WCT REPORT 2013 · Centurion 0046 Report compiled by: PROJECT MANAGER Prof Anna-Maria...

STELLENBOSCH UNIVERSITY

WCT REPORT 2013

MAPPING DISEASE RESISTANCE GENES IN WHEAT

Anna-Maria Botha-Oberholster PhD

15 July 2013

EXECUTIVE SUMMARY

The aim of the project is to provide a DNA-based marker system with high-throughput capabilities; that is background non-specific, “gene” based and trait-linked. We also strive to provide the wheat community with scientific information on bread wheat pest resistance, the Russian wheat aphid-bread wheat interaction, and Russian wheat aphid (D. noxia) biotypification. In order to achieve these goals we study the genetics/genomics of pest resistance in wheat; plant-aphid interactions; Russian wheat aphid biology, biotypes, and aphid endosymbionts. To date we have identified numerous genes that are important in the bread wheat (host) defense against the pest, as well as putative eliciting agents in the Russian wheat aphid. During this financial year we have achieved the following major objectives: (1) We have saturated the map that will enable us to sequence the 7D chromosome arm that

contains most of the Dn resistance genes in wheat. Currently, we are isolating the Dt7DS and Dt7DL chromosome arms using ditelosomic lines containing the Dn genes so that we can commence with the sequencing of Chr. 7 in RWA resistant wheat.

(2) We have succeeded with the transformation of wheat using two genes involved in aphid resistance, namely APX and GST, demonstrating proof-of-concept and will now be developing transgenic wheat lines with increased aphid and drought tolerance.

(3) As part of an international cooperative, we have completed sequencing the RWA genome – i.e., a representative sample from 11 different RWA biotypes from global RWA populations – and have achieved overall coverage of more than >10 times using several NextGen sequencing platforms, and thus are able to discern all D. noxia biotypes on DNA sequence (i.e., cryptic RWA biotypes), as alternative to virulence/differential screening.

Botha-Oberholster et al. 2013

Report compiled for:

Winter Cereal Trust PO Box 7088 Centurion 0046

Report compiled by:

PROJECT MANAGER Prof Anna-Maria Botha-Oberholster Department of Genetics Stellenbosch University Phone: (021) 808 5832 Fax: (021) 808 5833 Cell no.: +83-450-0630 Email: [email protected]

CO-INVESTIGATORS Dr. Leon van Eck (Postdoctoral Fellow, SU staff) Miss. Thia Schultz (Ph.D.) Miss. Anandi Bierman (Ph.D.) Mr. N. Francois V. Burger (M.Sc., SU staff) Mr. Kenneth Mbwanji (M.Sc.) Miss. Nadia Fischer (M.Sc.) Miss. Laura de Jager (M.Sc.) Mr. Wynand Cloete (M.Sc.) Mr. Marlon le Roux (B.Sc. Hons) Mr. Louis Steyn (B.Sc. Hons) Ms. Kelly Breedts (B.Sc. Hons) Mr. Teboho Tyholweni (Greenhouse assistance, WCT funded) Mr. Siyethemba Masikane (Laboratory assistance, WCT funded)

INTERNATIONAL COLLABORATORS

Prof. Nora L.V. Lapitan (Colorado State University, USA) Dr. Gary Puterka (USDA, Stillwater, USA) Prof. Mike Smith (Kansas State University, USA) Dr. El-Bouhssini, Mustapha (ICARDA) Dr. Ana Maria Castro (Argentina) Prof. Martina Zurovcova (Czech Republic) Dr. J. Peng (China) Dr. Joyce Malinga (Kenya)

NATIONAL COLLABORATORS

Dr. Astrid Jankielsohn, SGI Dr. Christelle van de Vuyver, IPB, Stellenbosch Unioversity

mailto:[email protected]


MAPPING DISEASE RESISTANCE IN WHEAT: NOVEL STRATEGIES FOR MARKER ASSISTED SELECTION

EXECUTIVE SUMMARY

The aim of the project is to provide a DNA-based marker system with high-throughput capabilities; that is background non-specific, “gene” based and trait-linked. We also strive to provide the wheat community with scientific information on bread wheat pest resistance, the Russian wheat aphid-bread wheat interaction, and Russian wheat aphid (D. noxia) biotypification. In order to achieve these goals we study the genetics/genomics of pest resistance in wheat; plant-aphid interactions; Russian wheat aphid biology, biotypes, and aphid endosymbionts. To date we have identified numerous genes that are important in the bread wheat (host) defense against the pest, as well as putative eliciting agents in the Russian wheat aphid. During this financial year we have achieved the following major objectives: (4) We have saturated the map that will enable us to sequence the 7D chromosome arm that

contains most of the Dn resistance genes in wheat. Currently, we are isolating the Dt7DS and Dt7DL chromosome arms using ditelosomic lines containing the Dn genes so that we can commence with the sequencing of Chr. 7 in the RWA resistant wheat.

(5) We have succeeded with the transformation of wheat using two genes involved in aphid resistance, namely APX and GST, demonstrating proof-of-concept and will now be developing transgenic wheat lines with increased aphid and drought tolerance.

(6) As part of an international cooperative, we have completed sequencing the RWA genome – i.e., a representative sample from 11 different RWA biotypes from global RWA populations – and have achieved overall coverage of more than >10 times using several NextGen sequencing platforms, and thus are able to discern all D. noxia biotypes on DNA sequence (i.e., cryptic RWA biotypes), as alternative to virulence/differential screening.


TABLE OF CONTENTS i. Executive summary ii. Project outputs August 2012 to July 2013

Peer-reviewed papers (August 2012/July 2013) Conference presentations/Invited lectures (August 2012/July 2013) Theses/dissertations completed during 2013.

iii. List of figures iv. List of tables

1. INTRODUCTION

1.1 Rationale and motivation 1.2 Emerging sector 1.3 Workplan (2010-2013/2014)

2. BUSINESS PLAN, WORKPLAN AND OUTPUT SUMMARY AS PROPOSED IN THE 2007 APPLICATION 2.1 Saturation of maps and map-based cloning of Dn1, Dn2 and Dn5. 2.2 Analysis of breeding lines for Industry. 2.3 Confirmation of association of genes with RWA resistance: Transformation of

selected wheat cultivars to elucidate the function of Ascorbate peroxidase (APX) and Glutathione transferase (GST).

2.4 Setting up of a high throughput system for mass screening of breeding lines and implementation of marker system into the different national breeding programmes

2.5 Russian wheat aphid.

3. ACKNOWLEDGEMENTS 4. APPENDIX


PROJECT OUTPUTS 2012-2013

Project outputs (book chapters/peer-reviewed papers) for August 2012/July 2013:

Botha A-M, Van Eck L, Jackson CS, Burger NFV, Shultz T (2012) Phloem Feeding Insect Stress and

Photosynthetic gene expression. In: Photosynthesis, Part 2, InTech - Open Access Publisher, ISBN 979-953-307-664-4.

Botha A-M (2013) A coevolutionary conundrum: The arms race between Diuraphis noxia

(Kurdmojov) a specialist pest and its host Triticum aestivum (L.). Arthropod-Plant Interactions 7: 359-372.

Conference presentations / Invited lectures (August 2012/July 2013): Botha A-M, Burger NFV, Castro A-M, El-Bouhsini M, Jankielsohn A, Lapitan NLV, Pearce F,

Puterka G, Smith CM, Surovcova M (2012) Next Generation sequencing of the genomes of 10 International RWA biotypes. Joint Meeting: 20th Biennial International Plant Resistance to Insects Workshop & Annual session of Western Extension/Education Research Activity-066 (WERA-066): Integrated management of Russian wheat aphid and other cereal arthropod pests, April 1-4, 2012, Minneapolis, Minnesota, USA. (POSTER)

Bierman A, Lapitan NLV, Botha A-M (2012) Mapping and characterization of selected Diuraphis

noxia resistance genes in Triticum aestivum. Joint Meeting: 20th Biennial International Plant Resistance to Insects Workshop & Annual session of Western Extension/Education Research Activity-066 (WERA-066): Integrated management of Russian wheat aphid and other cereal arthropod pests, April 1-4, 2012, Minneapolis, Minnesota, USA. (PAPER)

Schultz T, Botha A-M (2012) Silencing of Russian wheat aphid resistance response related genes

using viral induced gene silencing. Joint Meeting: 20th Biennial International Plant Resistance to Insects Workshop & Annual session of Western Extension/Education Research Activity-066 (WERA-066): Integrated management of Russian wheat aphid and other cereal arthropod pests, April 1-4, 2012, Minneapolis, Minnesota, USA. (PAPER)

Burger NFV, Botha A-M (2012) Transcriptomic changes in Diuraphis noxia when transferred

from preference to non-preference hosts. Joint Meeting: 20th Biennial International Plant Resistance to Insects Workshop & Annual session of Western Extension/Education Research Activity-066 (WERA-066): Integrated management of Russian wheat aphid and other cereal arthropod pests, April 1-4, 2012, Minneapolis, Minnesota, USA. (PAPER)

Luna E, Van Eck L, Lapitan NLV, Botha A-M, Leach JE, Tisserat N, Campillo T, Cilia ML (2013)

Novel species Enterobacteriaceae isolated from Russian wheat aphid (Diuraphis noxia). American Phytopathological Society Meeting: Austin TX, USA, 10–14 August 2013. (POSTER)

Van Eck L, Davidson R, Wu S, Zhao B, Leach JE, Lapitan NLV (2013) Rice genomics tools reveal a

WRKY53 transcriptional network in wheat. Plant & Animal Genome XXI: San Diego CA, USA, 12–16 January 2013. (POSTER)


Van Eck L, De Jager L, Burger NFV, Botha A-M (2013) Next-generation sequencing Buchnera

aphidicola genomes from 11 Diurahpis noxia biotypes. Plant & Animal Genome XXI: San Diego CA, USA, 12–16 January 2013. (POSTER)

Thesis/dissertation completed during 2013:

Schultz T (2013) Elucidating functional interactions between the Russian wheat aphid (D. noxia Kurdjumov) and bread wheat (Triticum aestivum L.). Ph.D. Thesis, Stellenbosch University, December 2013.


ii. LIST OF TABLES TABLE 1: Comparing mortality rate of isolated embryos — cultivars Gamtoos-S (Dn7-) and Gamtoos-R (Dn7+) — bombarded with constructs pUBI-GSTF6b and pUBI-APX when cultured on delayed selection. TABLE 2: A comparison between the mortality rate of isolated embryos — cultivars Gamtoos-S (Dn7-) and Gamtoos-R (Dn7+) — bombarded with construct pUBI-GSTF6b when cultured on early selection medium. TABLE 3: Comparison between RNA concentration and total yield obtained after extraction using the different D. noxia tissue types. TABLE A1: Genes of relevance to D. noxia defence that were significantly up- and down regulated upon RWA infestation after normalization with MAS5, RMA, GCRMA, PLM and VSN. Indicated is the GenBank accession number, Affymetrix probe set ID and target description as well as functional category. Also indicated is gene expression. Red = up-regulated; green = down-regulated.


ii LIST OF FIGURES

FIGURE 1: Amplified fragment length polymorphism (AFLP) screening with Tugela, TugelaDN and 32 resistant and susceptible genotypes. Shown are the products after selective amplification with primer combination M-CTT/E-ACC. Where lanes 1-14 = susceptible F3-plants; lane 15 = Tugela (susceptible NIL/parent); lane 16 = TugelaDN (Dn1 resistant NIL/parent); lanes 17-29 = resistant F3-plants; lane 30 = SA1684 (Dn1 progenitor); lane 31 = SA2199 (Dn2 progenitor); lane 32 = lane 32 = SA463 (Dn5 progenitor); lane 33 = TugelaDn2 (Dn2 resistant NIL); ); lane 34 = TugelaDn5 (Dn5 resistant NIL); lane 35-36 = Tugela (susceptible NIL/parent), and lanes 37-38 = TugelaDN (Dn1 resistant NIL/parent. FIGURE 2: 8% Polyacrylamide gel depicting resistant samples amplified with the microsatellite marker Xgwm111 where L= 100 bp ladder. The arrow depicts the 200 bp mark above which the 210 bp polymorphic band of Xgwm111 can be seen. FIGURE 3: Maps drawn from the linkage groups derived from dataset 1. FIGURE 4: Maps drawn from the linkage groups derived from dataset 2. FIGURE 5: A map drawn from the linkage groups derived from dataset 3. FIGURE 6: Metaphase chromosomes stained with Giemsa and separated before being visualized in Acetocarmine under 10 000x magnification.

FIGURE 7: Fluorescent image of labelled chromosomes counterstained with Hoescht. Possible fluorescent signal indicated by the arrow. One is unable to distinguish between cellular debris and chromosomes in this image. FIGURE 8: Fluorescent image of labelled chromosomes. Possible fluorescent signal indicated by the arrow. Chromosomal structure can be better distinguished in this image. FIGURE 9: Immature embryos isolated from wheat inflorescence. (A) Represents inflorescence of cultivar Gamtoos-R (Dn7+) at the beginning of anthesis. (B) Shows the entire seed before the embryo has been isolated. (C) Representing an isolated embryo that is still intact 11 to 14 days after anthesis. FIGURE 10: Different stages of tissue culturing: (A) Callus tissue on induction A medium for 14 days. (B-D) Shows embryonic tissue on regeneration medium for 10 days; (B) and (C) represents embryonic tissue differentiating into plantlets, i.e. Gamtoos-S (Dn7-) and Gamtoos-R (Dn7+) cultivars respectively; while (D) shows non-differentiating calli; (E) Plantlet on shoot elongation medium for 14 days in a 90 mm pedri dish; (F) Plantlet on shoot elongation medium for 14–20 days in a tissue culture flask. FIGURE 11: Different stages of plantlets on shoot elongation medium: (A and B) Plant transformed with construct pUBi-GST; (C) Plant transformed with construct pUBI-APX; and (D) Control (tissue culture derived plant).


FIGURE 12: Harding off: (A) transformed Gamtoos-S (Dn7-) pUBi-GST; (B) tissue culture derived control Gamtoos-R (Dn7+); and (C) transformed Gamtoos-S (Dn7-) pUBi-APX. FIGURE 13: (A) Harding off: Left: tissue culture derived control Gamtoos-R (Dn7+), Right: transformed Gamtoos-S (Dn7-) pUBi-GST; (B) Harding off: Left: tissue culture derived control Gamtoos-R (Dn7+), Right: transformed Gamtoos-S (Dn7-) pUBi-APX. FIGURE 14: Relative APX expression in the transgenic Gamtoos-S (Dn7-) pUBi-APX wheat. FIGURE 15: Global migration of Diuraphis noxia from its endemic regions in the Fertile Crescent to become an invasive pest species in Africa and the Americas. (A) Development and migration of D. noxia biotypes in the USA from 1980 (US1) to the development of eight biotypes in 2008 (US2-US8). (B) Development and migration of South African D. noxia biotypes (SA1, SA2 and SA3) from 1978 to 2008 (From Botha 2013)1. FIGURE 16: Process of gland isolation. (A) Whole aphid body; (B) Severed head; (C) Removed glands still attached to stylet and other unwanted tissue; and (D) Isolated gland subunits.

FIGURE 17: Whole RWA protein extraction, with 25 g of SAM and SA1 protein separated on a 12% SDS-PAGE gel, stained with colloidal coomasie. Ladder: PageRuler Pre-stained Protein Ladder Plus (Fermentas).

FIGURE 18: Salivary gland extraction separated on a 12% SDS-PAGE gel, stained with colloidal coomasie. Ladder: SDS Molecular Weight Marker (Sigma-Aldrich).

FIGURE 19: Relative fold change in transcript abundance of unknown transcript COO2 and Endo-1,4-β-xylanase between different tissue types.

1 Botha A-M (2013) A coevolutionary conundrum: The arms race between Diuraphis noxia (Kurdmojov) a specialist

pest and its host Triticum aestivum (L.). Arthropod-Plant Interactions 7: 359-372.


1. INTRODUCTION

RATIONALE AND MOTIVATION The role of a more productive, profitable wheat system that fosters food security and generate local employment, raising local incomes (GDP) and thus alleviating poverty, must not be underestimated. The prevalence of pests and diseases put pressure on this crop, resulting in yield losses. While breeding of resistant cultivars adapted to South African conditions proved effective in the control of diseases and pests, it is time consuming and has certain limitations. For example, selection of varieties to ensure desirable agronomic traits mostly results in a narrow genetic base. Also, combining/pyramiding resistance genes in a specific line/cultivar usually result in prolonged durability, stabilising crop yields during epidemics, but is difficult to confirm. Marker-assisted selection (MAS) can aid in this process; and dramatically shorten the time scale and the efficacy of a specific breeding program. However, thus far only 20 % of loci tightly linked to traits on genetic maps were converted into useful markers. Useful markers can be defined by linkage; should be co-dominant (differentiate between homo- and heterozygous individuals), genetic background non-specific, and reproducible. To address this problem, we propose a multi-disciplinary approached to marker development applying modern breeding tools, biotechnology and plant genomics. Markers developed in the project are co-dominant, genetic background non-specific and reproducible and thus, can potentially contribute significantly toward efforts in breeding for resistance. The rationale for this project can be summarized as follows: 1. Cereal crops are an important food source in Southern Africa; 2. Exchange of valuable information for breeders in South Africa; 3. Insects and pathogens are continually appearing and evolving (species/new

races/biotypes); unless cultivars with durable resistance can be provided to the farming community, losses due to pests and diseases will continue and most likely increase;

4. Large amounts of money are spent annually by farmers on pesticides to manage pest and disease outbreaks, which is both expensive and bio-hazardous; and

5. Export markets are becoming increasingly aware of products with chemical residues. EMERGING SECTOR Wheat is the second most important field crop in South Africa and together with secondary processing industries provides a large number of job opportunities. South Africa has about 3 800 to 4 000 commercial wheat farmers providing work opportunities to about 28 000 people (WCT Strategy document, April 2004). Annual wheat production in South Africa far exceeds that of other SACU countries (2.4 million tones for 2004, FAO Food outlook 1, April 2004). In 2004, production in the rest of the SADC countries amounts to 473 000 tons with Zimbabwe (13%), Tanzania (4%) and Zambia (3%), these figures have changed since then. However, demand exceeds production and the industry has to import wheat. Since the country is a net importer of wheat, wheat prices in South Africa tends to be based on import parity (landed cost of international wheat in South Africa, including the wheat tariff) (WCT strategy document, April 2004). Since there is a need for better quality wheat and higher yielding crop in arid


environments, significant efforts are invested into the identification of genes that can confer stress tolerance during biotic stresses.

Besides producing improved varieties for the wheat farmers, there also exists a need for a trained/skilled workforce. Therefore, training of previously disadvantaged individuals to become specialists in the field of Biotechnology and Breeding and expressing an interest in the wheat industry is a focal point in the programme. Also, since the project aims to improve the drought tolerance and pest resistance of South African wheat cultivars, it should benefit the emerging sector, wheat milling and baking industry and economy in the medium to long-term.

WORKPLAN (2010-2013/2014): o Saturation of maps and map-based cloning of Dn1, Dn2 and Dn5. o Analysis of breeding lines from the SGI, PANAR and SENSAKO. o Confirmation of association of the genes with RWA resistance through mapping and via

viral induced gene silencing (VIGS). o Setting up of a high throughput system for mass screening of breeding lines. o Implementation of marker system into the different national breeding programmes. o Transfer of technology to ARC, CSIR or other testing facility of choice. Alternatively,

screening can be done at US on a per cost basis. Proposed duration of the project: 2010-2013/2014.


II. BUSINESS PLAN, WORKPLAN AND OUTPUT SUMMARY AS

PROPOSED IN THE 2010 APPLICATION

2.1 Saturation of maps and map-based cloning of Dn1, Dn2 and Dn5.

The objective in the mapping experiments is to confirm linkage to the trait of interest (insect/disease resistance) and determine chromosomal location. The segregating population was prepared and phenotypically evaluated after infestation with the RWA using the 1-10 scale. DNA of F3/4 plants, as well as Tugela, TugelaDN and rogenitor species (i.e., the donor SA1684, SA2199, SA643, etc.) were extracted. DNA quality of samples to be used for mapping was determined through PCR with primers amplifying a 16S Ribosomal fragment. This screening process yielded a mapping population of 571 samples from the F3/4 population segregating for Dn1 and originating from a cross between Tugela and TugelaDN.

Amplified Fragment Length Polymorphisms (AFLP) using the high throughput NenR Model 4300 DNA-Analyzer (LI-COR BioSciences, Lincoln, Nebraska) is currently being used for mapping of the population. To date, 26 primer combinations (M-CAA/E-ACC; M-CAA/E-ACG; M-CAC/E-ACT; M-CAG/E-ACA; M-CAG/E-ACC; M-CAT/E-AGC; M-CAT/E-AGG; M-CTA/E-AGC; M-CTA/E-AGG; M-CTC/E-AAC; M-CTC/E-AAG; M-CTC/E-ACA; M-CTG/E-AAC; M-CTG/E-AAG; M-CTG/E-ACA; M-CTT/E-AAC; M-CTT/E-AAG; M-CTT/E-ACA; M-CAA/E-AAC; M-CAA/E-ACA; M-CAC/E-AAC; M-CAC/E-ACA; M-CTT/E-ACC; M-CTT/E-ACG; M-CTG/E-ACT; M-CTG/E-AGC) were screened in 2011 with a subset of two resistant and two susceptible samples from the F3/4 population, as well as the resistant TugelaDN and susceptible Tugela parents (Figure 1).

FIGURE 1: Amplified fragment length polymorphism (AFLP) screening with Tugela, TugelaDN and 32 resistant and susceptible genotypes. Shown are the products after selective amplification with primer combination M-CTT/E-ACC. Where lanes 1-14 = susceptible F3-plants; lane 15 = Tugela (susceptible NIL/parent); lane 16 = TugelaDN (Dn1 resistant NIL/parent); lanes 17-29 = resistant F3-plants; lane 30 = SA1684 (Dn1 progenitor); lane 31 = SA2199 (Dn2 progenitor); lane 32 = lane 32 = SA463 (Dn5 progenitor); lane 33 = TugelaDn2 (Dn2 resistant NIL); ); lane 34 = TugelaDn5 (Dn5 resistant NIL); lane 35-36 = Tugela (susceptible NIL/parent), and lanes 37-38 = TugelaDN (Dn1 resistant NIL/parent).


Marker sets with the highest number of polymorphisms (M-CTT/E-ACC; M-CTG/E-ACT; M-CAG/E-ACA) were selected to screen the entire mapping population. Marker data was generated using SAGATM GT/MX Automated AFLP analysis software (LI-COR BioSciences, Lincoln, Nebraska) to size fragments and place them into marker bins (Figure 1). Microsatellite and expressed sequence tag (EST) markers Microsatellite (SSR) markers known to be closely linked to the Dn1 resistance gene were selected and applied for mapping of the population. Mapping of SSR Xgw111 was completed during 2012, with a second and third marker, SSR Xgw44 and SSH1 (EST) still under way. Marker data for Xgwm111 was generated manually by visualizing the presence or absence of bands on 8% Polyacrylamide gels stained with Ethidium Bromide (Figure 2). Additional microsatellites and EST markers were selected from the GrainGenes database (http://wheat.pw.usda.gov), as well as Affymetrix data available within our research group. These markers were screened across Tugela, TugelaDN as well as resistant and susceptible offspring. Only one of these markers, an EST called SSH1, was shown to be polymorphic in the mapping population. This marker, along with Xgwm44, was fluorescently labelled and is being used to screen the mapping population on a high throughput system, adding to the saturation of the genetic map.

FIGURE 2: 8% Polyacrylamide gel depicting resistant samples amplified with the microsatellite marker Xgwm111 where L= 100 bp ladder. The arrow depicts the 200 bp mark above which the 210bp polymorphic band of Xgwm111 can be seen.

Genetic/Linkage Mapping JoinMap software v 4.1 (Kyazma B.V 1995) was used to calculate linkage groups and generate recombination frequencies between markers for the AFLP primer combinations M-CTT/E-ACC; M-CAG/E-ACA; M-CTG/E-ACT; the microsatellite Xgwm111 and the Phenotype. An EST marker (29/30) from another project was also added. The data was subdivided into six different data sets varying in the inclusion or exclusion of the AFLP markers or using Xgwm111 as co dominant marker data instead of dominant. Each dataset was subjected to linkage grouping using Independent LOD, as well as Recombination Frequency with both parameters of Maximum Likelihood and Regression Mapping used in each instance. Maps were drawn up for three of the datasets (Dataset 1, 5 and 6).

http://wheat.pw.usda.gov/


Dataset 1 contained all AFLP markers, microsatellite Xgwm111 dominant data, EST 29/30 and the phenotype. Maps were drawn up for one linkage group made using the Independent LOD. This linkage group had a LOD score of 4 and consisted of 23 markers totalling 706 cM. Two more maps were drawn up for linkage groups made using Recombination Frequency. These maps had recombination frequencies of 0.25 and totalled 117.6 cM for 4 markers and 35.4 cM for 2 markers. Some consistencies were observed between these maps in that the phenotype mapped 35.4 cM from an AFLP marker, E-ACA-M-CAG-0126, under both Independence LOD and Recombination Frequency; and an AFLP marker, M-CTG-E-ACT-270 mapped at 30.4 cM from Xgwm111 under both Independence LOD and Recombination Frequency (Figure 3).

FIGURE 3: Maps drawn from the linkage groups derived from dataset 1. Dataset 5 contained all AFLP markers, microsatellite Xgwm111 codominant data, EST 29/30 and the phenotype. Maps were drawn up for two linkage groups made using the Independent LOD. These linkage groups had LOD scores of 4 and consisted of 22 markers totalling 728.5 cM and 4 markers totalling 10079.6 cM. Two more maps were drawn up for linkage groups made using Recombination Frequency. These maps had recombination frequencies of 0.25 and totalled 109 cM for 4 markers and 35.4 cM for 2 markers. Some consistencies were observed between these maps in that the phenotype mapped 35.4 cM from an AFLP marker, E-ACA-M-CAG-0126, under both Independence LOD and Recombination Frequency in three of the maps and an AFLP marker, M-CTG-E-ACT-270 mapped at 22.3 cM from Xgwm111 under both Independence LOD and Recombination Frequency in two of the maps (Figure 4).

FIGURE 4: Maps drawn from the linkage groups derived from dataset 2.


Dataset 6 contained microsatellite Xgwm111 dominant data, EST 29/30 and the phenotype. A map was drawn up for one linkage group made using the Independent LOD. This linkage group had a LOD score of 3 and consisted of 3 markers totalling 173.3 cM (Figure 5).

FIGURE 5: A map drawn from the linkage groups derived from dataset 3.

Discussion and Conclusion Microsatellite maps have been constructed in wheat with markers evenly distributed along chromosomes to provide good coverage of the genome (Roder et al. 1998)2. Dn1, Dn2, Dn5, Dn6 and Dnx are resistance genes either allelic or tightly linked in a cluster on chromosome 7D. These genes have been closely linked to a microsatellite marker, Xgwm111, at distances of roughly 3 cM by numerous authors (Miller et al. 2001; Liu et al. 2001, 2002; Heyns 2005)3. Another microsatellite marker, Xgwm437 was closely linked to Dn2 at 2.8 cM by Miller et al. (2001)4. Numerous RAPD and RFLP markers have also shown close linkage to the Dn resistance genes in literature, however these marker systems often prove difficult to reproduce or convert to a more stable system for screening purposes, as in the development of SCAR markers from RAPDs (Myburg et al. 1998)5. Apart from microsatellites, Ma et al. (1998)6 stated that the inclusion of other marker systems such as AFLP may help to obtain more closely linked markers.

2 Roder MS, et al. (1998) A Microsatellite Map of Wheat. Genetics 149: 2007-2023.

3 Miller CA, Altinkut A, Lapitan NLV (2001) A Microsatellite Marker for Tagging Dn2, a Wheat Gene Conferring

Resistance to the Russian Wheat Aphid. Crop Science 41: 1584-1589; Liu M, Smith CM, Gill S (2002) Identification

of microsatellite markers linked to Russian wheat aphid resistance genes Dn4 and Dn6. Theoretical and Applied

Genetics 104: 1042-1048; Liu XM, et al. (2001) Microsatellite markers linked to six Russian wheat aphid resistance

genes in wheat. Theoretical and Applied Genetics 102: 504-510; Liu XM, et al. (2005) Molecular Mapping and Allelic

Relationships of Russian Wheat Aphid Resistance Genes. Crop Science 45: 2273; Heyns IC (2005) Mapping of

chromosome arm 7DL of Triticum aestivum L. University of Stellenbosch. 4 Miller CA, Altinkut A, Lapitan NLV (2001) A Microsatellite Marker for Tagging Dn2, a Wheat Gene Conferring

Resistance to the Russian Wheat Aphid. Crop Science 41: 1584-1589. 5 Myburg AA, et al. (1998) Development of RAPD and SCAR markers linked to the Russian wheat aphid resistance

gene Dn2 in wheat. Theoretical and Applied Genetics 96: 1162-1169. 6 Ma Z, et al. (1998) Genetic mapping of Russian wheat aphid resistance genes Dn2 and Dn4 in wheat. Genome 41:

303-306.


Generally an effective marker must be linked to the gene at a distance of less than 10 cM (Miller et al. 2001).7 Apart from the difficulty in obtaining markers closely linked to the Dn resistance genes, there is much controversy as to the chromosomal location of the genes with regards to the short or the long arm of chromosome 7D. Werner et al. (1992)8 showed that the physically or cytologically longer arm of 7D is actually the genetically shorter arm as it is homoeologous to 7AS and 7BS. In some instances, authors working with ditelosomic lines did not even have Dt7DL lines, they were working with Dt7DS. This was established using chromosome banding. Dn1, Dn2 and Dn5 have thus been wrongly mapped to 7DL by several authors (Liu et al. 2005)9. This highlighted the significant role cytological techniques can play in mapping studies. This project, to date, has yielded marker data for a mapping population of 373 samples segregating for the Dn1 resistance gene. The discrepancy between this figure and the stated 571 individuals in the original sampling population is due to the fact that not all samples yielded results for each and every marker tested. The marker data includes three AFLP primer combinations and a microsatellite marker known to segregate with the resistance gene. Preliminary genetic maps were constructed in April 2013 varying in the mapping parameters, sample sets and loci (Figure 3, Figure 4 and Figure 5). The phenotypic scores of samples were used along with the microsatellite marker, Xgwm111, as anchors since the Dn1 gene is known to be closely linked to this marker. The closest linkage obtained between the Dn1 resistance gene and Xgwm111 was 173.3 cM (Figure 5) when the dataset was decreased to exclude the AFLP markers. An AFLP marker, E-ACA-M-CAG-0126, was found with closer linkage to Dn1 though. This marker consistently mapped at 35.4 cM from Dn1 in all datasets tested (Figure 3 and Figure 4). An AFLP marker, M-CTG-E-ACT-270, was also found consistently associated with Xgwm111 at 22 – 30 cM depending on the dataset tested (Figure 3, Figure 4). In addition to the markers already mapped, twenty additional microsatellites and ESTs (GrainGenes database (http://wheat.pw.usda.gov)) were screened in March 2013 for polymorphism between resistant and susceptible plants. Only two markers proved informative: a microsatellite, Xgwm44, with known linkage to the Dn resistance genes in other populations and an EST, AMO000SSHL1, derived from previous work done by the Cereal Genomics group. These two markers will be screened across the entire mapping population at the end of July 2013 and added to the genetic map. Currently, markers derived from Affymetrix data compiled by the Cereal Genomics group, are also being screened for polymorphism in resistant and susceptible plants. Should any of these markers prove informative, then they will be screened across the entire mapping population and added to the genetic map.

Physical mapping To enable for the physical mapping of RWA resistance genes Dn1, Dn2 and Dn5, we are developing a novel approach to map-based cloning, based on the excision of the chromosome

7 Miller CA, Altinkut A, Lapitan NLV (2001) A Microsatellite Marker for Tagging Dn2, a Wheat Gene Conferring

Resistance to the Russian Wheat Aphid. Crop Science 41, pp.1584-1589. 8 Werner JE, Endo TR, Gill BS (1992). Toward a cytogenetically based physical map of the wheat genome. Proc Natl

Acad Sci U S A. 1992 December 1; 89(23): 11307–11311. 9 Liu XM, et al. (2005) Molecular Mapping and Allelic Relationships of Russian Wheat Aphid Resistance Genes. Crop

Science 45, p. 2273


arm and then sequencing the respective arm. To do this, we had to optimize our cytological techniques.

Cytology: chromosome squashes, and Fluorescent in Situ Hybridization Chromosome suspensions were prepared from root tips of Tugela and TugelaDN seedlings according to Vrana et al. (2000)10. Chromosome suspensions were stained with Giemsa in order to validate efficacy of the protocol (Figure 6). SSR markers (Xgwm111 and Xgwm44) known to be linked to the Dn1 resistance gene on chromosome 7D were fluorescently labelled using a 3' EndTag DNA Labeling System (Vector Laboratories; MB-9002) with Texas Red Maleimide Thiol reactive label (SP-1505; Vector Laboratories). In addition, telomeric probes were used. These probes were labelled using Fast-Tag DNA Labeling System (Vector Laboratories; MB-9002) with Fluorescein Maleimide Thiol reactive label (SP-1505; Vector Laboratories).

FIGURE 6: Metaphase chromosomes stained with Giemsa and separated before being visualized in Acetocarmine under 10 000x magnification. Fluorescent probes were subsequently hybridized to chromosome spreads prepared from Tugela and TugelaDN root tips and visualized with the help of the Physiology Department at the University of Stellenbosch (Figures 7 and 8).

10

Vrána J, Kubaláková M, Šimková H, Cíhalíková J, Lysák MA, et al. (2000) Flow sorting of mitotic

chromosomes in common wheat (Triticum aestivum L.). Genetics 156: 2033–2041.

Figure 4: Map 1 depicting a 9-marker map generated from linkage groups as discussed above. Marker no 126 is polymorphic.


FIGURE 7: Fluorescent image of labelled chromosomes counterstained with Hoescht. Possible fluorescent signal indicated by the arrow. One is unable to distinguish between cellular debris and chromosomes in this image.

FIGURE 8: Fluorescent image of labelled chromosomes. Possible fluorescent signal indicated by the arrow. Chromosomal structure can be better distinguished in this image.


Discussion and conclusion With a saturated genetic map in place, physical mapping can be carried out through map based cloning and chromosome walking. The size and complexity of the wheat genome11 makes this a monumental task which is why this project aims to isolate chromosome 7D through Fluorescent in Situ Hybridization and microdissection in order to decrease the background noise and scale down on the complexity of the genome. Chromosome suspensions (Figure 6) have proven to be good starting material for FISH.

Microsatellite markers (Xgwm111 and Xgwm44) labelled with TexasRed dye has been hybridized to chromosome suspensions placed on microscope slides (Figure 7 and Figure 8). Results show promise as red fluorescence is clearly visible on the blue background of the Hoescht stain. Additional probes for chromosome 7D were investigated to serve as controls and to help distinguish between chromosomes and cellular debris. Telomeric probes labelled with Fluorescein dye were incorporated into the FISH experiments in order to tag chromosome ends and aid in the differentiation from cellular debris. It is estimated that the microdissection of chromosome 7D will be completed between July and September 2013 followed by sequencing of the chromosome in October 2013. This will then be followed by the bioinformatic analyses of the obtained sequence data which include contiq assembly, sequence alignment and gene calling.

Envisage completion date: March 2013

2.2 Analysis of breeding lines for Industry.

RWA virulence screening for industry Due to the lack of suitable quarantine facilities we were unable to screen significant numbers of line with the highly virulent SAM Russian aphid biotype. However, facilities for this activity near completion and we will soon be able to commence with this activity at the Welgevallen Experimental Farm, Stellenbosch University.

2.3 Confirmation of association of genes with RWA resistance: Transformation of selected wheat cultivars to elucidate the function of Ascorbate peroxidase (APX) and Glutathione transferase (GST).

For many years increased resistance was bred using non-transgenic strategies such as marker assisted selection. Although this strategy has proven to work well, it is short lived due to the emergence of new aphid biotypes. With the ongoing evolutionary battle of adaptation and counter adaptation that exists between plant resistance (wheat) and aphid virulence (Russian Wheat Aphid) aphids overcome plant resistance, putting pressure on the plant to withstand invasion (Botha 2013)12. An alternative to this approach is transgenics whereby a single gene

11

Brenchley R, et al. (2012) Analysis of the bread wheat genome using whole-genome shotgun sequencing. Nature,

491: 705-710. Available at: http://dx.doi.org/10.1038/nature11650; Gill BS, et al. (2004) Workshop Report A

Workshop Report on Wheat Genome Sequencing: International Genome Research on Wheat Consortium. Genetics

168: 1087-1096. 12 Botha A-M (2013) A coevolutionary conundrum: The arms race between Diuraphis noxia (Kurdmojov) a specialist

pest and its host Triticum aestivum (L.). Arthropod-Plant Interactions 7: 359-372.

http://dx.doi.org/10.1038/nature11650


can be overexpressed to increase resistance of a particular plant defence response (Gurr & Rushton 2005)13. Genetic manupaltion revolutionised cell biology, enabling researches to investigate genetically controlled traits (Jones et al. 2005)14. The practical application of this technique is fundamental in understanding the molecular basis of plant-pathogen/pest interactions to improve agronomic important crops, this short-cuts the conventional breeding process which ranges between 6-10 years and thus the process can now reduced by 2 years (He et al. 2001)15.

Tissue culture and transformation via particle bombardment In order to obtain plant material susceptable to particle bombarbment, embryogenic tissue was required. For this, a bread wheat tissue culture system had to be put in place; where after embryonic tissue was bombarded and the transformation efficiency assessed (Tables 1 and 2). It can be seen from table 1, when comparing the hormones added to the selection mediums, that with the Zeatin-rich medium, two plantlets were obtained with a transformation efficiency of 3%. However, no plantlets were obtained with the BAP-rich medium. On the other hand, embryogenic tissue bombarded with the silencing construct pUBI-APX instead of pUBi-GST, produced a plantlet on the BAP-rich medium with a transformation efficiency of 2%. When comparing delayed selection against early selection, it was found that only bombarded tissue which underwent the delayed selection process generated plantlets. When comparing between the hormones under these conditions, we found that medium containing Zeatin (3%) gave higher transformation efficiency than the BAP-containing medium (2%). Thus it can be concluded that the delayed selection process together with the addition of Zeatin is more preferable for culturing bombarded tissues (Table 2). In order to test the association of the genes with RWA resistance in wheat, a tissue culture platform enabling transformation was required. For this, immature seed (Figure 9B) were obtained from 11 to 16 day old anthesis (Figure 9A). Gamtoos-R (Dn7+) and Gamtoos-S (Dn7-) cultivars were used to isolate immature embryos (Figure 9C). Embryos were pre-cultured on Induction A medium for 4 to 6 days in a dark growth room at 26°C. After 6 days of culturing in the dark, embryonic tissue were bombarded with pUBI constructs containing the APX and GSTF6b inserts respectively.

13

Gurr SJ, Rushton PJ (2005) Engineering plants with increased disease resistance: how are we going to express it?

Trends in Biotechnology 23: 283-290. 14

Jones HD, Doherty A, Wu H (2005) Review of methodologies and a protocol for the Agrobacterium-mediated

transformation of wheat. Plant Methods 1: 1-9. 15

He H, Deng W, Cassel MJ, Lucas JN (2001) Fluorescence in situ hybridization of metaphase chromosomes in

suspension. International Journal of Radiation Biology 77: 787–795

TABLE 1: Comparing mortality rate of isolated embryos — cultivars Gamtoos-S (Dn7-) and Gamtoos-R (Dn7+) — bombarded with constructs pUBI-GSTF6b and pUBI-APX when cultured on delayed selection.

TABLE 2: A comparison between the mortality rate of isolated embryos — cultivars Gamtoos-S (Dn7-) and Gamtoos-R (Dn7+) — bombarded with construct pUBI-GSTF6b when cultured on early selection medium.

Cultivar # Embryos isolated

Gene Construct

Regeneration Mediaa

Shoot Elongation Mediaa

Type of Selection

Selection Hormone

% Efficiency

- + - +

Gamtoos-S (Dn7-) 27 pUBI-GST 22 5 4 1 Delayed Zeatin 3% Gamtoos-S (Dn7-) 38 pUBI-GST 32 6 6 - Delayed BAP 0% Gamtoos-S (Dn7-) 22 pUBI-GST 19 3 3 0 Delayed BAP 0% Gamtoos-R (Dn7+) 30 pUBI-GST 21 9 8 1 Delayed Zeatin 3% Gamtoos-R (Dn7+) 44 pUBI-GST 44 - - - Delayed BAP 0% Gamtoos-R (Dn7+) 20 pUBI-GST 20 - - - Delayed BAP 0%

Total # plantlets obtained

181 158 23 21 2

Gamtoos-S (Dn7-) 50 pUBI-APX 38 12 12 0 Delayed BAP 0% Gamtoos-R (Dn7+) 47 pUBI-APX 41 6 5 1 Delayed BAP 2%


97 79 18 17 1

a. Where “–“ denotes no proliferation and “+” denotes proliferation on media

Cultivar Isolated Embryos

Gene Construct

Regeneration Mediaa

Shoot Elongation Mediaa

Type of Selection

Selection Hormone

% Efficiency

- + - +

Gamtoos-S (Dn7-) 40 pUBI-GST 37 3 3 0 Early BAP 0% Gamtoos-R (Dn7+) 53 pUBI-GST 36 17 17 0 Early BAP 0%


93 73 20 20 0

a. Where “–“ denotes no proliferation and “+” denotes proliferation on media

FIGURE 9: Immature embryos isolated from wheat inflorescence. (A) Represents inflorescence of cultivar Gamtoos-R (Dn7+) at the beginning of anthesis. (B) Shows the entire seed before the embryo has been isolated. (C) Representing an isolated embryo that is still intact 11 to 14 days after anthesis. Bombarded tissue (Figure 10A) underwent a series of subculturing on different mediums with various supplements. After bombardment embryogenic tissue were co-cultured on induction medium for period of 14 days in a dark growth room. Fourteen days later embryogenic tissue was tranferred to the regeneration medium in a 20°C light growth room covered with protective screens to avoid direct light. After 10 days on regeneration medium embryogenic tissue showing first leaf stage (as shown in figure 10C-F) were subcultured to shoot initiation medium for a period of 10 days. Followed by culturing the embryogenic tissue with its shoot to shoot elongation medium for a period of 14-20 days with direct light in a 26°C growth room. The medium contained the same supplements as that of the shoot initiation medium expect no geneticin was added to this medium.

FIGURE 10: Different stages of tissue culturing: (A) Callus tissue on induction A medium for 14 days. (B-D) Shows embryonic tissue on regeneration medium for 10 days; (B) and (C) represents embryonic tissue differentiating into plantlets, i.e. Gamtoos-S (Dn7-) and Gamtoos-R (Dn7+) cultivars respectively; while (D) shows non-differentiating calli; (E) Plantlet on shoot elongation medium for 14 days in a 90 mm pedri dish; (F) Plantlet on shoot elongation medium for 14–20 days in a tissue culture flask.

A B C


The next step entailed transfer of the surviving plantlets which generating roots (Figure 11A-D) on shoot elongation media to soil (Figure 12). The soil consisted of sand, palm peat/autoclaved potting soil and vermiculite. They were placed in a greenhouse with protective screens preventing direct sunlight exposure for a period of three days as shown in figure 4.

FIGURE 11: Different stages of plantlets on shoot elongation medium: (A and B) Plant transformed with construct pUBi-GST; (C) Plant transformed with construct pUBI-APX; and (D) Control (tissue culture derived plant).

In total, 371 immature embryos were isolated and bombarded from which 310 died on the regeneration medium and 58 died on the shoot elongation medium. From a starting total of 371 bombarded embryogenic tissues, only three were able to withstand the entire selection process. These putative transgenic plantlets are shown in figure 11.

FIGURE 12: Harding off: (A) transformed Gamtoos-S (Dn7-) pUBi-GST; (B) tissue culture derived control Gamtoos-R (Dn7+); and (C) transformed Gamtoos-S (Dn7-) pUBi-APX.

After a four week-period of harding off significant height differences between the control and transformed plants were observed. The control had a height of 30 cm (Figures 12 and 13) and the putative GSTF6b plant was only 14 cm long. This difference in height was also observed between the control and the putative APX transgenic plant.


FIGURE 13: (A) Harding off: Left: tissue culture derived control Gamtoos-R (Dn7+), Right: transformed Gamtoos-S (Dn7-) pUBi-GST; (B) Harding off: Left: tissue culture derived control Gamtoos-R (Dn7+), Right: transformed Gamtoos-S (Dn7-) pUBi-APX. To validate if these plants were transgenic, the next step was then to test for the presence of the transgene in the respective plants. To enable for this confirmation, qRT-PCR analyses were performed to test the expression levels of the transgenes (Figure 14). When comparing the relative APX expression measured in the control Gamtoos-S (Dn7-) to the transformed Gamtoos-S (Dn7-) pUBi-APX, the control had a relative APX expression level of 1.05 while that of the transformed Gamtoos-S (Dn7-) pUBi-APX plant was only 0.52, showing a reducing of approximately 50%, implying partial silencing in the transgenic Gamtoos-S (Dn7-) pUBi-APX.

Genotypes tested

Re

lative

AP

X e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

APX

Tis

sue c

ulture

d

Gam

toos-R

(D

n7

+)

Gam

toos-R

(D

n7

+)

Gam

toos-S

(D

n7

- )

Gam

toos-S

(D

n7

- )pU

Bi-

AP

X

Gam

toos-S

(D

n7

- )pU

Bi-

GS

T

FIGURE 14: Relative APX expression in the transgenic Gamtoos-S (Dn7-) pUBi-APX wheat.


Discussion and conclusion The entire process of transfornation, i.e. from tissue culture establisment to hardening off of transgenic plantlets is very laborious and time consuming. The success rate is also low (2-3 %). Even though many researches attempted transformation of bread wheat, the reported average transformation efficiencies remained low (1-5 %) (Abdul et al. 2010)16. This is mainly ascribed to bread wheat’s large genome size and the fact that wheat is a monocot, and thus reculcitrant to Agrobacterium transfromation. In our study, we succeded in the establishment of a tissue culture and transfromation platform and produced three putative transgenic plants. These were succesfully harden off and is rapidly growing. Using qPCR, the partial silencing was validated in the transformed pUBi-APX plant. The next step, is to infest these plants with aphids and then to measure the expression levels of APX using qPCR.

In conclusion, even though our obtained transformation efficiency is only between 2-3 % - it is comparable with that reported in literature. We also succeeded in producing transgenic plants within our first attempts.

2.4 Setting up of a high throughput system for mass screening of breeding lines and implementation of marker system into the different national breeding programmes

The list of defense-related genes that are associated with the RWA resistance in Dn1, Dn2 and Dn5-containing genotypes are presented in Table A1 (Appendix). These can be used to screen breeding material during MAS, however the costs involved is still unacceptably high and these are now screened for the presence of single nucleotide polymorphisms (SNPs) in order to obtain suitable markers.

2.5 Russian wheat aphid Diuraphis noxia Kurdjumov (Aphididae), generally known as the Russian wheat aphid (RWA), is a small, yellow-green or grey-green elongated (1.4-2.3 mm) phloem feeding insect with a host preference that includes cereal grasses, favouring mostly wheat and barley. It is suggested that RWA coevolved with triticale in the Fertile Crescent, and then distributed from western Asia to Africa17. The first RWA sighting in South Africa was reported in 1978 from which the aphid then spread to South America and Mexico and finally arriving in the USA and Canada in 1986 (Smith et al. 1992; Smith 2009)18.

16

Abdul R, Ma Z, Wang H (2010) Genetic Transformation of Wheat (Triticum aestivum L.): A Review. Triticeae

Genomics and Genetics 1(2): 1-7. 17

Botha A-M (2013) A coevolutionary conundrum: The arms race between Diuraphis noxia (Kurdmojov) a specialist

pest and its host Triticum aestivum (L.). Arthropod-Plant Interactions 7: 359-372. 18

Smith CM (2009) Global phylogenetics of an invasive species: Evidence for multiple invasions into North America.

Joint meeting of the Southwestern Branch of the Entomological Society of America and WERA066 (Western

Extension/Education Research Activity), February 2009, Stillwater, Oklahoma; Smith CM, Schotzko DJ, Zemetra RS,

Souza EJ (1992) Categories of resistance in plant introductions of wheat resistant to the Russian wheat aphid

(Homoptera: Aphididae). J Econ Entomol 85: 1480-1484


1978

1980

Major wheat producing areas

Diuraphis noxia’s endemic distribution

Duiraphis noxia global distribution as invasive pest species

1992

1986

2003-2008

US2-US8

SA1

SA2 SA3

2006-2008

SA2-SA3

A

B

FIGURE 15: Global migration of Diuraphis noxia from its endemic regions in the Fertile Crescent to become an invasive pest species in Africa and the Americas. (A) Development and migration of D. noxia biotypes in the USA from 1980 (US1) to the development of eight biotypes in 2008 (US2-US8). (B) Development and migration of South African D. noxia biotypes (SA1, SA2 and SA3) from 1978 to 2008 (From Botha 2013)19.

The economic losses attributed to this pest was estimated at around $893 million in the United States from 1987 to 1993 (Morrison and Pears 1998)20, after which these estimates increased significantly following 2003, with the development of many new RWA biotypes in the US (Botha et al., 201021). Currently, it is estimated that there are more than 20 RWA biotypes in the US alone (personal communication, Dr. Gary Puterka, USDA-ARS, Stillwater). In South Africa, we presently have three RWA biotypes (i.e., SA1, SA2 and SA3) and a highly virulent laboratory developed (mutant) RWA biotype (SAM). In South Africa, the “native” RWAs are distributed throughout the major wheat producing areas (i.e., Eastern and South-western Free State and Western Cape) and is even believed to be present in the smaller wheat producing areas (Burger and Botha, unpublished results). Although RWA has the ability to reproduce sexually and asexually, most instances of new introductions are believed to have happened by parthenogenesis (i.e., asexual reproduction). In fact, in South Africa, unlike other countries, RWA males have never been found. The absence of RWA males eliminates sexual recombination as a source of genetic variation, which explains the limited observed sequence variation between geographically

19 Botha A-M (2013) A coevolutionary conundrum: The arms race between Diuraphis noxia (Kurdmojov) a specialist

pest and its host Triticum aestivum (L.). Arthropod-Plant Interactions 7: 359-372. 20 Morrison WP, Pears FB (1998) Response model concept and economic impact, pp. 1-D11. In S S Quisenberry and F

B Peairs [eds.], A response model for an introduced pest - the Russian wheat aphid. Thomas Say Publications in

Entomology. Entomological Society of America, Lanham, MD. 21 Botha A-M, Swanevelder ZH, Lapitan NLV (2010) Transcript profiling of wheat genes expressed during feeding by

two different biotypes of Diuraphis noxia. Journal of Environmental Entomology 39: 1206-1231.


isolated RWA populations (Lapitan et al. 2007; Sufran et al. 200722). However, RWA populations from different geographically regions still express varying levels of virulence (Puterka et al. 1993; Smith et al. 199223). Virulence is the measurement of the ability of an aphid to overcome host resistance. Aphid populations from different geographical regions may also express different levels of virulence and are then classified as “biotypes”. Biotypes are intraspecific classifications based on biological, rather than morphological characteristics (Shufran et al. 200724) and the biotypic status of RWA is thus solely based on the phenotypic response of the plant as a result of the aphid’s feeding. Therefore, a new RWA biotype is a population, independent of geographic location, that is able to injure a cultivated plant containing a specific gene(s), that was previously resistant to known aphid populations (Basky 200325). Clearly, the development of new RWA biotypes enable for feeding on non-preference host varieties, and suggests towards an evolutionary adaptation to the biotic stresses preset by humans. This raises the question on how new RWA biotypes develop in such short time, if parthenogenesis is the only method of reproduction.

Sequencing of the Diuraphis noxia genome Diuraphis noxia were obtained from different locations across the globe to obtain a representative sample for genomic DNA sequencing (i.e., North America – US Biotypes 1-8; South America – Argentinia (Argentian Biotypes 1 and 2); European (Czech Biotypes); Asia (Syrian Biotype) and from Africa (SA Biotypes 1 to 3 and SAM). This was done to capture the diversity of global D. noxia populations (Figure 15) in order to understand the biotypification phenomenon. DNA was extracted from all the samples. The pooled sample and an individual sample were sequenced, for the first we used the Solid and Titanium sequencing platforms, for the latter we used the Illumina, IonTorrant and Titanium sequencing platforms. Currently we are conducting the bioinformatics analyses.

Transcriptome and proteome of RWA Biotypes When feeding the RWA secretes a proteinaceous salivary sheath that lines the stylet’s path, and also watery saliva that contains several enzymes, proteins and amino acids. The secretion of saliva is very important to the aphid’s feeding process, but it also contains certain bioactive

22 Lapitan NLV, Li Y-C, Walters RSG, Peng Y, Peairs FB, Botha A-M (2007) Limited nuclear and mitochondrial DNA

variation among Russian wheat aphid (Diuraphis noxia) biotypes from the United States and Africa, American

Entomological Society, San Diego, December 9-12. American Entomological Society, San Diego; Shufran KA,

Kirkman LR, Puterka GJ (2007) Absence of mitochondrial DNA sequence variation in Russian wheat aphid

(Hemiptera: Aphididae) populations consistent with a single introduction into United States. Journal of the Kansas

Entomological Society 80: 319-326. 23 Smith CM, Schotzko DJ, Zemetra RS, Souza EJ (1992) Categories of resistance in plant introductions of wheat

resistant to the Russian wheat aphid (Homoptera: Aphididae). Journal of Economic Entomology 85: 1480-1484;

Puterka GD, Black IV WC, Steiner WM, Burton RK (1993) Genetic variation and phylogenetic relationships among

worldwide collections of the Russian wheat aphid, Diuraphis noxia (Mordvilko), inferred from allozyme and RAPD-

PCR markers. Heredity 70: 604-618. 24 Shufran KA, Kirkman LR, Puterka GJ (2007) Absence of mitochondrial DNA sequence variation in Russian wheat

aphid (Hemiptera: Aphididae) populations consistent with a single introduction into United States. Journal of the

Kansas Entomological Society 80: 319-326. 25 Basky Z (2003) Biotypic and pest status differences between Hungarian and South African populations of Russian

wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae). Pest Management Science 59: 1152−1158.


components that can induce the plant’s defence response26. The two types of saliva secreted, have different compositions, and this has been a focus of investigation to elucidate their function and also understand plant-aphid interactions. Miles (1999)27 proposed that the aphid’s saliva contain an eliciting agent(s) and that it acts in the gene-for-gene model, later it was discovered this elicitor is of protein composition28. Elicitors are defined as molecules able to induce physiological and/or biochemical responses associated with the expression of resistance29. Even with recent progress no specific elicitors have been found to date30. One of the problems with current studies is that neither enzymatic activity of these salivary enzymes nor how the aphids are able to manipulate the plant’s defense is well understood. To be able to identify proteins that are important in the aphid’s feeding process one would require a technique that specifically enriches for these proteins. Samples required for accurate aphid salivary proteome analysis can be collected in one of two ways, namely collection of secreted saliva from artificial feeding media or by dissecting out the salivary gland. There are some drawbacks to collecting secreted saliva, since it doesn’t capture all the salivary proteins present in the gland. There is also the concern that the aphid may have a varying salivary protein composition based on its diet, and since it’s not a natural diet the results may not be accurate.

The second approach to study the aphid’s salivary proteome is to dissect out the salivary glands or use the head as a whole. These salivary glands are microscopic, and are composed of two primary glands and two accessory glands. One primary gland consists of only eight secretory cells. Salivary gland excision is the preferred technique since the salivary gland on its own delivers a protein sample of reduced complexity and more relevance compared to the head or body as the starting material. Furthermore a transcriptomics approach can also be used with the enriched sample to target salivary proteins being expressed. Here we report on the development of a novel technique to efficiently excise salivary glands out of the RWA through microdissection. Furthermore we show that these collected salivary glands can be used for accurate proteomic and transcriptomic studies. Protein were isolated from the glands and analyzed successfully with tandem mass spectrometry analysis. Furthermore salivary gland specific mRNA was isolated from the glands, and qPCR performed on these are more enriched compared to using the whole body as your starting material.

We developed a novel technique to isolate the RWA salivary gland through microdissection. The technique used is relatively fast to perform on a single RWA, and isn’t difficult to master. In this pilot study, we collected 206 glands per biotype (RWA SAM and RWA SA1) — gland(s) refers to the two primary and two accessory glands) for the proteomics part (Figure 16).

26

Cardoza YJ, Reidy-Crofts J, Edwards OR (2007) Differential interand intra-specific defense induction in Lupinus by

Myzus persicae feeding. Entomol. Exp. Appl. 117: 155–163. 27Miles PW (1999) Aphid saliva. Biol. Rev. 74: 41-85. 28 Lapitan NLV, Li YC, Peng J, Botha AM (2007) Fractionated extracts of Russian Wheat Aphid Eliciting defence

responses in wheat. Journal of Economic Entomology. 100: 990-999; Miles PW (1999) Aphid saliva. Biol. Rev. 74:

41-85; Brigham DL (1992) Chemical ecology of the Russian wheat aphid: host selection and phytotoxic effects. M.S.

thesis, Colorado State University, Fort Collins, CO. 29

Kogel G, Beiman B, Reisener HJ, Kogel KH (1988) A single glycoprotein from Puccinia graminis f. sp. tritici cell

walls elicits the hypersensitive response in wheat. Physiol Mol Plant Pathol. 33: 173-185. 30

Harmel N, Letocart E, Cherqui A, Giodanengo P, Mazzucchelli G, Guillonneau F, De Pauw E, Haubruge E, Francis

F (2008) Identification of aphid salivary proteins: a proteomic investigation of Myzus persicae. Insect Molecular

Biology. 17: 165-174.


FIGURE 16: Process of gland isolation. (A) Whole aphid body; (B) Severed head; (C) Removed glands still attached to stylet and other unwanted tissue; and (D) Isolated gland subunits.

For the proteomics approach the separation and visualization of the whole body protein extraction yielded distinct protein bands (Figure 17). Similarly the separation of salivary gland specific proteins also resulted in distinct bands (Figure 18). In this comparative study there were almost no difference in the number of bands seen for RWA SAM and RWA SA1 (Figure 18). Distinct bands can be seen above 42 kDa, but below this point the resolution was too low to accurately identify bands, the concentration of these proteins were most likely to low to accurately produce a band. Comparing the two extractions with one another, there isn’t a major difference in the amount of protein bands observed. The protein bands observed in the whole body extraction can occur in any part of the RWA body, while the bands observed in the salivary gland extraction is gland specific. The enrichment of the sample makes the resulting extraction more specific for salivary proteins, making it more accurate compared to the whole body protein isolation. Rare proteins have a higher abundance in this enriched sample, whereas in the whole body extraction these proteins may be lost because of the abundance of other proteins.

FIGURE 17: Whole RWA protein extraction, with 25 g of SAM and SA1 protein separated on a 12% SDS-PAGE gel, stained with colloidal coomasie. Ladder: PageRuler Pre-stained Protein Ladder Plus (Fermentas).


FIGURE 18: Salivary gland extraction separated on a 12% SDS-PAGE gel, stained with colloidal coomasie. Ladder: SDS Molecular Weight Marker (Sigma-Aldrich).

After a MS/MS analysis of the salivary gland protein extraction for both SAM and SA1, a total of 132 different peptides were obtained (data not shown) from SAM and only 114 were obtained from SA1. SAM had 56 unique peptides, SA1 53, and they share 76. This served only as a survey of the proteins present, and not a census, since this pilot study was not done in triplicate. It does however indicate that this technique is sensitive enough to detect many peptides, even if the concentration of the proteins in gel were low. Furthermore we know that all these peptides identified occur in the salivary gland, and the peptides identified that are hypothetical proteins, or has unknown functions can be further analysed. For the transcriptomics part we collected 250 salivary glands from US biotype 2 through microdissection. TABLE 3: Comparison between RNA concentration and total yield obtained after extraction using the different D. noxia tissue types.

Sample ID/Tissue type ng/µL A260 260/280 260/230 Constant Yield

Salivary Gland 31.78 0.79 1.66 0.14 40.00 1.59 µg

Whole Body 480.90 12.02 2.06 1.31 40.00 24.05 µg

From Table 3 it is evident that that the total RNA yield was much higher in the whole body compared to the salivary gland. This is expected since the glands are a much smaller sample and would therefore not contain as much RNA. However, to demonstrate the enrichment of low abundant transcript after gland extraction, qPCRs were conducted testing the abundance of unknown transcript COO2 and Endo-1,4-β-xylanase between different tissue types (Figure 19).


Diuraphis noxia tissue type

Re

lative

exp

ressio

n

0

100

200

300

400

500

COO2

Endo-1,4-xylanase

Whole body Salivary glands

FIGURE 19: Relative fold change in transcript abundance of unknown transcript COO2 and Endo-1,4-β-xylanase between different tissue types. The salivary glands of RWA are highly enriched for both transcripts. The unknown C002 transcript exhibited a 371.174-fold increase in abundance in salivary glands as compared to the whole aphid. RWA salivary glands are enriched 71.814-fold for endo-1,4-beta-xylanase transcripts (Figure 19). This result indicate that the enriched sample is more specific, meaning it is enriched for all the salivary specific transcripts, making it more accurate to detect rare transcripts compared to using the whole body where these transcripts may be lost in the overall sample.

Conclusion Here we have shown that it is possible to efficiently excise RWA salivary glands, to enrich the sample. The sample has reduced complexity and is of more biological relevance compared to the use of other tissue samples for the same purpose. The excision process is easy to master and it is cost efficient. In this pilot study we successfully did a proteomics analysis, and a transcriptomics analysis. We showed for both these approaches the gland sample is much more enriched. When dealing with aphid-plant feeding interactions this method will provide a great overall compilation of the proteins and transcripts that are present in the salivary glands. This serves as an excellent starting point to elucidate RWA-wheat interactions.

3. ACKNOWLEDGEMENTS The project manager and collaborators are expressing their sincere gratitude to the SGI for provision of genetic material. They would specifically like to thank Dr. Christelle van de Vuyver and Dr. Astrid Jankielsohn for their collaborations, as well as the international consortium members for their contributions.

4. APPENDIX

TABLE A1: Genes of relevance to D. noxia defence that were significantly up- and down regulated upon RWA infestation after normalization with MAS5, RMA, GCRMA, PLM and VSN. Indicated is the GenBank accession number, Affymetrix probe set ID and target description as well as functional category. Also indicated is gene expression. Red = up-regulated; green = down-regulated.

Genbank accession Affy ID Gene description

Genotypes

Tugela TugelaDN TugelaDn2 TugelaDn5

Photosynthesis and chloroplast related

BQ171501 Ta.14118.1.A1_at Transcribed sequence with weak similarity to protein pir:T51328 (A.thaliana) T51328 transcription initiation factor sigma5, plastid -specific (imported) - Arabidopsis thaliana

5.832137466 6.384730355 5.396705765 4.248990073

CA744411 TaAffx.104814.1.S1_at Transcribed sequence with weak similarity to protein pir:T51328 (A.thaliana) T51328 transcription initiation factor sigma5, plastid -specific (imported) - Arabidopsis thaliana

8.463657177 8.727875032 8.254216652 2.808351696

CK211804 Ta.22984.1.S1_x_at Transcribed sequence with moderate similarity to protein ref:NP_565786.1 (A.thaliana) photosystem II type I chlorophyll a b binding protein (Arabidopsis thaliana)

10.59431333 11.63223951 8.533082881 8.746275359

BJ283947 Ta.4061.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_565786.1 (A.thaliana) photosystem II type I chlorophyll a b binding protein (Arabidopsis thaliana)

9.444743998 9.481408497 9.100880159 8.606755827

CD454927 Ta.872.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_186922.1 (A.thaliana) thioredoxin f1 (Arabidopsis thaliana) 9.356197054 9.977978126 8.963297109 8.520823754

CD454927 Ta.872.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_186922.1 (A.thaliana) thioredoxin f1 (Arabidopsis thaliana) 9.356197054 9.977978126 8.963297109 8.520823754

CK152589 Ta.28541.1.A1_at Transcribed sequence with weak similarity to protein ref:NP_186922.1 (A.thaliana) thioredoxin f1 (Arabidopsis thaliana) 7.640766104 8.016814187 7.447543392 7.736608912

CK167182 Ta.3249.1.S1_at Transcribed sequence with moderate similarity to protein sp:P04777 (A.thaliana) CB21_ARATH Chlorophyll A-B binding protein 165180, chloroplast precursor

10.41632492 11.49057457 8.534325973 9.873746134

BQ171842 Ta.27013.1.S1_at Transcribed sequence with moderate similarity to protein sp:P04777 (A.thaliana) CB21_ARATH Chlorophyll A-B binding protein 165180, chloroplast precursor

7.02117828 7.684056411 6.416830473 6.821148736

BJ306896 Ta.7717.1.S1_x_at Transcribed sequence with moderate similarity to protein ref:NP_190750.1 (A.thaliana) chlorophyll synthetase (Arabidopsis thaliana) 8.224684701 8.206241861 8.400435702 8.761103027

BJ306896 Ta.7717.1.S1_x_at Transcribed sequence with moderate similarity to protein ref:NP_190750.1 (A.thaliana) chlorophyll synthetase (Arabidopsis thaliana) 8.224684701 8.206241861 8.400435702 8.761103027

CA722522 Ta.2402.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_191049.1 (A.thaliana) chlorophyll ab-binding protein (Arabidopsis thaliana)

9.104677156 9.591754246 8.38319607 8.704952597

CA722522 Ta.2402.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_191049.1 (A.thaliana) chlorophyll ab-binding protein (Arabidopsis thaliana)

9.104677156 9.591754246 8.38319607 8.704952597

BJ301728 Ta.28468.2.S1_x_at Transcribed sequence with moderate similarity to protein ref:NP_191049.1 (A.thaliana) chlorophyll ab-binding protein (Arabidopsis thaliana)

8.590602359 8.658283075 7.779221328 7.916521697

BJ301728 Ta.28468.2.S1_x_at Transcribed sequence with moderate similarity to protein ref:NP_191049.1 (A.thaliana) chlorophyll ab-binding protein (Arabidopsis thaliana)

8.590602359 8.658283075 7.779221328 7.916521697

CD934365 Ta.12788.1.S1_x_at Transcribed sequence with moderate similarity to protein ref:NP_178547.1 (A.thaliana) putative ferredoxin-thioredoxin reductase (Arabidopsis thaliana)

10.16816421 10.05346491 10.11348218 10.81717593


10.16816421 10.05346491 10.11348218 10.81717593


10.16816421 10.05346491 10.11348218 10.81717593

CD894372 Ta.1166.2.S1_at Transcribed sequence with moderate similarity to protein ref:NP_175032.1 (A.thaliana) fructose 1,6-bisphosphatase, putative (Arabidopsis thaliana)

9.026963203 9.26895029 8.628731304 8.457894666


CA737897 TaAffx.50270.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_175032.1 (A.thaliana) fructose 1,6-bisphosphatase, putative (Arabidopsis thaliana)

6.044253584 5.881114628 4.115416187 4.576508333

BJ318319 Ta.3590.2.S1_s_at Transcribed sequence with weak similarity to protein ref:NP_565765.1 (A.thaliana) putative chloroplast 50S ribosomal protein L28 (Arabidopsis thaliana)

8.067991906 8.132937375 7.843326545 3.624128248


8.067991906 8.132937375 7.843326545 3.624128248


8.067991906 8.132937375 7.843326545 3.624128248

BQ171831 Ta.13241.2.S1_at Transcribed sequence with weak similarity to protein ref:NP_564560.1 (A.thaliana) ATP-dependent Clp protease proteolytic subunit 7.301282714 7.283431892 7.496857223 6.251961516



CA649317 Ta.20705.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_565892.1 (A.thaliana) putative non-green plastid inner envelope membrane protein (Arabidopsis thaliana)

7.370554684 7.588554825 7.866139423 8.083237788

CA707977 Ta.9795.1.A1_at Transcribed sequence with weak similarity to protein ref:NP_178547.1 (A.thaliana) putative ferredoxin-thioredoxin reductase (Arabidopsis thaliana)

8.3362749 8.444326516 8.370575884 9.093195064


8.3362749 8.444326516 8.370575884 9.093195064


8.3362749 8.444326516 8.370575884 9.093195064

CK154963 Ta.6901.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_182281.1 (A.thaliana) photolyaseblue-light receptor 8.374118141 8.661552058 8.68576038 8.987599331

CK158867 Ta.13250.2.S1_x_at Transcribed sequence with weak similarity to protein sp:O65282 (A.thaliana) CH1C_ARATH 20 kDa chaperonin, chloroplast precursor 9.120716265 8.953244138 9.50793959 9.681643861

Signal transduction

CK195120 Ta.5029.2.A1_a_at Transcribed sequence with weak similarity to protein pir:T04831 (A.thaliana) T04831 probable serinethreonine-specific protein kinase 6.454439783 6.613728935 7.155349178 5.167105434

CK195120 Ta.5029.2.A1_a_at Transcribed sequence with weak similarity to protein pir:T04831 (A.thaliana) T04831 probable serinethreonine-specific protein kinase 6.454439783 6.613728935 7.155349178 5.167105434

CA684267 Ta.3087.2.A1_at Transcribed sequence with moderate similarity to protein ref:NP_176371.1 (A.thaliana) postsynaptic protein CRIPT, putative (Arabidopsis thaliana)

6.08492927 5.90388265 7.140613186 9.262340299

CA684267 Ta.3087.2.A1_x_at Transcribed sequence with moderate similarity to protein ref:NP_176371.1 (A.thaliana) postsynaptic protein CRIPT, putative (Arabidopsis thaliana)

8.311515559 8.107668902 8.634388601 8.959531191


6.08492927 5.90388265 7.140613186 9.262340299


8.311515559 8.107668902 8.634388601 8.959531191


6.08492927 5.90388265 7.140613186 9.262340299


8.311515559 8.107668902 8.634388601 8.841832495


6.08492927 5.90388265 7.140613186 9.262340299

CA596503 Ta.1422.2.S1_at Transcribed sequence with moderate similarity to protein ref:NP_193486.1 (A.thaliana) ras-related small GTP-binding protein RAB1c (Arabidopsis thaliana)

7.374706956 7.368347879 7.538622264 4.920701416

CA596503 Ta.1422.2.S1_x_at Transcribed sequence with moderate similarity to protein ref:NP_193486.1 (A.thaliana) ras-related small GTP-binding protein RAB1c (Arabidopsis thaliana)

7.590120781 7.631437838 7.82483392 5.773716537


7.374706956 7.368347879 7.538622264 4.920701416


7.590120781 7.631437838 7.82483392 5.773716537



7.374706956 7.368347879 7.538622264 4.920701416


7.590120781 7.631437838 7.82483392 5.773716537

CD909308 Ta.4416.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_197502.1 (A.thaliana) RAN2 small Ras-like GTP-binding nuclear protein 6.81299788 6.386474708 7.272363423 7.372052172

CD454106 Ta.25563.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_190267.1 (A.thaliana) GTP-binding protein Rab11 (Arabidopsis thaliana) 4.196084837 4.427587808 4.457033987 5.197730113

CD454106 Ta.25563.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_190267.1 (A.thaliana) GTP-binding protein Rab11 (Arabidopsis thaliana) 4.196084837 4.427587808 4.457033987 5.197730113

CD933162 Ta.29629.1.S1_s_at Transcribed sequence with moderate similarity to protein ref:NP_178291.1 (A.thaliana) putative receptor-like protein kinase (Arabidopsis thaliana)

4.061591744 3.832658046 4.774839714 4.852025273

BJ226675 Ta.1763.2.S1_at Transcribed sequence with moderate similarity to protein ref:NP_175056.1 (A.thaliana) GTP-binding protein 7.492125489 7.649409574 7.990888446 5.964789557

BJ226675 Ta.1763.2.S1_at Transcribed sequence with moderate similarity to protein ref:NP_175056.1 (A.thaliana) GTP-binding protein 7.492125489 7.649409574 7.990888446 5.964789557

CK207280 Ta.7158.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_189083.1 (A.thaliana) protein kinase, putative (Arabidopsis thaliana) 3.90459379 3.85677683 5.339900599 10.48962551




CD877105 Ta.1908.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_187383.1 (A.thaliana) GTP cyclohydrolase I (Arabidopsis thaliana) 7.456766375 7.630834408 7.526305336 2.276844951



CA664699 Ta.20015.1.A1_x_at Transcribed sequence with weak similarity to protein ref:NP_197141.1 (A.thaliana) AMP-binding protein (Arabidopsis thaliana) 9.1114749 8.718347235 8.835030502 5.878486954



CK159198 Ta.4257.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_198720.1 (A.thaliana) ABC transporter -like protein (Arabidopsis thaliana)

7.386589411 7.293731053 7.338281257 7.953828714


7.386589411 7.293731053 7.338281257 7.953828714


7.386589411 7.293731053 7.338281257 7.953828714

BQ241030 Ta.3765.2.A1_x_at Transcribed sequence with moderate similarity to protein pir:T04442 (A.thaliana) T04442 ABC-type transport protein T18B16.180 - Arabidopsis thaliana

5.790690841 5.522540991 5.820518738 6.634159071

Reactive Oxygen Species (ROS)

CA658987 Ta.975.1.S1_a_at Transcribed sequence with weak similarity to protein pir:T51415 (A.thaliana) T51415 Carboxylesterase-like protein - Arabidopsis thaliana 11.85028159 11.55543684 10.95588902 11.24263097


CK217120 Ta.975.2.S1_at Transcribed sequence with weak similarity to protein pir:T51415 (A.thaliana) T51415 Carboxylesterase-like protein - Arabidopsis thaliana 10.20102108 9.871763492 9.082648263 9.692139491

CK217120 Ta.975.2.S1_x_at Transcribed sequence with weak similarity to protein pir:T51415 (A.thaliana) T51415 Carboxylesterase-like protein - Arabidopsis thaliana 10.15621314 9.907118934 9.035588017 9.605144844

CA658793 TaAffx.28555.1.S1_s_at Transcribed sequence with weak similarity to protein pir:T51415 (A.thaliana) T51415 Carboxylesterase-like protein - Arabidopsis thaliana 9.606729815 10.281919 8.573745231 8.795484159



CA660275 Ta.22403.1.S1_at Transcribed sequence with weak similarity to protein pir:T51415 (A.thaliana) T51415 Carboxylesterase-like protein - Arabidopsis thaliana 6.75081361 7.947296054 6.900552603 5.766769969

CK213957 Ta.21505.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_196153.1 (A.thaliana) peroxidase (Arabidopsis thaliana) 11.75227884 12.06952052 11.09001373 12.1874845

BJ250276 Ta.7524.1.A1_at Transcribed sequence with weak similarity to protein ref:NP_196153.1 (A.thaliana) peroxidase (Arabidopsis thaliana) 9.205760259 9.810068871 8.157556927 8.319271794

CK213957 Ta.21505.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_196153.1 (A.thaliana) peroxidase (Arabidopsis thaliana) 11.75227884 12.06952052 11.09001373 12.1874845

AF092524.1 Ta.1944.1.S1_at Triticum aestivum manganese superoxide dismutase (SOD) mRNA, nuclear gene encoding mitochondrial protein, complete cds. /PROD=manganese superoxide dismutase /FL=gb:AF092524.1

8.284227849 8.278331308 8.38431404 2.892388666


8.284227849 8.278331308 8.38431404 2.892388666


8.284227849 8.278331308 8.38431404 2.892388666

AJ440792.1 Ta.14632.1.S1_at Triticum aestivum mRNA for glutathione transferase F3 (gstf3 gene). /PROD=glutathione transferase F3 5.364506808 5.785703978 5.825818227 7.872764232

AJ440792.1 Ta.14632.1.S1_x_at Triticum aestivum mRNA for glutathione transferase F3 (gstf3 gene). /PROD=glutathione transferase F3 5.628528457 6.121114572 5.843916189 7.843723923





AJ440795.1 Ta.23704.1.S1_s_at Triticum aestivum mRNA for glutathione transferase F6 (gstf6 gene). /PROD=glutathione transferase F6 8.065823053 9.126731846 8.666363589 9.340586463



AJ440795.1 Ta.23704.1.S1_s_at Triticum aestivum mRNA for glutathione transferase F6 (gstf6 gene). /PROD=glutathione transferase F6 8.065823053 9.126731846 8.666363589 9.340586463

CD876924 Ta.12472.1.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_196291.1 (A.thaliana) peroxidase (Arabidopsis thaliana) 10.4670637 10.61850027 10.1859374 8.300181579



CA640533 Ta.18681.1.S1_x_at Transcribed sequence with weak similarity to protein pdb:1FSI (A.thaliana) A Chain A, Crystal Structure Of Cyclic Nucleotide Phosphodiesterase Of Appr>p From Arabidopsis Thaliana

7.422266168 7.244086398 7.069098906 4.67960104

CA640533 Ta.18681.1.S1_x_at Transcribed sequence with weak similarity to protein pdb:1FSI (A.thaliana) A Chain A, Crystal Structure Of Cyclic Nucleotide Phosphodiesterase Of Appr>p From Arabidopsis Thaliana

7.422266168 7.244086398 7.069098906 4.67960104

Defense related

CD452587 Ta.1607.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_176151.1 (A.thaliana) viral resistance protein, putative (Arabidopsis thaliana) 6.99274836 7.053846954 7.159044971 7.81638908



CA627615 Ta.2294.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_173768.1 (A.thaliana) Pto kinase interactor, putative (Arabidopsis thaliana) 9.971358032 10.05208685 10.12836865 10.74343882




CD492047 Ta.17948.1.A1_s_at Transcribed sequence with moderate similarity to protein ref:NP_195325.1 (A.thaliana) thaumatin-like protein (Arabidopsis thaliana) 4.305884177 4.888049375 4.665137982 4.874972654

CK206212 Ta.3346.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_178341.1 (A.thaliana) putative phloem-specific lectin (Arabidopsis thaliana) 7.204190396 7.249192017 7.284266975 6.481087038



CA650490 Ta.1207.1.S1_s_at Transcribed sequence with weak similarity to protein ref:NP_177794.1 (A.thaliana) 12-oxophytodienoate reductase 2.835970283 3.364713445 3.876416676 5.386249807



CD453420 Ta.27016.1.A1_x_at Transcribed sequence with weak similarity to protein ref:NP_177795.1 (A.thaliana) 12-oxophytodienoate reductase 10.87207932 10.54139845 10.78277678 9.850224995


CD453420 Ta.27016.1.A1_s_at Transcribed sequence with weak similarity to protein ref:NP_177795.1 (A.thaliana) 12-oxophytodienoate reductase 10.86937923 10.4736358 10.63389794 9.692527674


Stress related

BG907881 Ta.27671.1.A1_at Transcribed sequence with weak similarity to protein ref:NP_172740.1 (A.thaliana) heat shock factor protein hsf8, putative (Arabidopsis thaliana)

5.730380611 5.712385259 5.941886807 6.340099904

BG907881 Ta.27671.1.A1_at Transcribed sequence with weak similarity to protein ref:NP_172740.1 (A.thaliana) heat shock factor protein hsf8, putative (Arabidopsis thaliana)

5.730380611 5.712385259 5.941886807 6.340099904

CK216436 Ta.5600.1.S1_a_at Transcribed sequence with weak similarity to protein ref:NP_172094.1 (A.thaliana) salt-tolerance protein (Arabidopsis thaliana) 8.824799661 9.147736938 8.247783654 8.186571364

CK216436 Ta.5600.1.S1_s_at Transcribed sequence with weak similarity to protein ref:NP_172094.1 (A.thaliana) salt-tolerance protein (Arabidopsis thaliana) 9.992746981 10.36384779 9.398892553 9.262035008

BQ789411 Ta.5600.2.S1_a_at Transcribed sequence with weak similarity to protein ref:NP_172094.1 (A.thaliana) salt-tolerance protein (Arabidopsis thaliana) 8.542853159 8.84362914 8.017881669 7.908607783



CA611316 Ta.7442.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_172094.1 (A.thaliana) salt-tolerance protein (Arabidopsis thaliana) 9.442729571 9.797363519 8.858579962 9.406314752

CA619808 Ta.6344.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_172094.1 (A.thaliana) salt-tolerance protein (Arabidopsis thaliana) 9.430782207 9.888159539 8.962576421 9.067428469

CA620810 Ta.1971.1.S1_a_at Transcribed sequence with weak similarity to protein ref:NP_172094.1 (A.thaliana) salt-tolerance protein (Arabidopsis thaliana) 11.25467444 11.75546818 11.03276866 11.28005912

AY093953.1 Ta.9453.1.S1_at Biostress-resistance-related protein mRNA, partial cds /DEF=Triticum aestivum biostress-resistance-related protein mRNA, partial cds. 10.51088173 10.42319745 10.65150669 10.98114583

AY093953.1 Ta.9453.1.S1_at Biostress-resistance-related protein mRNA, partial cds /DEF=Triticum aestivum biostress-resistance-related protein mRNA, partial cds. 10.51088173 10.42319745 10.65150669 10.98114583

BJ309786 Ta.14519.1.S1_x_at Transcribed sequence with moderate similarity to protein pir:T51387 (A.thaliana) T51387 UVB-resistance protein-like - Arabidopsis thaliana

7.941739555 8.058194342 8.202222115 8.417922753

CK164215 Ta.545.1.S1_x_at Transcribed sequence with moderate similarity to protein ref:NP_188548.1 (A.thaliana) metalloprotease, putative (Arabidopsis thaliana) 9.203105876 9.395838601 9.154197033 8.599541291

CK217217 Ta.22952.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_193139.1 (A.thaliana) selenium-binding protein like (Arabidopsis thaliana)

10.38100101 10.33329324 10.60118733 10.97814942



10.38100101 10.33329324 10.60118733 10.97814942


10.38100101 10.33329324 10.60118733 10.97814942


10.38100101 10.33329324 10.60118733 10.97814942

CA721628 Ta.22094.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_188286.1 (A.thaliana) translationally controlled tumor protein-like protein (Arabidopsis thaliana)

8.943726991 8.675793062 8.362659833 5.052219415


8.943726991 8.675793062 8.362659833 5.052219415


8.943726991 8.675793062 8.362659833 5.052219415

Senescence

CD861726 Ta.22475.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_565154.1 (A.thaliana) 1-aminocyclopropane-1-carboxylate oxidase, putative (Arabidopsis thaliana)

8.006642069 8.223252751 8.514543475 3.603280338


8.006642069 8.223252751 8.514543475 3.603280338


8.006642069 8.223252751 8.514543475 3.603280338

CK171474 Ta.5227.3.S1_a_at Transcribed sequence with moderate similarity to protein ref:NP_188365.1 (A.thaliana) putative s-adenosylmethionine synthetase (Arabidopsis thaliana)

8.79079928 9.397592829 9.237516576 9.262329075

CA731654 Ta.22237.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_178476.1 (A.thaliana) putative nonsense-mediated mRNA decay protein (Arabidopsis thaliana)

8.554642255 8.778344848 8.529054391 9.055679879

CA731654 Ta.22237.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_178476.1 (A.thaliana) putative nonsense-mediated mRNA decay protein (Arabidopsis thaliana)

8.554642255 8.778344848 8.529054391 9.055679879

CD863459 Ta.5490.1.S1_s_at Transcribed sequence with weak similarity to protein ref:NP_182296.1 (A.thaliana) putative auxin-responsive protein (Arabidopsis thaliana)

8.951577367 8.855032654 8.782802242 9.568021474

CD863459 Ta.5490.1.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_182296.1 (A.thaliana) putative auxin-responsive protein (Arabidopsis thaliana)

9.101292134 9.046865779 9.048467894 9.709808755


8.951577367 8.855032654 8.782802242 9.568021474


9.101292134 9.046865779 9.048467894 9.709808755


8.951577367 8.855032654 8.782802242 9.568021474


9.101292134 9.046865779 9.048467894 9.709808755

CD875153 Ta.1151.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_568651.1 (A.thaliana) senescence-specific cysteine protease SAG12 (Arabidopsis thaliana)

9.938353623 9.966512809 10.2697719 10.92565529

CD875153 Ta.1151.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_568651.1 (A.thaliana) senescence-specific cysteine protease SAG12 (Arabidopsis thaliana)

9.938353623 9.966512809 10.2697719 10.92565529

CD453722 Ta.23069.1.S1_at Transcribed sequence with weak similarity to protein sp:Q38822 (A.thaliana) AXI3_ARATH Auxin-responsive protein IAA3 4.431493139 4.453658876 4.831450719 5.932976458



BJ224514 Ta.7339.3.S1_a_at Transcribed sequence with weak similarity to protein sp:Q9FVC1 (A.thaliana) SVP_ARATH SHORT VEGETATIVE PHASE protein 7.316623225 7.112921633 7.249750535 6.449143415

BJ224514 Ta.7339.3.S1_a_at Transcribed sequence with weak similarity to protein sp:Q9FVC1 (A.thaliana) SVP_ARATH SHORT VEGETATIVE PHASE protein 7.316623225 7.112921633 7.249750535 6.449143415

CA676673 Ta.28290.1.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_569030.1 (A.thaliana) senescence-associated protein (Arabidopsis thaliana) 7.185470406 6.977374614 6.616955877 6.265155529


CA676673 Ta.28290.1.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_569030.1 (A.thaliana) senescence-associated protein (Arabidopsis thaliana) 7.185470406 6.977374614 6.616955877 6.265155529

Transcription factors

BQ171259 Ta.24806.2.S1_at Transcribed sequence with weak similarity to protein pir:T48034 (A.thaliana) T48034 bZIP transcription factor-like protein - Arabidopsis thaliana

9.940182922 9.70830275 9.770187973 6.882320577


9.940182922 9.70830275 9.770187973 6.882320577


9.940182922 9.70830275 9.770187973 6.882320577

BJ264278 Ta.30607.1.A1_at Partial mRNA for putative MADS-box transcription factor (madsno. 11 gene) 8.082527828 8.029806976 7.772032421 3.598974273



CA627693 Ta.3273.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_565507.1 (A.thaliana) putative CONSTANS-like B-box zinc finger protein (Arabidopsis thaliana)

8.665079557 9.249348014 8.452704606 8.447145825

CA627693 Ta.3273.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_565507.1 (A.thaliana) putative CONSTANS-like B-box zinc finger protein (Arabidopsis thaliana)

8.665079557 9.249348014 8.452704606 8.447145825

CK213855 Ta.2909.1.S1_x_at Transcribed sequence with moderate similarity to protein pir:T45593 (A.thaliana) T45593 small zinc finger-like protein TIM9 - Arabidopsis thaliana

8.280334233 8.06618951 8.472980802 8.835525306

CK213855 Ta.2909.1.S1_x_at Transcribed sequence with moderate similarity to protein pir:T45593 (A.thaliana) T45593 small zinc finger-like protein TIM9 - Arabidopsis thaliana

8.280334233 8.06618951 8.472980802 8.835525306

BQ170258 Ta.11513.1.S1_s_at Transcribed sequence with weak similarity to protein ref:NP_177672.1 (A.thaliana) bZIP transcription factor ATB2, putative (Arabidopsis thaliana)

6.417918489 7.324272907 6.99547181 6.26148884

CA709110 Ta.2880.1.S1_at WESR4 mRNA for Zinc-finger motif, partial cds 8.353031981 8.125641343 8.335366237 8.869582529

Lipid metabolism

CA632594 Ta.1337.2.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_568904.1 (A.thaliana) nonspecific lipid-transfer protein precursor - like (Arabidopsis thaliana)

7.660969222 7.192229172 7.707552959 4.350789705

CK164223 Ta.1337.1.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_568904.1 (A.thaliana) nonspecific lipid-transfer protein precursor - like (Arabidopsis thaliana)

10.04272909 9.698234631 10.1214955 8.271092924


7.660969222 7.192229172 7.707552959 4.350789705


7.660969222 7.192229172 7.707552959 4.350789705

BJ265183 Ta.27482.1.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_198633.1 (A.thaliana) lipid transfer - like protein (Arabidopsis thaliana) 8.365773799 7.916040818 8.387138556 9.626495667

BJ265183 Ta.27482.1.S1_at Transcribed sequence with weak similarity to protein ref:NP_198633.1 (A.thaliana) lipid transfer - like protein (Arabidopsis thaliana) 7.974311456 7.527669864 8.101936269 9.421293556





CK157489 Ta.23813.1.S1_at Transcribed sequence with moderate similarity to protein ref:NP_176896.1 (A.thaliana) glyoxalase I, putative (Arabidopsis thaliana) 9.280393995 9.234191726 9.176781341 8.630346092




CA681065 Ta.14727.1.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_177561.1 (A.thaliana) putative glycerophosphodiester phosphodiesterase (Arabidopsis thaliana)

4.170208367 4.966138334 4.341480896 3.134773519

CK192982 Ta.7488.1.S1_a_at Transcribed sequence with moderate similarity to protein pir:T00580 (A.thaliana) T00580 probable (acyl-carrier-protein) S-malonyltransferase

5.178510663 5.268283669 5.409764868 5.860705447

CK214819 Ta.30191.1.A1_s_at Transcribed sequence with weak similarity to protein ref:NP_197498.1 (A.thaliana) lipophosphoglycan biosynthetic protein - like (Arabidopsis thaliana)

10.98751359 11.10619939 10.20486067 10.32897502

BJ227883 Ta.9635.1.A1_at Transcribed sequence with moderate similarity to protein pir:B71415 (A.thaliana) B71415 probable phosphocholine cytidylyltransferase - Arabidopsis thaliana

6.370832791 6.35854867 6.45372155 7.348219374


6.370832791 6.35854867 6.45372155 7.348219374


6.370832791 6.35854867 6.45372155 7.348219374

CD888399 Ta.10542.1.S1_a_at Transcribed sequence with moderate similarity to protein ref:NP_188147.1 (A.thaliana) putative 3-hydroxybutyryl-CoA dehydrogenase (Arabidopsis thaliana)

9.027226585 8.862993646 9.027506117 8.535563544

CD888399 Ta.10542.1.S1_a_at Transcribed sequence with moderate similarity to protein ref:NP_188147.1 (A.thaliana) putative 3-hydroxybutyryl-CoA dehydrogenase (Arabidopsis thaliana)

9.027226585 8.862993646 9.027506117 8.535563544

CA655589 Ta.28669.1.S1_a_at Transcribed sequence with weak similarity to protein ref:NP_564165.1 (A.thaliana) protein phosphatase type 2C, putative (Arabidopsis thaliana)

9.064627141 9.405967552 9.732884518 9.939924151

BJ254182 Ta.28669.2.S1_x_at Transcribed sequence with weak similarity to protein ref:NP_564165.1 (A.thaliana) protein phosphatase type 2C, putative (Arabidopsis thaliana)

8.518839789 8.758246358 9.142541602 9.509860654


9.064627141 9.405967552 9.732884518 9.939924151


9.064627141 9.405967552 9.732884518 9.939924151

CA729626 Ta.19311.2.S1_at Transcribed sequence with weak similarity to protein ref:NP_175238.1 (A.thaliana) protein phosphatase-2C, putative (Arabidopsis thaliana)

7.223291363 6.961579571 6.972867738 6.706307104

Secondary metabolism

CK163877 Ta.9332.2.A1_at Transcribed sequence with weak similarity to protein pir:T45624 (A.thaliana) T45624 flavonoid 3-hydroxylase-like protein (imported) - Arabidopsis thaliana

4.821229131 6.245574859 6.509854418 6.914136234


4.821229131 6.245574859 6.509854418 6.914136234

CK213911 Ta.9332.3.S1_x_at Transcribed sequence with weak similarity to protein pir:T45624 (A.thaliana) T45624 flavonoid 3-hydroxylase-like protein (imported) - Arabidopsis thaliana

4.188232263 4.95416207 5.061047788 5.649475637


4.821229131 6.245574859 6.509854418 6.914136234

CA650542 Ta.5623.2.S1_a_at Transcribed sequence with moderate similarity to protein sp:Q42524 (A.thaliana) 4CL1_ARATH 4-coumarate--CoA ligase 1 6.765246535 7.492539761 7.70144496 7.585814301

CK197632 Ta.5623.1.S1_x_at Transcribed sequence with moderate similarity to protein sp:Q42524 (A.thaliana) 4CL1_ARATH 4-coumarate--CoA ligase 1 7.847102028 8.376174282 8.490935498 8.457382369





WCT REPORT 2013 · Centurion 0046 Report compiled by: PROJECT MANAGER Prof Anna-Maria...

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Transcript of WCT REPORT 2013 · Centurion 0046 Report compiled by: PROJECT MANAGER Prof Anna-Maria...