Lecture 2 - Breeding Technology - Casson

Question 1

Give examples of crop genomes

Answer 1

Wheat - can be hexaploid
Soya - complex genome
Makes breeding difficult!

Question 2

What are breeders interested in?

Answer 2

TRAITS. They do not care about genes. Purely what it brings to the plant.

Question 3

What is an elite line?

Answer 3

Plant line with high numbers of positive traits. These can be monogenic such as resistance. Or polygenic (most are) such as height.

Question 4

What is the plant breeding cycle?

Answer 4

Diagram. 
Identify plant with desirable trait. 
Combine with existing variety.
Identify improved progeny.
Test over multiple generations and environments. 
Repeat.

Question 5

Describe the dev pipeline for plants

Answer 5

Large number of plants (150,000) in a few locations. Identify plants with good traits, try them in more locations. Ends up with a few plants in many locations.
This can take 10 years, GM would take 13.
Hence only a few new varieties per year

Question 6

Why did the Green Revolution come about?

Answer 6

Borlaug was tasked with increasing wheat yield in Mexico.

Many countries turned from importers to exporters of grain.

Question 7

Describe NB’s process

Answer 7

NB grew plants in two locations - northern (short days) and central highlands (long days).
Many crops at the time were only long day. These plants were being selected under two day lengths. Eventually selected for photoperiod-insensitive, and climate-tolerant crops.

Question 8

What problems did NB run into?

Answer 8

Need for high amounts of N fertiliser - Haber process allowed this.
Stem breakages - developed semi-dwarf variety.
Allowed more food to be produced, and population boom.

Question 9

Describe increased agricultural productivity

Answer 9

Increased yield, but land usage has stayed the same.

Question 10

What are some limits to plant breeding?

Answer 10

Development of potato resistant to late blight took 30 years.
Rust-fungus-resistance in rice cannot be transferred to other plants by traditional breeding methods.
Unintentional gene transfer- see next qu

Question 11

Describe the problem with unintentional gene transfer + give an example

Answer 11

Traits are linked to others, and you do not want a negative trait spreading to progeny.
Southern Corn Blight. Male corn must be emasculated to prevent selfing - labour intensive (detassling)- so a sterile strain was used.
80% of species were derived from male sterile version, which was susceptible to new corn blight.
The sterility was LINKED to susceptibility to new version of blight.

Question 12

Give three types of modern breeding technologies

Answer 12

1. Marker Assisted Breeding
2. Mutation breeding
3. TILLING

Question 13

Describe marker assisted breeding

Answer 13

You have an elite line with lots of good traits. You have a donor line with one good trait, that you would like to introduce to your elite line. You cross them, and perform a series of backcrosses with your initial elite line to enrich the progeny with elite alleles. You use known polymorphisms in the DNA as your marker, this could be the allele itself or nearby polymorphisms. The progeny are checked at each generation, the markers are being used to follow the process. By 6 BCs, 99% elite DNA in progeny.That 1% is ideally the donor trait.

Question 14

How has marker assisted breeding improved recently?

Answer 14

• DNA sequencing methods make it easier to generate new markers specific to a particular cultivar
• Increased scale + automated DNA extraction makes it much more likely to generate the end product you want

Question 15

Give an example of marker assisted breeding

Answer 15

SUB1 rice
SUB1A locus identified in submergence-tolerant strain of rice, and was introduced to high-yielding Swarna strain.
The actual gene responsible wasn’t discovered until 2006, , but the Swarna-Sub1 line had already been developed.

Question 16

What are the limitations of marker assisted breeding?

Answer 16

You can only breed the trait if it exists already. You cannot introduce a trait if it doesn’t exist in your species already (in the pangenome of your species)

Question 17

What technique increases variation by making new alleles?

Answer 17

Mutation breeding

Question 18

How did mutation breeding begin?

Answer 18

Natural mutation rate is low, so breeders decided to use radiation and chemical mutagenesis to create variation.
This was partly due to efforts after WW2 to find peaceful uses for atomic energy.

Question 19

Is mutation breeding GM?

Answer 19

NO, it is not introducing any extra genetic material. It is allowed in countries where GM is banned

Question 20

Limitations of mutation breeding?

Answer 20

It is not targeted. The mutagen will target the whole genome, therefore there is no predictability about where it will work.

Question 21

What is TILLING?

Answer 21

Targeting Induced Local Lesions in Genomes. Technique for identifying mutations in a desired gene.

Question 22

Describe TILLING

Answer 22

Two step process:

1. Mutagenise population (non-targeted)
2. Analyse specific gene in ALL progeny

Question 23

Is TILLING GM?

Answer 23

No. The mutagenesis is not targeted. Mutation events are fairly random, TILLING is scanning for mutations in a region.

Question 24

EcoTILLING - what is it?

Answer 24

Wild relatives of crops are analysed for sequence variants in gene of interest.
This offers the potential to introduce these new alleles into our crops (using marker assisted breeding)

Question 25

Describe process of TILLING

Answer 25

1. Must know sequence of gene of interest (reverse genetics)
2. Develop a mutagenised population of crop (500-5000 plants). EMS - G/C to A/T transition. Sodium azide - A/T to G/C.
3. Self the mutagenised plants to generate homozygous M2 seed
4. Extract DNA from individual M2 plants. M3 seed is archived
5. Pool samples - reduces population for screening (screening method determines pool size)
6. PCR specific region from samples and analyse

Question 26

What do you do with all the DNA samples?

Answer 26

TILLING analysis is based on the formation of heteroduplexes between WT and mutant strands of DNA.
If you take a region of DNA, mix WT and mutant, denature and reanneal, some heteroduplexes will form between WT/mutant.
Then use a mismatch nuclease eg CELI to cleave the heteroduplex at the mismatch site.
Different methods now used…

Question 27

Describe advances in TILLING using high-throughput techniques

Answer 27

1. Non CELI (nuclease) based identification of heteroduplexes
   Conformation Sensitive Capillary Electrophoresis (CSCE)
   High Resolution DNA Melting analysis (HRM)
2. Next Generation Sequencing (NGS)
   Significant increase in pool size (requires pooling strategy and bioinformatics pipeline to resolve)
   Allows greater number of genes to be surveyed

Question 28

Give an example of where TILLING is used today

Answer 28

Starch is 75-80% amylopectin and 20-25% amylose. High amylose content wanted in wheat for consumption, high amylopectin wanted in eg potatoes for the adhesives industry.

Question 29

READING: What is the CIMMYT?

Answer 29

International Maize and Wheat Improvement Centre (in Mexico)

Question 30

Why was GR success not seen in Africa?

Answer 30

The GR strategy was not appropriate where population densities were low and market infrastructure was poor.
The poor also often depended on orphan crops as opposed to the three main staple cereals.

Question 31

What is meant by an ‘agricultural renaissance,’ and how has private investment enabled it?

Answer 31

Huge advances being seen in agriculture worldwide. Private RnD and supply chains have been main driver behind dev of Bt cotton production across Asia and S.America.
CIMMYT in partnership with Monsanto to develop drought-tolerant maize for Africa

Question 32

What is HTTP?

Answer 32

High Throughput Phenotyping Platforms.
Our ability to phenotype has limited our full use of new molecular tools. Advances in phenotyping needed. HTTPs eg European Plant Phenotyping Network - fully automated faciliites in greenhouses or growth chambers. Remote sensing techniques to assess plant growth and performance.