Lecture 7 - Exploiting natural and induced genetic variation in crops - part 1

Question 1

How can the segmented colinearity of genomes be estimated?

Answer 1

By using molecular marker systems based on low copy sequences

E.g. Brassica napus and arabidopsis involved sequenced RFLP markers

Question 2

What is the degree of colinearilty in protein coding genes of plants?

Answer 2

Plants typically show high degree of short range colinearity (conserved synteny) between the genomes of closely related species

[transposon related sequences vary much more than protein coding genes]

Question 3

What is observed in the genomes of ancient polyploids?

Answer 3

Extensive interspersed gene loss

Question 4

How are the genome evolutions of arabidopsis and brassica different?

Answer 4

• Arabidopsis is simple
• Last polyploidy event 35mya ago and since then have had a continual process and stabilisation and diploidisation.
• Crops tend to be more complex
• 3 rounds of polyploidy and diploidisation since the shared ancestor followed by a hybridisation process for B. napus
• In arabidpopsis have regions of the genome related to one another through the duplication event (orthologous relationships)
• however in Brassica there are 12 related sections

Question 5

How can restriction fragment length polymorphism markers be used to estimate the segmental colinearity of genomes?

Answer 5

RFLP markers

Sequences of these markers are often in genes

Can be related to genome sequences through comparative genomics

To determine the structure/organisnisation of unknown species with models e.g. arabidopsis

RFLPs arranged in order of the model genome

e.g. brassica napus and arabidopsis show coserved colinearity - typically observed in dicots

Question 6

How is the genome structure typically observed in dicots?

Answer 6

conserved segmental colinearity between related species in low copy sequences

Question 7

Why is it easy to identify the arabidopsis genome?

Answer 7

because the arabidopsis genome has been fully sequenced

Question 8

How can the genome structure of species be identified without the whole genome sequence?

Answer 8

Base instead on a set of overlapping backbones (RFLP)

Question 9

What do the lines linking genomes in a colinearity plot mean?

Answer 9

Which genes in one genome (e.g. brassica) have sequence similarity to those in the model genome (e.g. arabidopsis)

Question 10

How is colinearity shown through a colinearity genome plot?

Answer 10

• As genes of the species of interest is arranged in order on the model species can see a colinearity between the genomes
• can establish where there are orthologous relationships of gene between the two species

Question 11

What is typically shown in between plant genomes of closesly related sequences in colinearity plots?

Answer 11

Typically plant genomes shows a high degree of short range colinearity (conserved synteny) between the genomes of closely related species

• genes are in the same order in crop species as in model species (only for protein coding genes)

Also show extensive interspered gene loss in the geomes of ancient polyploids

Question 12

How do the structures of protein coding genes differ from transposon related genes?

Answer 12

Transposon related genes: vary much more than protein coding genes

Question 13

How does gene content vary between species/genomes?

Answer 13

• variation in copy number of tandem gene repeats (common)
  
  • decrease/increase in different species related to the ancestral species
• instances of genome-specific deletion
  
  • e.g. between arabidopsis and b.napus/rapa/oleraceae, after the formation of the tetraploid. B. rapa has a gene missing but is present in oleracae and napus.
  • Sometimes in progenitor species gene may have been lost even though it is seen in the polyploid
  • as polyploids derived from a specific individual and what we have as a representative may significantly differ in genome composition
• Species-specific genome deletion occurs but is rare

Question 14

Why is there variation of gene content between species/genomes?

Answer 14

Because polyploid genomes are dynamic

Question 15

What is a common problem when interpreting the colinearity of genomes?

Answer 15

Interpret comparisons relative to the model system (e.g. arabidopsis) as if it represents the ancestral genome. The two species share a common ancestor.

e.g. may see that a gene would have been shared in the whole genome sequence of arabidopsis but since divergence arabidopsis has lost it.

This occurs when comparing polyploid genomes that have evolved through a diploidisation processes with a corresponding model gene

Question 16

How does polyploidy affect genetic diversity?

Answer 16

Polyploidy represents a major bottleneck to genetic diversity.

Question 17

Why does polyploidy represent a major bottleneck to genetic diversity?

Answer 17

The variation represented by polyploids when they are first formed is only that which is generated by the direct parents and any mechanisms which occur after. Crops therefore lack genetic diversity in relation to the wild species that exist.

Question 18

Why do crop plants appear to lack genetic diversity in comparison to wild relatives?

Answer 18

Crops are a domesticated version of the wild species with a number of important characteristics having been selected for, and everything that is genetically linked to the important loci in these characteristcis tends to be selected along during domestication reducing the available diveristy.

• Selecting for domestication events and linkage drag

Question 19

Give some exmaples of traits that have been selected for during the domestication of crop plants

Answer 19

1. Shatter resistance - cereals (SH1)
   
   • seeds stay on the plant instead of breaking off and being dispersed
2. Inflorescence architechure (tb1)
   
   • selected to have different morphology to wild species e.g. teosinte branched in maize reducing the branching and permitting the formation of large ears on the plant
3. Determinate growth habit (TFL1)
   
   • control of determinancy
   • Instead of growing/flowering/producing seed indefinately this is stopped e.g. teminal flower orthologs
   • seeds that have set can go onto maturity instead of having the resources divested to produce more and more seed that will never ripen

Question 20

How can resynthesis of the polyploid increase diversity for crop breeding?

Answer 20

Exploits the genetic base of hybrid (brassica napus) by exploiting the genetic variation available across its progenitor species (B. rapa and B oleraceae)

Question 21

How is resynthesis mostly achieved?

Answer 21

By embryo rescue

Question 22

How does resynthesis add additional diversity that may not have been present in the origianl hybrid plant?

Answer 22

Will have new representatives of the ancestral parent plants and so will bring in new genetic diversity that can be crossed with the polyploid crop

Eg. in brassica form a fully fertile tetraploid b. napus from ancestral species

Question 23

What is fixed heterosis?

Answer 23

The enhanced vigour shown by resynthesised hybrid progenitor species e.g. resynthesised B. napus.

Synomymous to F1 vigour as seen in hybrid plants

Question 24

How was fixed heterosis shown to be advantageous?

Answer 24

Hybridisation of B. rapa with B.oleracea. Young plants grown at the same time, resynthesised B. napus shows increased vigour

Question 25

Why are resynthesised hybrids not easy to work with?

Answer 25

Two genomes have come together for the first time in the new plant Events occur that attempt to stabilise the new genome which has 2Xgenetic material of the ancestral species

Question 26

What are the common mechisms of stability of resynthesised plants?

Answer 26

1. Altered genome methylation 2. Homeologous exhanges 3. Variation in copy number of genes leading to off types by chromosome abnormalities * Polyvalent chromosome * Abberent chromosome fragments

Question 27

Why might a resynthesised plant change the methylation pattern of DNA at the beginning of the stabilisation process?

Answer 27

To shut down excess copies of gene

Question 28

How can the process of methylation for stabilisation be observed?

Answer 28

With southern blots 1. Hybridise with RFLP probes/gene specific probes after digestion of genomic DNA with different restiction enzymes * one sensititve to blocking of methylation of the target site * One which is not sensititve 2. Where methylation occurs it results in a shift in the band on the southern blot to indicate a methylation event compared to that of the earlier generations.

Question 29

What is homeologous exchange?

Answer 29

Mechanism of genome stabilisation following resynthesis of a hybrid crop Innapropriate pairing of chromosomes resulting in an exchange of homeologous regions of the 2 genomes

Question 30

Give an example of homeologous exchange in resynthesised B.napus

Answer 30

1. Southern blot hybridisation with RFLP marker 2. 3 markers arranged down the arm of one chromosome 3. expected to hybridise A chromosome and C chromosome (expect to see sum of the A and C chromosomes) 4. Expected pattern seen 5. Also incidence on chromosome 17 where loss of the A genome and intensification of the C genome (where C genome was duplicated and replaced the A genome on the other homolgog) 6. Incidence on chromosome 3 where loss of C genome and intensification of the A genome bands

Question 31

What do homeologous exchanges result in?

Answer 31

Phenotypic variation * Different plants generated by multiple generations of the same resynthesised plant can show different characteristics

Question 32

Give an example of the phenotypic variation generated through homeologous exchange

Answer 32

1. **Flowering times** * B.rapa has early flowering times * B oleracea has late flowering times * over time see an increasing spread derived from hybridisation because of a variation in the cope number of genes * lots of opportunities for selection

Question 33

Give an example of a crop that been developed from the resynthesising approach

Answer 33

**Resynthesised B. napus vegetable Hanakkori** 1. Saishin (AA) crossed with Broccoli (CC) 2. Formed Hanakkori genome (AACC) * Followed up at the chromosome level and shown had created a new type of vegetable with intermediate morphological phenotypes

Question 34

What seed off types were observed in the generation of the resynthesised B. napus vegetable Hanakkori?

Answer 34

* On type * Large lobes * Waxless * No lobes * Anomaly

Question 35

Why are off types produced during resynthesis?

Answer 35

When looking for a particular morphology Anything that is a resynthesised polyploid get variation giving a range of morpholgy Occurs during the breeding process e.g. Waxless leaf morphology v common in brassica - has 40 chromosomes whereas B. napus should have 19 pairs (38 chromosomes). Waxless has two extra. Stained chromosomes with 45S rDNA (red) and CentBr2 (green) showed had 3 and a half copies of a chromosome where should have had 2 copies

Question 36

What is the general characteristic of the chromosomes of off-type morphologies of resynthesised hybrids?

Answer 36

Normally have more/less than the normal chromosome number of the hybrid **however** even in individuals with the correct number of chromosome get a variation

Question 37

How can variation in resynthesised hybrid morphologies occur when the number of chromosomes is as expected?

Answer 37

1. **Polyvalent chromosomes** * pairing of genomes not with the chromosome from the correct genome but from another genome * leads to instability 2. **Aberrent chromosome fragments** * ​​Seen in telophase 2 * Sections of chromosomes floating around that may/may not be inherited by particular chromosomes

Question 38

Aside from the use of resynthesised crops, how else can genetic diversity be increased in polyploid crops?

Answer 38

**Alien introgression**

Question 39

What is alien introgression?

Answer 39

Crossing plant with a species that is outside the normal germplasm of the crop

Question 40

Give an example of alien introgression in B. napus

Answer 40

1. Take the crop you want to increase the genetic diversity of - B. napus. 2. Cross with anoth brassica species e.g. B. carinata (BBCC) - which has essentially half the genome of napus (AACC) and a B genome which is completely unrealted 3. Crossed to produce an F1 with a mixture of all the different genomes 4. Repeat multiple backcrosses with Napus, selecting individuals more related to Napus than carinata and are fertile and produce seeds 5. This series of backcrosses back to the plant of interest means in each generation maintain a smaller and smaller proportion of the alien genome that is being captured into the genome of interest 6. end up with A and C and the genome variation from the other species - a small amount of captured genome

Question 41

Why is alien introgression used?

Answer 41

Long term process used on a high value trait e.g. the ability to make hybrid crops efficiently e.g. the F1 veg hybrids bought to demonstrate heterosis

Question 42

What are the examples of alien introgression in Brassica species

Answer 42

* CMS restorer function from R. sativus to B. napus * Stem canker resistance from B. nigra (B genome) to B. napus * Clubroot resistance from B. nigra to B. napus and from Raphanus sativus (radish) to B. oleracea * Alernaria blight resistance from Sinapis alba to B.napus

Question 43

What is the purpose of the male sterility system?

Answer 43

So that the parents of hybrids make males that do not produce pollen and pollen can be taken from another plant and pollinate the hybrid.

Question 44

What is the normal technique of the commercal male sterility system?

Answer 44

Normally done with cytoplamsic male sterility. Based on problems with cytoplasmic systems e.g. mitochondria Then complement with a restorer system with a male bought into the cross so that fertility is restored and subsequence plants produce seed

Question 45

Why are crops generally more susceptible to disease than their wild relatives?

Answer 45

Have a lack of diversity in disease resistant loci Especially problematic as crops are essentially grown in a monoculture

Question 46

Outline the use of the alien introgression technique and the cytoplasmic male sterility system in brassica

Answer 46

1. Used all traditional methods 2. Chromosome C9 in brassica shown against the results of sequencing an individual brassica plant and mapping the sequence reads onto the chromosome 3. Colour coding dependent on whether information from the A or the C genome (Yellow - A genome; Cyan - C genome; Magenta - radish genome) 4. In normal brassica napus have A and C genomes so looks green 5. Radish sequenced and mapped onto chromosome C9 to show a differentiation between radish and brassica genome, ordered according to the order of genes in brassica 6. Brassica breeding material (oil seed rape production system): female plant produced by breeding with cytoplasmic male sterility system included. 7. Female series of indiviual plants then mapped onto the C9 brassica chromosome (as have A and C genome get green colour throughout) 8. The result of the introgression of male restorer from radish (Raphanus sativus) into B. napus aligned to the same chromosome shows a combination of magenta on top of the cyan. Has replaced the C region with the blue region representing same half of the genome. 9. Most oil seed rape crop is hybrid developed used this system 10. No change in morphology