAH-14-5 HEMA

240bp 220bp

F M H F M H bnlg 1520 bnlg 1621 F M H F M H

MAH-14-5 HEMA

240bp 220bp

Plate 13: Diversity analysis- Linear tree

Plate 11: Summary of identified SNPs

SSR ANALYSIS

Table 2: Summary of polymorphism and allele size in base pairs of different markers

Plate 9: Banding pattern for two hybrids with different SSR markers

Primer MAH-14-5 HEMA

F M H F M H

Bnlg 1520 220 240 220,240 Monomorphic

Umc 1288 220 200 220,200 Monomorphic

Phi 053 Monomorphic 120 140 120,140

Bnlg 1621 Monomorphic 220 240 220,240

Bnlg 1014 Monomorphic 180 160 180,160

Bnlg 1185 120 100 120,100 100 110 100,110

Umc1594 140 120 140,120 140 130 140,130

Plate 8 : Leaf- undulation of margin of blade.

Hema MAH-14-5

Inte

rmed

iate

Str

ong

MAH-14-5 Hema

Slig

ht

Plate 7:Anthocyanin coloration at the base of glume

Str

ong

Total plants True to type Offtypes Genetic purity

(%)

400 372 28 93.0

GOT results: Table 1: Summary of plant count in GOT

Plate 6: Anthocyanin coloration at the silk.

Med

ium

Stro

ng

NAI 137(F) Hema

Plate 5: Angle between blade and stem

Med

ium

Larg

e

CAL-1443(F) MAH-14-5

44388

73.7%

12391

20.6%

3424 5.7%

MAH-14-5 Hema

Plate 12: Heatmap for kinship analysis

0

20

40

60

80

100

120

R 1 2 3 4 5 6

% heterozygosity

% homozygosity

Plate 14: Zygosity percentage

1 – MAH-14-5

2 – CAL- 1443 (F)

3 – CML- 451 (M)

4 – NAI- 137 (F)

5 – MAI-105(M)

6 - Hema

0.5

R

2

4

5

6

1

3

Plate 10: Lot testing using Phi-053 for Hema

140bp 120bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

140bp 120bp

56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 10 offtypes/100

Table 3: Comparison of lot testing by

various methods

References

Plate 1: Representation of SSRs and SNPs

Hybrid - Hema

No. of plants - 400

Spacing - 25 × 60 cm

No. of rows - 10

Plate 3: DNA extraction by protocol stated by Doyle and Doyle (1990)

Plate 4: ddRAD sequencing and SNP identification

Screening of SSR markers by PCR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 L 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 L

71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

39 40 412 42 43 44 45 46 47 48 49 50 51 52 L 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

11 offtypes/100

68 69 70

Plate 15: Lot testing using Tetra1 for Hema

190bp 170bp

41

Category GOT SSR

Marker

SNP

Marker

Total Plants 400 400 400

True to Type 372 352 350

Offtypes 28 48 50

Genetic

purity %

93 88 87.5

Table 4: Cost comparison between GOT and

GBS

Field GOT SSR (in rupees) SNP (in rupees)

Cold storage of

lot for a year-

3,000- 5,000/lot

Primer

development –

900/primer

Initial screening-

6000-10,000/

hybrid

Sequencing cost

– 15,000/sample

Marker

development –

12,000/marker

Lot testing-

1,800/lot

Lot testing –

1,900/ lot

Lot testing –

1,200/lot

Acknowledgement : Directorate of

Research, UAS, for funding the project to

NN and ICAR for JRF-fellowship to SS.

Plate 2: Field of GOT

Raw Data

dDocent Pipeline

Variant Filtering

TASSEL

RE double digest

Add adapter A &B

Size select &

amplify library

Sequence and Analyze