Maize Lethal Necrosis

Molecular breeding

CIMMYT

MLN – Molecular breeding

MLN – Two viruses + interaction effect

Transmission by many insect vectors

Seed transmission

Alternative vectors – sugarcane, sorghum?

MCMV interact with other poty viruses too

Therefore genetic control is the more effective

way to control MLN

Understanding the genetic architecture of the

MLN is crucial and here molecular breeding

come into picture

Discovery – Discover genomic regions associated with

MLN resistance

Validation – Validate the results

Deployment – Integrate into breeding program

Major genes - MABC

Minor genes / quantitative trait - MARS or GWS

MLN – Molecular breeding

Maize Lethal Necrosis - Discovery

Association studies

QTL Mapping

Validation of AM results with QTL Mapping

Genomic prediction

Implementation - MABC

4

Association mapping panels - Two

IMAS panel – 382 lines genotyped

with GBS,

Artificial inoculation – 3

DTMA panel – 235 lines - GBS,

Artificial inoculation - 3

Natural inoculation - 2

Maize Lethal Necrosis

IMAS panel DTMA panel

Phenotypic analysis – Across environments

MLN_86DAP Environment Mean (range) σ²G σ²GxE σ²e h²

IMAS panel

Narok12 2.84 (1.00-4.99) 0.36** - 0.27 0.73

Narok13 3.20 (1.00-5.00) 0.34** - 0.32 0.68

Naivasha13 2.83 (1.14-5.00) 0.25** - 0.60 0.40

Across Env 2.98 (1.15-4.85) 0.25** 0.08** 0.38 0.73

Phenotypic analysis - AM panel

DTMA panel

Naivasha13 3.66 (2.01-5.00) 0.16** - 0.19 0.63

Naivasha14 3.29 (1.99-4.25) 0.11** - 0.22 0.68

Narok13 3.61 (2.31-5.00) 0.12** - 0.34 0.41

Across Env 3.53 (2.51-5.00) 0.09** 0.05** 0.23 0.62

Across Across Env 3.00 (1.00-5.00) 0.19** 0.07** 0.45 0.78

Genotype

MLND scores (scale 1-5) Seed

color

Breeding Program

Env1 Env2 Env3 Across Env

CLRCY039 1.18 0.92 1.14 1.17 Yellow CIMMYT lowland tropics

CPHYS138 1.02 1.49 1.42 1.32 Yellow CIMMYT Physiology

CLRCY034 1.14 1.62 1.71 1.48 Yellow CIMMYT lowland tropics

CLWN270 1.36 1.80 1.41 1.52 Yellow CIMMYT lowland tropics

CKL05003 1.12 1.58 2.12 1.62 White CIMMYT Kenya

SM-189-75 1.07 1.57 2.26 1.69 Orange KALRO, Kenya

CLWQ251 1.42 2.28 1.60 1.81 White CIMMYT lowland tropics

CML494 1.33 2.30 1.92 1.83 White CIMMYT Gene bank

SM-189-38 1.73 1.88 1.63 1.86 White KALRO, Kenya

CPHYS159 1.97 1.50 2.15 1.86 White CIMMYT Physiology

CLYN261 1.87 1.49 2.19 1.87 Yellow CIMMYT lowland tropics

SM-189-78 1.86 2.75 1.14 1.88 Orange KALRO, Kenya

CLYN231 1.08 2.03 2.48 1.90 Orange CIMMYT lowland tropics

SM-189-69 1.23 1.98 2.59 1.99 Yellow KALRO, Kenya

Genome-wide Association mapping

Quality screening of GBS markers

Select the SNPs with MAF of > 0.01

Remove the SNPs with >5% of the missing

Select the lines which has both genotypic and

phenotypic data

For IMAS the final set has 381 lines and 259,476

SNPs

For DTMA the final set has 235 lines and 260,000

SNPs

PCA and density distribution plots

MLM based GWAS results

DTMA-AM panel

P<0.00001

MLM based association mapping results

Chr - SNP

MLM_P values

Pg

(%) MAF

M

allele FAE

Donors

86DAP

Lowland

Midland

Highland

S1_52083545 1.06E-05 7.36 0.22 A 0.09 CLRCY039

CLRCY034

DTPYC9-F46-

1-2-1-2-B

CLWN270

CLWQ251

CML494

CLYN261

CLYN231

LaPoSeqC7-

F78-2-1-1-1-B

CML543

BSC-9

BSC-12

BSC13

KTN71-3 S2_8916323 7.03E-06 7.99 0.03 A -1.06

S2_38324933 3.97E-06 8.39 0.32 G -0.35

S2_211771737 2.51E-06 8.38 0.03 G -1.38

S3_44062810 1.66E-06 8.36 0.36 T 0.02

S3_169349054 7.53E-06 7.47 0.07 A -0.57

S3_202831000 5.25E-06 8.09 0.02 C 0.00

S3_203868724 1.10E-06 7.88 0.02 G 0.00

S5_162012868 1.00E-06 9.56 0.02 A -1.21

S5_198780420 9.38E-06 7.57 0.48 C 0.05

S6_70013961 5.92E-06 6.79 0.01 A 0.00

S9_2825540 3.19E-06 8.42 0.26 T -0.42

S9_152057703 8.63E-06 7.78 0.47 G -0.07

S10_9212960 9.66E-06 7.37 0.17 A -0.02

S10_103937258 6.83E-06 8.04 0.02 T -0.73

Total 59.0

SNP Chr Position

(Mba)

MLM-P

values

Pg (%) Putative

candidate genes

Predicted function of

candidate gene

S1_46411896 1 46.4 6.78E-05 15.26 GRMZM5G814718 Oxidoreductase activity

S1_269037989 1 269.0 2.86E-05 16.16 GRMZM2G047055 Actin cross link

S5_16837972 5 16.8 5.04E-06 17.91 GRMZM2G077828 Unknown

S5_16839191 5 16.8 3.79E-06 18.44 GRMZM2G018943 Translation initiation factor eIF-2B

delta subunit

S5_95192724 5 95.1 6.26E-06 18.81 GRMZM2G109805 Hypersensitivity

S5_199371477 5 199.3 3.43E-05 14.91 GRMZM2G376067 MAIZE Putative uncharacterized

protein

S6_84786872 6 84.7 4.29E-06 18.42 GRMZM2G139073 MADS-box transcription factor

S7_15457394 7 15.4 4.13E-05 14.67 GRMZM2G480050 Response to freezing

S10_148638187 10 148.6 5.11E-05 15.30 GRMZM2G074754 Transporter activity

Total 41.93

MLM based association mapping results

Association mapping - Conclusions

Best 15 significant SNPs explained individually 7 to

10% and together up to 59% of the total genotypic

variance for MLN resistance

MLN is controlled by few major and many minor

genes

The best performing lines can be used as a donors

Validation of significant SNPs is crucial

Pop1 – CML444 x CML539,

pop size 184, Locations - 3

Pop2 – CML444 x CML543,

Pop size 203, Locations – 3

Pop3 – LaPostaSeqC7-F71-1-2-1-2-B-B-B-B. x CML543,

Pop size 229, Locations – 3

Pop4 – M37W x CML144 – Pop size -130

Pop5 – J80W x CML144 - Pop size -156

DH Pop – CML494 x CML550 - Pop size -236

Maize Lethal Necrosis – QTL mapping

Maize Lethal Necrosis – CML539xCML444

MLN2_Bom MLN2_Nai MLN2_Ole MLN2_3loc

12

34

5

Dis

ea

se

se

ve

rity

sca

le (

1-5

)

CML444 CML539

86DAP

CML444 2.71

CML539 (CZL3014) 3.35

CML543(CML543) 2.32

La Posta Seq C7-F71-1-2-1-2-B-B-B-B-B 3.27

Maize Lethal Necrosis – CML539xCML444

Epistatic QTL – CML539xCML444

Maize Lethal Necrosis – CML543xCML444

86DAP

CML444 2.71

CML543(CKL5003) 2.32

Populations Environ σ2G σ2

GE σ2error h2

CML444 X CML543 Bomet13 0.08 - 0.28 0.36

Naivasha13 0.18 - 0.27 0.57

Naivasha14 0.03 - 0.14 0.31

Across Env 0.07** 0.02* 0.24 0.60

Main and Epistatic QTL – CML543xCML444

Pop3 - CML543xLa Posta Seq C7-F71

86DAP

La Posta Seq C7-F71-1-2-1-2-B-B-B-B-B 3.27

CML543(CKL5003) 2.32

Environments σ2G σ2

GE σ2error h2

Bomet13 0.21** - 0.25 0.62

Naivasha13 0.17** - 0.32 0.52

Naivasha14 0.02** - 0.12 0.29

Across Env 0.06** 0.06** 0.23 0.52

CML543xLa Posta Seq C7-F71

CML494xCML550 DH pop

Chromosome 1

15.7Mbp

Chromosome 3 Chromosome 7 Chromosome 9

Conclusion

In each Pop and DH – best 3 major QTL are explained ~40-

50% of the total phenotypic variance

Major additive QTL detected on Chr 3 was consistent across

scorings and locations in two populations

The presence of major QTL(s) on chromosome 3 and 6 are

planned to use in MABC to improve the resistance in the

elite lines

Prediction of MLN performance based on random

and random with linked SNPs

Inclusion of MLN resistance associated SNPs can

significantly increase the prediction accuracy

Future direction for discovery phase

MLN --- SCMV + MCMV + Interaction

SCMV – Oligogenic – Major genes

MCMV – Oligogenic or polygenic

Interaction effect – Complex and non-genetic

Selecting resistance for individual viruses, either

in same or in different lines, to avoid interaction

effect and able to manage MLN effectively

26

MCMV1 MCMV2 MCMV3 MCMV4

Geno_Var 0.09 0.12 0.25 0.13

Block_Var 0.01 0.02 0.05 0.00

Res_Var 0.06 0.09 0.15 0.24

Herritability 0.73 0.73 0.77 0.52

Trait Marker Chr P-value Pg (%)

MCMV2 S1_273096577 1 6.86E-06 10.22

MCMV2 S1_273036576 1 6.93E-06 9.39

MCMV2 S1_240766785 1 4.72E-05 7.85

MCMV2 S2_63372985 2 9.12E-05 7.64

MCMV2 S4_30928389 4 3.49E-05 8.16

MCMV2 S5_143970340 5 2.35E-05 9.36

MCMV2 S6_161804207 6 9.29E-05 7.52

MCMV2 S7_29253328 7 5.60E-05 8.23

MCMV2 S8_139630981 8 3.72E-05 8.37

MCMV2 S8_166902808 8 7.47E-05 7.63

MCMV – screening and GWAS

Trait Marker Chr p Pg(%)

MCMV2_pot S5_117936895 5 7.40E-07 22.19

MCMV2_pot S2_5005955 2 1.62E-06 21.72

MCMV2_pot S2_1353042 2 8.72E-06 16.99

MCMV2_pot S4_12909642 4 9.71E-06 16.60

MCMV2_pot S7_67826176 7 1.20E-05 18.14

MCMV2_pot S2_94276895 2 1.49E-05 16.10

MCMV2_pot S10_81442902 10 1.77E-05 17.50

MCMV2_pot S4_56969341 4 1.83E-05 16.55

MCMV2_pot S2_63374956 2 2.16E-05 15.60

MCMV2_pot S5_163417521 5 2.44E-05 15.23

MCMV2_pot S5_9655176 5 2.63E-05 16.05

MCMV2_pot S1_29258797 1 3.88E-05 14.54

MCMV2_pot S6_146216747 6 4.06E-05 15.00

MCMV2_pot S6_151455727 6 4.11E-05 14.56

MCMV2_pot S3_201545312 3 4.87E-05 14.61

MCMV2_pot S3_206692477 3 4.87E-05 15.47

Thank you