Characterization of Markers Linked to Resistance Motifs against ...

InnovativeAgricultureResearchInitiative(iAGRI)GraduateResearchWorkshop

May5‐6,2016

Characterization of Markers Linked to Resistance Motifs against Maize Lethal Necrosis Disease in Tanzanian Maize

Germplasms

INOCENT P. RITTEM. EGNIN P.M KUSOLWA

Graduate Research WorkshopMay 5‐6, 2016

INTRODUCTION Maize (Zea mays) is among the world’s major crop for food andincome generation In Tanzania – Major staple foodAmong the biological constraint facing maize production inTanzania is the severe occurrence of maize lethal necrosisdisease (MLN) MLN- is a disease complex caused by dual infection of maize byMCMV and any of the Poty virus infecting cereals e.g. SCMV

FAOSTAT, 2013; MAFSC, 2011; IITA, 2010.


INTRODUCTION…. Singular infection with either of the two viruses produces milder symptoms The host range for MCMV is limited to members of the Gramineae maize is the natural host MCMV transmission occurs through insect vectors such as maize thrips, maize flea beetles and the cereal leaf beetles Insecticides: not an efficient way to manage MLN in Tanzania Use of resistant maize varieties Effective way to manage MLN

Drake and Roy, 2011; Nelson et al., 2011


DISEASE PREVALENCE Major MLN epidemics:

.Peru (1974)

.Kansas (1976)

.Hawaii (1990) In E/Africa MLN was first reported in Kenya (2011) and later confirmed in Tanzania (2012), Uganda, Rwanda and Sudan

Scheets, 2000; Wangai et al., 2012


OBJECTIVES OF THE STUDY Identification of MLN resistant genotypes and molecular markers that may be associated to resistance of maize to MLND in TZ maize germplasms for use in breeding for resistance.

Specific Objectives To perform phenotypic screening through artificial inoculation of Tanzanian maize germplasm with SCMV and MCMV isolates

Assessment of MLN disease symptoms and severity of Tanzanian corn germplasms infected with maize MLN

To develop and characterize preliminary AFLP markers in known resistant and susceptible maize for molecular identification of MLN resistant germplasm.


MATERIALS AND METHODSPlant materials 152 gene bank accessions [NPGRC] collected from different agro-ecological zones in Tanzania

33 Tanzanian inbred lines from SARI

24 CIMMYT inbred lines with known background on MLN reaction

4 USA lines with resistance and susceptibility background to MLN

4 EA hybrid cultivars known to be MLN susceptible


MATERIALS AND METHODS…Experimental plot layout Planting date Nov. 20, 2014 (Season 2014B) Two separate trials laid in CRD with 2 replica

i. TZ maize landracesii. TZ maize inbred lines

Trials were planted with P fertilizer (DAP) Spacing: 0.25m within and 0.75m apart rows 13 plants in single rows, each row 3M long Top dressing w/ N fertilizer (CAN) 2 weeks after emergence Sprinkler irrigation to limit drought stress.


MATERIALS AND METHODS…MLN Phenotyping

Virus isolates (MCMV &SCMV)

Inoculum production (2 separate screen houses)

Inoculum preparation [1g leaf materials/20mls 1X P buffer]

Homogenized in a blender and sieved w/ cheese cloth

MCMV and SCMV extracts were mixed in [1:4] ratio

Carborundum added in inoculum extracts at a rate of 1g/L

Plants potentially challenged to MLN in single & double infection at 4‐5 leaf

Symptoms evaluated fortnightly for landraces & at weekly intervals for inbred lines by 1‐5 Scale (1=No MLN symptoms and 5=Complete plant necrosis)


MATERIALS AND METHODS…

MLN phenotyping activities at Naivasha maize lethal necrosis screening facility. (A) Screen house inoculum production (B) Leaves sampling (C, D & E) Inoculum

preparation and inoculation (F) MLND evaluation


MATERIALS AND METHODS…Laboratory Investigation (SUA) Seeds of selected landraces under artificial inoculation, CIMMYT lines and SARI lines were germinated in screen house

Young maize leaves were sampled from each genotype at 2‐3 leaf stage and stored at ‐20o C

gDNA was isolated from frozen leaf samples as according to the protocols by Egnin (1998, 2010)

gDNA was quantified by spectrophotometer (ND)‐2000

AFLP analysis was performed as described in (Vos et al., 1995) with minor modifications of Invitrogen AFLP Kit


AFLP ANALYSIS 500ng of gDNA was simultaneously digested w/ MseI and EcoRI at 37ᴼC for 2:30 hours followed by incubation at 70oC for 15min MseI/EcoRI adapters were ligated at 20ᴼC for 2hrs. [4-fold dilution] Pre-amplification conditions: 94°C/30s, 56°C/60s and 72°C/60s (20 cycles). Then 4-fold dilution was made Selective amplification: 1 cycle at 94°C/30 s; followed by 65°C/30s and 72°C/60s, followed by 13 cycles of touch down PCR 23 cycles at 94°C/30 s, 56°C/30 s and 72°C for 60 s. The reaction was then stored at -20oC Equal volume of 2X TBE-Urea Dye was added to each PCR reaction, denatured by heating at 95oC for 3 minutes Products were separated in 6% denaturing polyacrylamide gel.Adawy et al., 2008; Vos et al., 1995; Tuskegee University Biotech Lab


SEQUENG OF AFLP MARKERS Amplified AFLP allelic fragments were estimated manually scored (1/0, presence/absence) exported to spreadsheet to generate binary matrices

Genetic similarities were estimated by UPGMA procedure in cluster analysis to develop a dendrogram

Polymorphic fragments w/ strong intensity were eluted from the gel, precipitated and re-amplified w/ the corresponding primer pairs and confirmed on 2% agarose gel

Confirmed eluted fragments (61) were subjected to sequencing by Beckman Genomics


DATA ANALYSIS Phenotypic data were subjected to ANOVA to test for significant differences among the genotypes For AFLP data; gel images with amplified fragments were manually scored (1/0) 1/0 data matrices were analyzed in NTSYSpc software

Sequence data

Bio Edit sequence alignment editor (Version 7.2.5)

Compared with sequences of Z. mays (NCBI)

Sequences with significant identities of >80%

Bio Edit sequence alignment editor (Version 7.2.5)

Molecular Evolutionary Genetics Analysis (MEGA, version 6.0)Hall, 1999; R Core Team, 2013; Rohlf, 2000; Tamura et al., 2013


RESULTSMLN Phenotyping [ANOVA]

Results showed significant differences among the landraces genotypes (P=0.05)

Statistically, no differences were observed between the inbred lines (P=0.05)

All inbred lines attained the mean score between 4.5 and 5.0

Resistant check CML494, slightly differed from inbred line test materials. Obtained mean score of 3.75


RESULTS…Responses of maize landraces


RESULTS…Maize lethal necrosis symptoms on maize landraces

(A) mild leaf chlorosis 14dpi, (B) higher density of chlorotic spots 28dpi, (C) Visible symptoms of necrotic tissues and complete plant death 42dpi(D,E and F) 72dpi.


RESULTS…Responses of inbreed lines


RESULTS…Maize lethal necrosis symptoms on inbred lines

(A) mild leaf chlorosis 7dpi, (B) higher density of chlorotic spots 14dpi, (C) necrotic tissues developed from leaf margins to the mid-ribs 21dpi (D)

complete plant death 52dpi.


RESULTS…Maize lethal necrosis disease severity

Evaluation Scale (1‐5)1 = No MLN symptoms, 2 = Fine chlorotic streaks on lower leaves3 = Chlorotic mottling throughout plant, 4 = Excessive chlorotic mottling and dead heart and5 = Complete plant necrosis.


RESULTS…AFLP analysis 16 primer combinations [11 combinations were reproducible]


RESULTS…

AFLP Gel Output pattern of 8 TZ maize biotypes derived from 2 sets of primer pair combinations. Arrow indicates polymorphic band.


RESULTS…Cluster analysis

UPGMA showing genetic relationships among the 22 maize genotypes generated based on Jaccard’scoefficient and the AFLP binary matrix data


SEQUENCING RESULTSSequencing results showed that; Out of 61 samples

37 samples successfully sequenced

24 samples were not successful

BLAST search results also revealed that 19 sequence data had high identity to Z. mays

18 were not maize related and however, they are of importance in

this study


BLAST SEARCH RESULTSAFLP MARKERS LINKED TO D’SE RESISTANCE


BLAST SEARCH RESULTS…

Nucleotide sequence alignment of 3 AFLP markers that tag for Z. mays rust resistance protein rp3‐1(rp3‐1) gene. * = identical base and ‐‐ =gaps. M‐CAA/E‐ACG218, M‐CAA/E‐ACG372 and M‐CAA/E‐ACG362


BLAST SEARCH RESULTS…5 pathogenesis related protein genes AFLP markers M-CAA/E-ACG265, M-CAA/E-ACG348, M-CAA/E-ACG258, M-CAA/E-ACG274 and M-CAA/E-ACG379.


OTHER OBSERVATIONSM-CAA/E-ACG308

Studies suggests that long non coding RNA participate in stress responsive regulation (Zhang et al., 2014).


CONCLUSION152 TZ maize accessions and 33 inbred lines were evaluated vs. MLND ANOVA results detected significant differences (P=0.05) among maize accessions at 72 dpi No significant differences (P=0.05) observed among inbred lines AFLP analysis performed w/ 11 primer combination and average polymorphism 62.46% obtained Cluster analysis discriminated potentially MLN promising and susceptible genotypes AFLP nucleotide sequence and BLAST search led to ID of 10 AFLP markers; 8 markers associated to disease resistance and 1 to drought response. The rest – not characterized.


WAY FORWARD Studies involving molecular markers to continue screening maize genotypes available in TZ

Platform such as GBS would be efficient method to genotype the available materials

Pyramiding pot virus resistant genes in commercial hybrid cultivars would help to lessen severe yield losses associated to MLN

Conducting studies to understand MLN epidemiology and host/vector/pathogen interaction in TZ

Re-phenotyping of materials which attained mean score of 4.0 and below

Characterization of TZ MCMV isolate


ACKNOWLEDGEMENTS Dr. Marceline Egnin – Major Adviser (USA)

Dr. Paul Kusolwa – Co‐Adviser (Tanzania)

Graduate students at TU‐Plant Biotech and Genomics Research Lab. [Mr. Steven Samuels and Mr. Chris Bernard]

Lab Technologists (Mr. Deogracious Massawe and Ms Sylvia Mlemba) ‐ SUA Plant Molecular Biology Laboratory

SARI‐Arusha (Mr. Kheri Kitenge)

TPRI[NPGRC]‐Arusha (Dr. Margareth Mollel)

CIMMYT‐Kenya (Dr. G. Mahuku, Dr. B. Das and Ms. J. Njeri)


THANK YOU FOR YOUR ATTENTION

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