# Presentation: 079961

## Molecular markers and genomics: interfacing pre-breeding and commercial wheat breeding

**Molecular markers and genomics: interfacing pre-breeding and commercial wheat breeding**

**Dr Michael Francki**
**Manager- Biotechnology**
**Plant and Animal Genome XVI**
**Town and Country Hotel, San Diego, CA**
**12 January 2008**

**Notes:**
Thanks Perry for giving me opportunity to present

Good opportunity to present how my program is streamlining R&D to ensure that outcomes are attractive for adoption by commercial breeding programs.

My program is funded by a range of agencies including VAWCRC, MPBCRC and GRDC and my program is housed at the SABC at Murdoch University

## Pre-breeding and commercial breeding

**Pre-breeding and commercial breeding**

**Notes:**
Wheat breeding in Australia has changed in the past 5 years with the conversion of public sector to commercial breeding.  There are three commercial wheat breeding programs in Australia that operate in competitive environments to deliver varieties specifically for WA.

However, wheat R&D is still funded in the public domain, is pre-competitive and outcomes are delivered to commercial breeding prorgams.   

My program is focused in the pre-breeding, pre-competitive environment where R&D outcomes are delivered to breeding programs for developing commercial varieties in Western Australia

We are involved in developing a range of technologies and strategies for delivering R&D outcomes for breeding programs to improve disease resistance, abiotic stress tolerance and grain quality traits

The delivery of R&D outcomes is either through new technologies and resources that can be directly implemented through marker assisted selection 
or
through germplasm developed with novel alleles fixed in relevant backgrounds and attractive for commercial breeding programs to adopt.

## Target traits for pre-breeding of spring wheat in Western Australia

**Disease:**
*Stagonospora nodorum* (leaf and glume resistance)
- New sources of rust resistance
**Abiotic Stress:**
- Salt tolerance- exploitation of wild relatives
**Grain quality:**
- Genetic analysis and QTL in adapted germplasm:  Flour (b*) colour, milling yield, water absorption, non-null4A GBSS FSV
**Adaptation:**
- Allelic determinants of *Ppd *and *Vrn* genes for adaptation of Australian wheat  to major grain production zones

**Notes:**
Western Australia produces 40-50% of Australia’s wheat productions and targets wheat for the premium export market in Asia and the Middle East

Many of the traits we target are for commercial breeding programs to develop varieties to meet the demands of Western Australian growers and end-product markets

Go through traits

I will focus mainly on the disease resistance and abiotic stress tolerance 

## S. nodorum- QTL mapping

***S. nodorum- QTL mapping***

- Adult plant resistance from spring and winter wheat sources
- Polygenic control- independent flag leaf and glume resistance
- QTL and flanking markers identified
- 	Flag Leaf: chromosome  2DL  (Shankar et al, in press)
- 	Flag Leaf: chromosome 2AL (unpublished)
- 	Glume:  chromosome 2DL (Uphaus et al, 2007. Crop Sci 47:1813)
- 	Glume:  chromosome 4BL (Shankar et al, in press)
- Phenotyping in multiple locations (local environments, USA and Australia)
- Each QTL accounts for 10-38% of total phenotypic variation
- Heritability:  0.77-0.81

**Notes:**
S. nodorum is a major fungal pathogen in WA and their has been no capacity to specifically breed for resistance.

In the past 5 years, my program has spent a  considerable amount of time in identifying sources of resistance and QTL mapping that can be used for breeding for resistance

Go through slide

The efforts in determining the genetic control and QTL identification has placed us in a good position to deploy resistance in wheat germpalsm adaptable to WA environments

## S. nodorum- Adoption through germplasm development

**Donor Parent**
- leaf (2DL) and glume (4BL) resistance from spring wheat 6HRWSN125
- glume (2DL) resistance from winter wheat P91193D1
**Choice of recurrent parents**
- Stage 3&4 crossbreds and released varieties- hard, soft and noodle classes with acceptable quality standards
- Minimal standards for rust resistance
- Marker polymorphism flanking QTL
- Same *Rht1 *and *Rht2* alleles
- Background resistance to *S. nodorum*
**Backcrossing and marker screening**
- Generate BC1F2 (spring wheat donor) and BC2F2 lines (winter wheat donor)
- SSR screening at each generation using Multiplex Ready TechnologyTM (MRTTM)

**Notes:**
We have used the parents of mapping populations for QTL analysis as donor parents for germplasm development  in backcrossing

The choice of recurrent parents was rather selective to ensure that new germplasm would be rapidly adopted by commercial breeding programs

Go through choice of parents 

The backcrossing strategy depends largely on the donor parent being used
We developed BCI when using spring wheat source as donor parents
BC2 when using winter wheat as donor source to retain as much spring wheat background as possible
Marker selection was done at each backcross using MRT developed by Matt Hayden at the University of Adelaide

## S. nodorum- MAS using MRT

- Selection of heterozygous lines for QTL at BC1F1/ BC2F1 using single SSR
- Selection of homozygous lines for QTL at BC1F2/ BC2F2 using flanking SSRs

***S. nodorum- MAS using MRT***

**Notes:**
We have developed 14 BC populations for germplasm development to fix resistant alleles in backgrounds relevant to WA

Selection of individuals during germplasm development has relied on MRT technology that provides a HTP capability.

For each QTL, a single marker was used to select heterozygous BC1F1 and flanking markers to select homozygous  BCF2 lines

This is a typical MRT chromatogram for a screening of SSR markers at BC1F2.

Go through chromatogram

Results from screening 6 populations at at each backcross stage using spring wheat donor parent.  A minimal set of markers are used to screen at BC1F1 to select heterozygous individuals for selfing and increasing the numbers of markers for screening homozygotes at BC1F2.

Selections that will be phenotypes for resistance in 2008
BC2 populations using winter wheat source is ongoing and expected to achieve in excess of 30,000 marker assays to select comparable number of lines 

## Novel stem rust resistance (SrR)

***SrR+***** identified on chromosome 1RS from ‘Imperial’ rye in 1DL.1RS translocation (**_**not**_** *****Sr31*****)**
**Linkage between secalin and rust resistance broken in 1DS recombinants**
**T6-1:  *****SrR******+****** Sec-1******-***
**T49-7:***** SrR******-****** Sec-1+***

**Small  introgression**

**Rye DNA probe**

**Rye DNA probe**

**Rye DNA probe**

**pAS1 + Rye DNA probe**

**Notes:**
I now would like to draw your attention to some of the work that my program has been involved in developing novel sources of stem rust resistance.

One source of Sr resistance has  been identified on 1RS from Imperial rye and known to be different from Sr31 since SrR is effective in Australia whereas Sr31 has broken down.

Over the past 30 years has seen the development of transocations, primary and secondary recombinants to break linkage between secalins that are thought to contribute to detrimental dough quality characteristics but retain SrR.  
Two tertiary recombinants have been developed T6-1 and T49-7 (explain)

The disease reaction can clearly be seen where T6-1 has retained a resistant reaction  whereas T49-7 is susceptible

Analysis of SDS-protein gels have shown that T6-1 is missing whereas T49-7 has retained secalin proteins, indicating that T6-1 is ideal for deploying novel SrR in Australian germplasm.

ISH analysis has identified T49-7 as having a large rye introgression. Although we could detect chromosome 1D using pAS1, we could not detect a rye segment in T6-1.  Therefore, this segment appears to be very small and ideal for germplasm development

## Deployment of SrR

**PCR marker, AW2-5, developed for tracking rye segment  **
**Deploying *****SrR***** on rye segment in WA adapted germplasm**
**5 BC****4****F****2**** populations using hard, soft and noodle varieties as recurrent parents**
**Selection at each backcross generation using AW2-5**
**Grain quality evaluation at BC****4****F****3**** to confirm no quality defects**

***Deployment of SrR***

**Notes:**
To effectively deploy the SrR in germplasm, we have developed a PCR-based marker to track the rye segment in T6-1. 

This gel shows the marker associated with the rye segment in T6-1 and in primary and secondary recombinants sharing the same segment.  However, it is clearly absent in susceptible recombinant lines that do not contain the same rye segment 

T6-1 is in an undesirable wheat background, so we have begun a backcrossing strategy using 5 varieties and crossbreds to deploy SrR.  We are using AW2-5 to screen at each generation to select individuals as female parents for recurrent backcrossing.

We are currently at the BC3 stage and progressing to BC4 for grain evaluation to ensure that quality characteristics have not been compromised when deploying SrR in adaptable backgrounds

## Salt tolerance from wild relatives

**Na****+**** exclusion from tall wheatgrass introgressed into wheat**
**Homoeologous group 3 substitution lines account for ~50% of Na+ exlcusion **(Mullan et al 2007 MGG *277***:**199)
**Induce homoeologous recombination with wheat**
**Marker development (EST-SSR) for tracking small wheatgrass segments **(Mullan et al 2005, Genome *48***:**811)
**Classify karyotypes according to different introgressed segments **(Mullan et al, unpublished)

**Notes:**
I now would like to move onto the final area where we are making significant progress in developing salt tolerant wheat through the exploitation of genes for tall wheatgrass 

Go through slide

The diagram shows a summary of the wheat-wheatgrass recombinants developed with varying amounts of whetagrass chromatin.  This was achieved by the analysis of molecular markers specifically for wheatgrass chromatin and also presence and absence of wheat loci.

The analysis of Na exclusion show that one line, 524-568 is similar to the group 3 substitution lines and shows a reduction in Na accumulation by about 50% compared with CS.

We have confirmed that 524-568 has a small introgression at the distal end of chromosome 3A using ISH and this line will be used as donor parent for deploying in germplasm development.

## Salt tolerance-comparative genomics and candidate genes

**Functional genes for Na****+**** transport in Arabidopsis**
**	**** *****SOS1*****, *****NHX1*****, *****NHX5*****, *****AVP1*****, *****AVP2*****, *****HKT1*****, *****LCT1***
**Comparative genomics to identify orthologs in wheat and wheatgrass using rice as a bridging species**

**Notes:**
We have taken the opportunity to expand this area of research and use comparative genomics to identify candidate genes on wheat and wheatgrass chromosomes that account for the reduction in Na accumulation.

We have taken 7 Arabidopsis genes where their function in Na transport has been well defined and used comparative genomics to identify orthologs in wheat and wheatgrass.

SOS 1 is a typical example where we have traced orthologs from Arabiopsis to wheat using rice a s a bridging species.  A comparison of intron-exon structure and conserved functional functional domains across species have identified their true orthologs.

In addition we have mapped the sequences to wheat and wheatgrass chromosomes.  SOS1 maps to the short arms of wheat 3A and 3D and to a similar position on wheatgrass 3E. 

However, the position of SOS1 on the short arm does not appear to be responsible for low Na accumulation since the introgression is on the long arm   

## Salt tolerance- gene location and expression

**Chromosomal assignment of wheat and wheatgrass orthologs for** ***SOS1*****, *****NHX1*****, *****NHX5*****,  *****AVP1*****, *****AVP2*****, *****HKT1*****, *****LCT1***

**Notes:**
Therefore, what wheat ortholog maps to the long arm of group 3 chromosomes?

We have mapped all seven wheat orthologs in a similar manner.  None of the remaining 6 orthologs mapped to the long arm of the group 3 chromosome.  
The orthologs were distributed across the wheat and wheatgrass chromosomes with most having homoeologous positions with some rearrangements noted.

Do any of these orthologs contribute to low Na accumulation?

We had done gene expression studies in different tissue for all orthologs between wheat and group 3 substitution lines in presence or absence of high salt. 
A notable feature is that for most orthologs, there was alternative splicing across tissue indicating these genes are regulated by complex mechanisms.  A notable feature is that HKT1 showed differences in transcripts in some substitution lines indicating  a complex interaction of wheat and wheatgrass genes involving post-transcriptional regulation on wheatgrass 3E with orthologs on other chromosomes.    

## Summary

***Summary***

**Notes:**
I begun this presentation with this slide showing you the areas of research, methodologies and strategies to deliver R&D outcomes to commercial wheta breeding programs.

Most of the outcomes will be delivered through germplasm development as a validation component to ensure that the R&D outocmes can be readily adopted by commercial breeding

In doing so, we have made significant advances in germplasm development for Stagonospora and rust resistance and we are making heading down the germplasm development approach for improving slat tolerance in wheat

## Acknowledgments

**DAFWA**
- Esther Walker
- Steve Brown
- Ghader Mirzaghaderi
- Allison Crawford
- Manisha Shankar
- Hossein Golzar
- Rob Loughman
**Intergrain Pty Ltd**
- Robin Wilson
- Iain Barclay
- Robyn McLean
- Chris Moore

***Acknowledgments***

**Purdue University**
- Jim Uphaus
- Herb Ohm
**The University of Western Australia**
- Daniel Mullan
- Tim Colmer
**University of Adelaide**
- Ian Dundas
- Ratna Anugrahwati
- Ken Shepherd
- Dawn Verling
- Matt Hayden

## Thankyou...........

***Thankyou...........***