PLNT 3140 Introductory Cytogenetics -2024

Genome Evolution - or Making Sense of the Genome

In introductory genetics, we are presented with linkage maps that make the eukaryotic genome look like it is cast in concrete. Indeed, perhaps the most difficult thing for many geneticists to swallow about transposition was the 'Pandora's box' of genomic chaos that it implied. Transposition relegated the gene from its status as a house built upon a rock, to more of a molecular mobile home. Perhaps it is not entirely a coincidence that not long after McClintock's publications of transposition, that the model of plate tectonics shook the science of geology like an earthquake. The eukaryotic genome has never been the same since. In retrospect, we can see that evolution requires a fluid, dynamic genome. Without change, there is no evolution. We can now make several generalizations regarding genome evolution:

• Genome sizes and karyotypes can differ drastically even among closely-related species. This necessarily demands that fundamental changes in genome structure can occur over relatively short evolutionary times.
• Most of the changes occur in the middle repetitive fraction of the genome.
• Most of these changes are in non-coding DNA.

Genome size can change relatively quickly in evolution

The sizes of chromosomes in Crepis species are drawn to scale, accompanied by drawings of florets and achenes, also to scale. This example illustrates how drastically genome size and chromosome number can change in short evolutionary times. It also illustrates something often observed, which is that the sizes of plant organs such as leaves, stems or fruit are often larger in species with larger chromosomes.

Fig. 9-19. Species of the genus Crepis showing the size relations of chromosomes, florets (lacking ovaries), and achenes, all drawn to the same magnification so that the sizes are relative to each other. A. C. sibirica; B, C. kashmirica ; C, C. conyzaefolia; D, C. mungierii; E, C. leontodontoides ; F, C. capillaris; G, C. suffreniana; H, C. fuliginosa (rearranged from Babcock, 1947). from Cytogenetics - the chromosome in division, inheritance and evolution. Carl P. Swanson, Timothy Merz and William J.Young. 2nd Edition. Prentice-Hall. Chapter 9. Evolution of the karyotype. achene (əkēn') , dry, simple, one-seeded fruit with the seed attached to the inner wall at only one point. Achenes are indehiscent, i.e., they do not split open at maturity. The so-called seed of a sunflower is an achene; the shell is the wall of the fruit, and the true seed lies within. A strawberry consists of many achenes embedded in a fleshy receptacle.

Genome Size is Related to Life History

The time required for mitosis and meiosis increases with genome size.

Annuals tend to have smaller genomes and shorter mitotic cycles. Perennial plants tend to have larger genomes and longer cell cycles.

Annuals have to grow very rapidly in spring. Therefore, ecological competition forces annuals to have smaller genomes.

Question: Is the perennial habit more tolerant of a longer mitotic cycle? What is the advantage of a larger genome or longer mitotic cycle?

Bigger organs means more biomass utilized, which means more biomass needed. Big genomes are probably better tolerated  when habitat is not limiting eg. food, water, light, space. The prediction would be that small genomes are favored under limiting conditions. This may be why our crops have big genomes. We pamper them with a good environment. Domesticated crops are poor competitors outside of cultivation.

Take home lesson: Genome evolution can be constrained by life history.

Allopolyploids are the result of speciation by interspecific hybridization

evolution - The change in allele frequencies within a population. This definition could encompass any genetic change, from single point mutations, to major chromosomal rearrangements.

speciation - The evolutionary process by which two populations diverge, resulting in decreased ability to hybridize. As populations diverge genetically, reproductive barriers arise, preventing mating, interfering with meiosis, or resulting in hybrids that are less fit than progeny from within-population matings. When fertile progeny can no longer result from matings between two populations, speciation can be said to be complete.

However,  taxonomists often define phenotypically distinct populations as species, even when reproductive barriers are not complete.  On the one hand, the greater the genetic divergence between two species, the lower the fertility rate will be in interspecific hybrids.  On the other hand, when interspecific hybridization does occur, it can often result in the creation of a new species, distinct from the parental species.

The creation of allopolyploid species is through interspecific hybridization between morphologically distinct diploid species, with retention of both chromosome complements. This effectively doubles the genome size C and the chromosome number  n. (amphidiploidy= allopolyploidization)

Determination of ancestral species can be made based on geographical distribution, morphological features, chromosome counts, karyotype analysis, isozyme banding patterns and molecular studies.

Putative ancestors can be verified through interspecific hybridization, synthesis of allopolyploids, comparison of morphological traits, meiotic pairing and fertility in the allopolyploids and their hybrids.

Example: the genus Brassica

The genus Brassica consists of three elementary or basic genomes Brassica campestris, or Brassica rapa (A; n=10), B. oleracea (C; n=9), and B. nigra (B; n=8). These diploids are secondary polyploids from an extinct species with a basic chromosome number of x=6, based on pachytene chromosome analysis in the haploid genomes. If 1-6 represent the ancestral basic six chromosomes, the following represents individual diploid genomes' complement of these basic six chromosomes.

Evolution of chromosomes

Based on what we have already discussed in previous lectures, we can summarize the chromosomal changes that contribute to genome evolution:

• amplification and deletion of repetitive sequences
• deletions
• duplications
• inversions
• translocation
• transposition
• polyploidy

All of these may contribute to mispairing of hybrids in meiosis, which represents a reproductive barrier. Reproductive barriers therfore drive speciation. Once two populations are reproductively isolated, each population will accumulate further mutations, until hybridization is no longer possible.

Chromosomal rearrangements seem to characterize differences between closely related species

Fig. 3-D from Schröck, E. et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273: 494-497.

Even between primates such as gibbon and man, a large number of chromosomal rearrangements are evident. In the figure, chromosome painting of gibbon chromosomes with a human chromosome painting kit shows that all gibbon chromosomes hybridize with human sequences. However, most chromosomes exhibit many colors, indicating that sequences homologous to several human chromosomes are present in each gibbon chromosome. This experiment illustrates the point that genomes can differ substantially, even between very closely-related species.

The longer two species are reproductively isolated, the more chromosomal rearrangements each species will accumulate. Thus, chromosomal rearrangements are an important component of genome divergence, as species diverge.  

As speciation progresses and species diverge, chromosomal rearrangements, deletions and duplications cause genomes to diverge. Given enough time, one would expect that the order and location of genes would become completely different, between distantly related species. However, even between distantly-related species, short regions of conserved chromosomal segments, or microsynteny, can be recognized. A comparison of several grass genomes showed substantial microsynteny near the adh1 locus in sorghum, maize and rice. While gene order was conserved, the intervening DNA showed numerous insertions/deletions of both genes and non-coding DNA. These results show that it is possible for gene synteny to be maintained even though the distances between genes can get larger or smaller.

Take home lesson: As two species become more and more diverged, meiotic pairing becomes more and more complex. This results in decreased fertility of hybrids.

1. Microsynteny - conservation of genes over short chromosomal regions

As speciation progresses and species diverge, chromosomal rearrangements, deletions and duplications cause genomes to diverge. Given enough time, one would expect that the order and location of genes would become completely different, between distantly related species. However,  even between distantly-related species, short regions of conserved chromosomal segments can be recognized.

Bennetzen JL, Ramakrishna W (2002) Numerous small rearrangements of gene content, order and orientation diffferentiate grass genomes. Plant Mol. Biol. 48:821-827.

A comparison of several grass genomes showed substantial microsynteny near the adh1 locus in sorghum, maize and rice. While gene order was conserved, the intervening DNA showed numerous insertions/deletions of both genes and non-coding DNA. These results show that it is possible for gene synteny to be maintained even though the distances between genes can get larger or smaller.

# - gene lost from maize adh1, relative to sorghum
* - genes acquired by sorghum adh1 region
Boxes with arrows indicate known or predicted genes

Take home lesson: Drastic gain or loss of interspersed repetitive sequences can take place between genes, without disturbing the order of genes on a chromosome.

2. Comparison of the Drosophila and mosquito genomes shows large blocks of synteny, and partial polyploidization events.

Evgeny M. Zdobnov,^1* Christian von Mering,^1* Ivica Letunic,^1* David Torrents,¹ Mikita Suyama,¹ Richard R. Copley,² George K. Christophides,¹ Dana Thomasova,¹ Robert A. Holt,³ G. Mani Subramanian,³ Hans-Michael Mueller,¹ George Dimopoulos,⁴ John H. Law,⁵ Michael A. Wells,⁵ Ewan Birney,⁶ Rosane Charlab,³ Aaron L. Halpern,³ Elena Kokoza,⁷ Cheryl L. Kraft,³ Zhongwu Lai,³ Suzanna Lewis,⁸ Christos Louis,⁹ Carolina Barillas-Mury,¹⁰ Deborah Nusskern,³ Gerald M. Rubin,⁸ Steven L. Salzberg,¹¹ Granger G. Sutton,³ Pantelis Topalis,⁹ Ron Wides,¹² Patrick Wincker,¹³ Mark Yandell,³ Frank H. Collins,¹⁴ Jose Ribeiro,¹⁵ William M. Gelbart,¹⁶ Fotis C. Kafatos,¹ Peer Bork¹ (2002) Comparative Genome and Proteome Analysis of Anopheles gambiae and Drosophila melanogaster  Science 298: 149-159.

A - 1:1 orthologs. Gold and green lines indicate matchups between orthologous genes in Dm chromosome arms 2L and 2R with Ag chromosome 3R. This is a gene-by-gene comparison between chromosomes (ie. each line represents a gene for gene match).

B - microsynteny blocks. Blocks of known loci conserved between Drosophila and Anopleles are indicated in gold and green lines. Blocks  indicate groups of genes that are in the same order when compared between two chromosomes (ie. each line is a match between microsyntenic blocks).

• Both arms of Drosophila chromosome 2, designated 2L and 2R, have a largely conserved gene order, when compared to chromosome 3R of Anopheles.

• The fact that many of the lines cross indicates probably at least two ancient inversion events. This is easier to see in B.

• The fact that both 2L and 2R are roughly colinear with 3R suggests that Drosophila chromosome 2 resulted from a Robertsonian translocation* of one copy of chromosome 2 to another.

• Although gene order is largely preserved between Ag3R and the two arms of Dm2, the distance between genes appears to be greater. This suggests that either Ag3R gained a lot of repetitive DNA, interspersed between genes, or Dm2 lost a lot of interspersed repetitive DNA.

*Robertsonian translocation - chromosomal rearrangements that result from the fusion of the entire long arms of two acrocentric chromosomes
Zhao et al. (2015) Robertsonian Translocations: An Overview of 872 Robertsonian Translocations Identified in a Diagnostic Laboratory in China  PLoS One. 2015 May 1;10(5):e0122647. doi: 10.1371/journal.pone.0122647

C - More complex relationships between Ag arms 2L and 3L with Dm arms 3L and 2R. Each block of orthologous loci is indicated by red, green, purple or magenta lines.

• Gray  bars indicate gene density in a sliding window of 1 Mb.

• Anopheles chromosomes have larger gene-poor regions than Drosophila chromosomes

• Microsynteny between Ag2L and Ag3L, with Dm3L and Dm2R indicate even further duplications of chromosome arms.

Eukaryotic genomes often contain lots of hidden polyploidy! 

C. Allopolyoploidization can cause reproducible changes in genome size, not accounted for by the adding the sizes of the two genomes together

H. Ozkan, M. Tuna, and K. Arumuganathan (2003) Nonadditive Changes in Genome Size During Allopolyploidization in the Wheat (Aegilops-Triticum) Group. Journal of Heredity 94:260-264

The accompanying table shows data from an experiment in which various relatives of wheat were crossed, followed by selfing, to create amphiploid hybrids. The amount of DNA in nuclei was measured by flow cytometry of chromosomes. DNA amounts are given in picograms (pg) per nucleus.

Key observations:

a) The observed DNA value in hybrids is always about 2 pg less in hybrids than would be expected by simply adding the genome sizes of the parents.

b) The loss of 2 pg seems to be consistent and reproducible, in many crosses. This implies some sort of programmed response

Possible mechanisms:

• non-homeologous pairing resulting in chromosome breakage, followed by loss of part of one or more chromosomes.
• Nulli/tetrasomy
• mobilization of transposable elements

Evolution of interspersed repetitive DNA

Flavell, R.B., Rimpau, J. and Smith, D.B. (1977) Repeated sequence DNA relationships in four cereal genomes. Chromosoma 63:205-222.

We can think of the process of speciation as beginning with the reproductive or geographical separation of two populations of a species. Initially, the two populations have essentially identical genomes. Over time,  mutational events, ranging from point mutations to chromosomal aberations to polyploidization occur. As two species diverge from a common ancestor, the percentage of sequences shared by both species decreases, as each species independently gains some sequences and loses others.

C₀t analysis is a powerful method for measuring the percentage of sequences shared between two genomes. If DNA from a single species is allowed to reanneal, essentially all of the DNA should reanneal if hybridization is allowed to go to completion. If DNA from two closely-related species is hybridized, one would expect less than 100% reannealing. The more distantly -related two species are, the lower the percentage of the DNA that should reanneal.

Specific subsets of repetitive DNA can be amplified or deleted

The relationships betwee four cereal species, in order of divergence from a single common ancestor, are shown at right. For example one speciation event separated the ancestor of oats, from the common ancestor of barley, wheat and rye. A later speciation event separated the ancestor of modern barley from the common ancestor of wheat and rye. A third speciation event separated wheat and rye into distinct species. Once they diverge as separate species, genomic sequences accumulate mutations. As well, some repetitive sequences are lost, while new repetitive sequences may arise. Thus, the set of repetitive sequences in these genomes would be expected to become more and more different, with time since speciation.

We can define several categories of genomic sequences in cereal species: 

Group I sequences - present in all cereals
Group II - barley, wheat & rye, but not oats
Group III - wheat & rye
Group IV - wheat only
Group V - rye only
Group VI - barley only
Group VII - oats only

The more sequences two DNA samples share in common, the higher the percentage of DNA that should hybridize, between two species. We can take advantage of this to calculate the percentage of each of the Group I through VII sequences in each of the four species.

Experimental design

An excess of unlabled DNA from each of the four species was hybridized with a range of labeled probes. The probes consisted of in-vivo labeled DNA, extracted from cells in culture which were fed radio-labeled nucleotides. Labeled DNA was sheared to a range of size classes, up to 1000 bp. In separate C0t experiments, each probe was hybridized with unlabeled DNA from one of the four species

An interesting observation from this experiment was that percent duplexed DNA (an indication of relatedness) increased with an increase in fragment size. Researchers interpreted this to mean that repetitive DNA was interspersed with single-copy DNA.

"Since duplexes formed from randomly-sheared fragments frequently have single stranded tails, better estimates of the proportions of the labelled DNAs in the renatured conformation come from extrapolations of the curves in Figure 2 to the ordinate" [Davidson, et al., (1973) J. Mol. Biol. 77:1-23.]

If single-stranded tails are present, you would be overestimating the amount of duplexed DNA. Thus, extrapolating to 0 length fragments solves this problem.

Based on the known ancestries of the four species, we can say in advance which classes of repetitive families are found in each:

To calculate the percentage of each class in a given species, we subtract the inter-species hybridization result, as measured at the Y-intercept, from the within-species result.

"For example, the intercept value when labelled wheat was hybridized to unlabelled oats DNA is 22%. Group I in wheat is therefore 22%. The intercept value in the labelled wheat+unlabelled barley DNA curve is 32%. Group II in wheat is therefore 32-22=10%. The intercept in the labelled wheat+rye DNA curve is 58%. Group III in wheat is therefore 58-32=26%. The intercept in the labelled wheat+unlabelled wheat DNA curve is 74%. Group IV in wheat is therefore 74-58=16%."

Some specific families of middle repetitive sequences show high variation

Between species of the same genus, certain middle repetitive families can vary greatly. In Drosophila, most individual species possess a different combination of the middle repetitive families, with the effect that very few families are present in all the species. In an experiment, D. melanogaster cloned repetitive sequences were used as a probe against DNA from other Drosophila species.

Figure 1. Restriction fragments of genomic DNA from adult D. melanogaster (D. mel), D. erecta (D. ere), D. yakuba (D. yak), D. simulans (D. sim), and D. mauritiana (D. mau) hybridized to 32P-labeled repetitive cloned segments of D. melanogaster, pDm 366, pDm 274, and pDm 73. Total genomic DNA was digested with Eco R1, fractionated by electrophoresis on an 0.8% agarose gel, and transferred to nitrocellulose. Numbers on the left give the length in kb of HindIII fragments of phage lambda. From: Dowsett, A. P. (1983) Closely related species of Drosophila can contain different libraries of middle repetitive DNA sequences. Chromosoma 88:104-108.

In fact, some of these middle repetitive elements are transposons:

Figure 2 A,B. Restriction fragments of adult DNA from five species digested with HinfI and hybridized to clones containing copia (A) and 412 (B). In situ hybridization showed that these stocks possess the following number of copies of copia: D. melanogaster , 10±2; D. simulans, 4±1; D. mauritiana, 4±1; D. yakuba, 0; and D. erecta, 0; and the following number of copies of 412: D. melanogaster, 24±4; D. simulans , 12±2; D. mauritiana, 9±2; D. yakuba, 3±1; and D. etecta, 0. The two lanes of D. yakuba DNA are from different stocks. Numbers on the left give the length in kb of HaeIII fragments of Phi-X 174

Transposons play a larger role in evolution than originally thought

Let's return to transposons, which we introduced a little while ago in the section on changes in chromosome structure. Transposons are mobile elements that can essentially copy and insert themselves into different places in a chromosome, along with the help of some activator gene. First discovered in maize by Barbara McClintock, they usually make up a large percentage of plant and animal genomes.

Transposons are a major component of plant genomes

• Transposable elements make make up a large percentage of most plant genomes, although there is a great deal of variation between species.
• Class I elements go through an RNA intermediate.  They are transcribed into RNA, reverse-transcribed into dsDNA, and then inserted into random sites in the chromosomes. 
  
• Class II elements transpose as dsDNA.
• In most plant species, Class I elements are more prevalent than Class II elements
• The examples of Cotton and Foxtail Millet demonstrate that even between different lines of the same species, there can be substantial differences in the transposon content of the genome. This suggests that major gain or loss of transposable elements can occur in a very short time.

Transposons mobilize in short evolutionary time frames

Gabriel (Gabriel A, Dapprich J, Kunkel M, Gresham D, Pratt SC, Dunham MJ (2006) Global Mapping of Transposon Location. PLoS Genet 2(12): e212)

As a proof of concept experiment, the authors developed an approach to use microarrays to detect the locations of transposons in yeast. This method was tested on two yeast strains whose genomes have been fully sequenced, and the locations of transposons identified from the sequence. Microarrays were designed with oligonucleotide probes from unique sequences, spaced roughly every 300 bp throughout the genome. The results verify that the the locations of almost all annotated transposons were correctly identified.

Step One: Isolate regions flanking transposable element insertions

1. Digest yeast genomic DNA with a restriction enzyme known to cut within transposons Ty1 and Ty2.

2. DNA samples from 2 strains RM11 and S288c.

3. Many fragments have a partial transposon at one end and regions flanking the insertion at the other end.

4. Denature DNA and use transposon-specific primer to synthesize DNA containing biotinylated nucleotides. Biotinylated fragments are purified by mixing with streptavidin-coated magnetic beads. Biotinylated DNA is bound by streptavidin, and magnetic beads are washed to remove unbound DNA. After washing, biotinylated DNA is released from the beads by washing in a buffer. The resulting purified sequences are enriched for unique sequences which flank transposon insertions.

5. Sequences are labeled with fluorescently labeled nucleotides: RM11 - Cy3 (green); S288c - Cy5 (red)

6. Mix labeled DNAs and hybridize with a yeast microarray containing unique yeast probes. Probes have been designed so that they do not contain Ty1 and Ty2 sequences any other repetitive sequences Consequently, probes will only hybridize based on the unique flanking regions from the labeled DNA.

7. Results are superimposed on chromosome maps.

Red Peaks: Ty1 or Ty2 unique to S288c
Green Peaks: Ty1 or Ty2 unique to RM11
Black circles: full length Ty1 or Ty2 elements identified from S288c genomic sequence
Triangles: full length Ty2 elements identified from RM11 genomic sequence
1,2,3,4 - false negatives
5,6 - false positives

Step Two: Identify differences in transposon location between yeast strains

These data show that even between strains, tremendous rapid changes in the locations of transposons can occur. The differences in the locations of transposons between the two strains provide evidence that locations and numbers of transposons in yeast can change, genome wide, over very short evolutionary time scales.

Transposons are activated by stress

In most species, transposition is suppressed by epigenetic imprinting. Methylation of carbon residues prevents mobilization of transposons, which results in genome stability across generations. Methylation patterns are known to be inherited from one generation to the next in most higher eukaryotic species. Barbara McClintock first observed that what we now know to be the suppression of transposition is relaxed during stress, resulting an a de-repression of transposition.

Table 2 shows that there are many stress conditions which can mobilize transposons,  including:

• heat stress
• UV light
• desiccation
• allopolyploidization
• passage through tissue culture
• osmotic stress (salt stress)
• irradiation
• wounding
• microbial infection

As well, mutations in methyltransferases and even the genetic background of plants in a cross can induce mobilize transposons.

Why have so much repetitive DNA?

The amount of repetitive DNA present in eukaryotic genomes seems almost excessive. What's the point of having so much repetitive DNA? Especially the repetitive DNA that is non-coding! Although repetitive DNA (particularly highly-repetitive fractions) does not contribute by coding for a protein, it has other functions for the eukaryotic cell.

Middle repetitive DNA makes it easier to generate genetic diversity while decreasing the potential risks of recombination

Genetic diversity can be generated by unequal crossing over. If there are two copies of a gene, some crossover events can lead to duplication of the gene on one chromosome, and deletion on the other. Successive duplication or deletion of copies can lead to increases or decreases in the size of multigene families.

If a gene duplication does occur, one copy of the gene is free to mutate, while the other can retain its original function. This allows evolution to proceed without the risk of a deleterious mutation, while still being able to take advantage of beneficial mutations, should they occur.

Unequal crossing over could have deleterious effects if it occurs within a coding region. If all recombination occurred in coding regions, then you would have a high frequency of inactivation of genes. This may be tolerated in unicellular organisms (bacteria and fungi) that have very little repetitive DNA. In this case their high reproductive rates my be such that, a) even if a fraction of individuals gets deleterious mutations due to recombination, the rapid growth rate is sufficient to prevent a population bottleneck: b) Furthermore, there is probably a selective advantage for having a small genome if you have a rapid growth rate.

The roles of some middle repetitive DNA are still unclear

Other proposed functions for middle repetitive DNA are a little more speculative. By definition, things like matrix attachment sites, and perhaps sites (if they exist) that govern higher levels of chromatin packaging, must be present in hundreds or thousands of copies. Therefore, such sites fall into the middle repetitive fraction of the genome.

On the other hand, middle repetitive DNA could simply be selfish DNA. Some sequences may just be more efficiently duplicated by the cellular machinery that duplicates sequences. Those sequences that lend themselves to being duplicated by the DNA replication machinery will tend to propagate throughout the genome. Darwinian selection operates even at the molecular level. Transposons are a good example of selfish DNA.

Middle repetitive DNA might play no significant role at all. Not everything has to have a selective advantage to get fixed in the population. Even if a particular trait or structure in the genome is selectively disadvantageous, it may take time to lose it after speciation occurs (speciation usually implies a population bottleneck). However, most of the domesticated species of plants and animals are not at equilibrium. In many cases, their genetic diversity is greatly limited by artificial selection. We have to be careful about inferring much about evolution from domestic species.