The Better Breeding Blog Breed Better Animals

People have always understood that characteristics of parents can be passed to offspring. But how this was actually done was a mystery. One theory — the now defunct Blending Inheritance theory — held that progeny inherited an average, or blending, of both parents’ features. Yet this didn’t adequately explain many observable results. For example, why does red hair or blue eyes skip generations? Why are most haemophiliacs male, and why is the disease as debilitating in them as in their ancestors, rather than lessening with each generation? Where did a solitary black spot on an otherwise pure white Merino sheep, with generations of pure white ancestors behind it, come from?

The breakthrough in understanding began in 1858, when Austrian monk Gregor Mendel began experiments breeding garden (edible) peas (Pisum sativum). He published his findings in 1866, but the significance of his work went unrecognised until rediscovered in 1900, sixteen years after his death in 1884. His paper described what came to be known as Mendelian Inheritance, and today he is universally acknowledged as the father of modern genetics.

What were his insights that had eluded everyone else? Mendel, with his (unusual for the time) background in both biology and mathematics, had deduced from his well-planned meticulous work and careful observations that inheritable characteristics aren’t blended, but are instead comprised of heredity units. He called these units “factors”, but you’d know them as genes.

Mendel set out to study seven characteristics of peas: seed shape, seed colour, seed coat colour, pod shape, pod colour, flower position, and stem length.

He began his experiments by first creating pure-breeding plants for each of those seven traits: plants that bred true generation after generation. Any plant that didn’t breed true was removed. This first step alone took a few years, as pea plants are seasonal and produce one generation a year.

At the end of this first step he had the following purebred strains:
Seed shape:
purebred round and purebred wrinkled

Seed colour:
purebred yellow and purebred green

Seed coat colour:
purebred coloured and purebred white

Pod shape:
purebred inflated and purebred constricted

Pod colour:
purebred green and purebred yellow

Flower position:
purebred axial (along the stem) and purebred terminal (at end of stem)

Stem length:
purebred long and purebred short

With these lines established, step two was to cross the purebred strains for each characteristic. His approach was very methodical, in that he not only focused on only one or two characteristics at a time, but he also undertook many identical matings so as to produce a large, statistically more meaningful data set.

With, for example, purebred round seed lines and purebred wrinkled seed lines established, only then did he cross round seed strains with wrinkled seed strains. Or green pod strains with yellow pod strains. If characteristics were indeed “blended”, the resulting offspring should have exhibited a seed shape partly round and partly wrinkled, or some indeterminate yellow-green pod colour. But they did not: every single seed was as round as the purebred round seed lines, and pods were as green their green pod parents. Mendel observed similar behaviour for the other characteristics studied (eg yellow seed strains crossed with green seed strains produced all yellow seeds), and called a character that was the only one to appear in a first cross generation dominant.

Step three was to cross the first cross generation with each other. From these results he noted that, for example, the round seed (dominant) phenotype appeared in roughly three quarters of the second cross generation, with the remaining quarter producing wrinkled seed. Mendel called the wrinkled phenotype recessive, as it had receded from a generation, or skipped a generation as we’d say today.

Mendel went further still, and crossed the second cross generation with each other to produce a third cross generation. He had found from his first crosses that, for example, short-stemmed plants were recessive to long-stemmed plants, and that yellow pods were recessive to green pods. In the third cross generations, plants with recessive phenotypes produced only plants with those same recessive phenotypes. The offspring of short-stemmed plants were always short-stemmed, and plants with yellow pods only ever produced yellow pods.

Mendel wrote his results in a lengthy paper called Experiments in Plant Hybridization, which was published in 1865 in Verhandlungen des naturforschenden Vereines in Brünn, the official journal of the Natural History Society in Brno (Brünn). You can read it here, and see where he drew on his mathematical background, if of interest. I will step through the results of Mendel’s crossings in more detail in the next post, hopefully in a way more clear and understandable!

This post will step through in greater detail all of Mendels results mentioned briefly in the It All Began With Gregor Mendel post.

To recap, we know that he first established the following pure lines of garden pea:
Seed shape:
purebred round and purebred wrinkled

Seed colour:
purebred yellow and purebred green

Seed coat colour:
purebred coloured and purebred white

Pod shape:
purebred inflated and purebred constricted

Pod colour:
purebred green and purebred yellow

Flower position:
purebred axial (along the stem) and purebred terminal (at end of stem)

Stem length:
purebred long and purebred short

He then crossed each line, eg purebred yellow seed with purebred green seed, purebred coloured seed coat with purebred white seed coat, etc.

When two organisms that are pure-breeding for a character are crossed, the parental generation is called the ‘P’ generation. The progeny from this cross is called the first filial generation, from the Latin word filius, a son. ‘F₁’ is the shorthand way of referring to the first filial generation. Crossing an F₁ generation with itself produces an ‘F₂’ generation (second filial generation). Crossing F₂ with itself produces an ‘F₃’ generation (third filial generation), and so on.

His results for this, first, parental cross were:

Instead of the F₁ generation showing “blends” of the parents’ characters, every one of the progeny exhibited in full exactly one of the two characters that had been crossed. Mendel called these dominant, and this is the word we still use today. From the F₁ Outcome column in the table above, Mendel found that round seeds are dominant over wrinkled seeds, yellow seeds are dominant over green seeds, and so on.

He termed the characters that did not appear as recessive. Wrinkled seeds, green seeds, etc, all receded from the F₁ generation.

Mendel next crossed the F₁ round seed types with themselves, the F₁ yellow seed types with themselves, etc, and recorded the resulting F₂ results:

Note how the results are roughly 3 to 1 ratios of dominant character to recessive character. This was the crucial discovery behind Mendel’s insight that these characters (‘factors’ as he called them, or genes as they became known) are units of heredity rather than a blend. He explained his discovery mathematically in his 1866 paper, but in 1905 British geneticist Reginald Punnett introduced his Punnett square as a teaching aid that explained breeding outcomes more simply.

The Punnett square is extremely effective at visually describing or predicting the results of Mendelian inheritance. Let’s introduce some terminology here before delving into how a Punnett square works.

Each characteristic (or trait) Mendel studied had two characters (or phenotype), eg the seed shape trait had two phenotypes: round and wrinkled; and the seed colour trait had two phenotypes, yellow and green. Round phenotype was found to be dominant to wrinkled phenotype, and yellow phenotype was found to be dominant to green.

In genetics the dominant phenotype is represented by an uppercase version of (usually) the first letter of the word that describes that dominant phenotype. For pea seed shape, round is the dominant character and so we denote this as ‘R’. Wrinkled phenotype is recessive, which we write as ‘r’, the lowercase form. Note we always use the same letter for the recessive phenotype as for the dominant phenotype, regardless of what word describes the recessive phenotype. If the dominant round pea shape is ‘R’, the corresponding wrinkled shape is always ‘r’, never ‘w’ for wrinkled, or any other letter. This shows at a glance which genes belong together. In Mendelian inheritance there is always a dominant gene and a recessive gene for each genotype: that is in fact the definition of Mendelian inheritance.

One last thing before getting back to the Punnett square — how those genes actually end up in the progeny.

I appreciate that most readers will already have the understanding that each parent carries two genes for each trait, and that the progeny inherits one of those genes from each of its parents to end up with two genes itself. Of those two genes at least one will be dominant and forms the phenotype seen in the progeny. (Please note that this is Mendelian inheritance — there are other patterns of inheritance that don’t follow these rules, which we will cover later.)

But most people don’t fully grasp the details of how only one gene per parent is passed onto progeny. I will cover these in a series of posts later, but for now this basic knowledge — that one parent contributes one gene and the other parent contributes a second gene for the same trait — will be more than enough to understand a Punnett square, so let’s move straight on.

Mendel began his experiments with pure-breeding lines, so let’s take the purebred round and the purebred wrinkled seed shapes as our example. As they are purebred, a round seed phenotype plant can only contribute ‘round’ genes and a wrinkled seed phenotype plant can only contribute ‘wrinkled’ genes. Because each plant has two genes for the ‘seed shape’ trait, we denote the purebred round seed genotype as ‘RR’ and the purebred wrinkled seed genotype as ‘rr’.

A Punnett square is simply a grid with one parent’s genes (genotype) across one side and the other parent’s genes (genotype) down the other. The inside squares represent all possible pairings that could arise when the two are crossed.

We can represent Mendel’s breeding of the purebred round seed plants he established his lines with like so:

You can see at a glance how all progeny can only ever inherit an ‘R’ gene from each parent, becoming pure ‘RR’ in turn and maintaining the pure round seed line.

Similarly, the breeding of the purebred wrinkled seed plants to maintain that line can be shown like this:

Again you can see how all progeny can only ever inherit an ‘r’ gene from each parent, becoming pure ‘rr’ in turn and likewise maintaining a pure wrinkled seed line.

Things become far more interesting once different lines are crossed. With a Punnett square we can actually predict the outcome of crossing a purebred round seed shape with a purebred wrinkled seed shape, as we know each parent’s genotype:

It’s very clear that every single outcome of this cross will produce progeny with an identical ‘Rr’ genotype. ‘R’ is the dominant gene and represents the round shaped seed phenotype. All progeny from such crosses — though they carry the recessive ‘r’ gene for wrinkled seed — will always have a round seed phenotype, exactly as Mendel observed. It’s also easy to see — and understand — why the recessive ‘r’ phenotype appears to have disappeared from the progeny. It didn’t disappear — it’s there but hidden.

Mendel then crossed these F₁ progeny. Again with a Punnett square we can predict the outcome he did in fact observe:

This F₂ generation comprises of one ‘RR’ genotype, two ‘Rr’ genotypes and one ‘rr’ genotype. (‘Rr’ and ‘rR’ in the square are the same genotype and it is more correct to write both as ‘Rr’.) We know ‘R’ (round seed) is dominant to ‘r’ (wrinkled seed), and thus from the above square we can see that crossing the F₁ generation results, statistically, in three dominant phenotypes for every one recessive phenotype. The three to one ratio Mendel’s own results resembled.

I say statistically because this is the mathematically determined, not absolutely guaranteed outcome. Mendel’s own observed ratios, after many many matings, never attained a perfect 3:1. They did come very close though, ranging from 2.82:1 to 3.14:1, helped by his large sample sizes.

(We see such things all the time — statistically there is a 50% chance a baby will be a boy or a girl, yet we all know of large families with all girls or all boys, though we’d expect equal numbers of both.)

The above Punnett square also explains how recessive genotypes reappear after skipping a generation (or more in some cases). it is only in an F₁ cross that a recessive gene can pair with its recessive partner to form the recessive phenotype. Even so there is only a one in four chance of this occurring.

Another point I wish to make concerns the one ‘RR’ and two ‘Rr’ genotypes. All three express the dominant phenotype, but only the ‘RR’ genotype is ‘pure’. This genotype is called homozygous, while the ‘Rr’ genotype is heterozygous. We’ll discuss the significance of these in later posts.

Mendel went further still and crossed the F₂ generations with each other. The possible crossings were:
RR with RR
RR with Rr
RR with rr
Rr with Rr
Rr with rr
rr with rr

By now you can probably picture these as Punnett squares and do them in your head! You can see how the first three crossings in the list will always produce all ‘R’ phenotypes, the next two will produce both ‘R’ and ‘r’ phenotypes, whilst the last one will always produce all ‘r’ phenotypes. Mendel had observed from his first crossings the existence of dominant and recessive phenotypes, but it took these third crossings to show the one thing that hadn’t been observable until then — that recessive phenotypes when crossed will only produce recessive phenotypes in the offspring. A dominant phenotype will never appear when recessive ones are crossed. This was a significant finding that will be covered in more detail later.

One last note: Mendel did cross plants with the purpose of studying two traits simultaneously, and I’ll summarise one example here to show how a Punnett square can be used for those more complicated situations.

Consider a pea plant with round, yellow seeds. As ‘round’ and ’yellow’ are both dominant phenotypes we shall label them ‘R’ and Y’ respectively. Now further consider that this plant is heterozygous for both traits. This means it also contains the ‘r’ gene for wrinkled seed and the ‘y’ gene for green seed. Its genotype can be written RrYy.

Cross this RrYy plant with another RrYy plant in a Punnett square to predict the results. Remember that a parent only contributes one of R and r, and one of Y and y, so make your gene combinations accordingly:

Hopefully you now have a good understanding of Mendelian inheritance, and can predict accurately any crossings of traits that consist of a clear dominant and clear recessive phenotypes.

The next post will wrap up this mini series on Mendel with a discussion of the three Laws he devised, and lead into a discussion into what it is at the cellular level that results in an organism inheriting a particular set of genes as opposed to some other set of genes.

From Mendel’s pioneering research on peas discussed here and here he devised three laws:

The First Law: the law of segregation
Mendel observed that inheritance was not some blend of parental characters. For example, the progeny from a purebred plant with green seeds when crossed with a purebred plant with yellow seeds had yellow seeds, not some yellow-green blend. Crossing the F₁ generation, which were all yellow to the eye, produced mostly yellow seeds, but sometimes green as well. From this he determined that there were units of heredity he called ‘factors’ — in this case there was a ‘factor’  which produced yellow seeds and a ‘factor’  which produced green seeds. These ‘factors’  (we now call them genes) segregated in some way such that a parent, which carried two such ‘factors’, would pass just one of its two to its offspring, with the other parent also contributing just one of its two.

The Second Law: the law of independent assortment
When Mendel crossed, for example, RrYy plants with each other — plants that visibly contained round, yellow seeds but were not purebred for those — he noticed that offspring could be any combination of round or wrinkled and yellow or green. We can see the combinations in the Punnett square below:

The round (or wrinkled) unit a parent passed on to its progeny had no influence on whether a yellow (or green) unit was also passed on. The units that determined seed shape were sorted independently of the units determining seed colour, and all were passed on independently of each other.

The Third Law: the law of dominance
Each characteristic Mendel studied comprised of two characters. For example, the characteristic of seed colour had two characters: yellow and green. For each pair of characters, one was always dominant to the other in that it was always expressed even when in the presence of the other character. Plants that had both the yellow seed character and the green seed character always produced yellow seeds. Yellow was dominant to green, without exception. The non dominant green was recessive by nature, and recessive when bred to recessive produced only recessive, always. Green (recessive) seeded plants when bred together only ever produced green seeded plants, never (dominant) yellow seeded ones.

Mendel’s work was truly groundbreaking. Yet while he intuited that units and not blends were behind inheritance, he never knew what those units actually where, or how they segregated and independently sorted themselves. That knowledge would come many years later, when scientists rediscovered his long-forgotten work, reproduced his results, and began to look deeper into the mechanisms at a cellular level. It is these mechanisms we shall cover in upcoming posts.

We know from Mendel’s experiments that each parent contains two copies of a ‘factor’ (gene). Let’s call these two copies a gene pair.

We know from Mendel’s Laws that these gene pairs segregate and independently sort themselves inside each parent in some way, such that each parent transfers just one copy of a gene pair to its offspring. The offspring in turn ends up with its own two copies of that gene, one from each parent, and the cycle repeats.

But what is that ‘way’? What ensures an offspring ends up with a pair of genes itself, and not a lone copy, or pairs of pairs, or any other number other than two copies?

You’d know that an animal is made up of cells. Each cell contains a nucleus (there are exceptions such as the red blood cells of most mammals), and inside this nucleus is the entire genetic material of that animal spread amongst multiple strands of deoxyribonucleic acid (DNA). A DNA strand is a very long molecule containing many genes along its length. A strand of DNA is packaged into a structure called a chromosome.

The number of chromosomes differs from species to species. Humans have 46 chromosomes, while dogs have 78, as do chickens. Cattle and goats have 60. There’s no real significance to this, it’s just fun to know!

What’s more meaningful to know is that chromosomes exist in pairs. This is why genes also exist in pairs — one of each gene pair resides on one of each chromosome pair.

We express the number of chromosomes in an animal as 2n, where n is the number of pairs, which varies from species to species. Dogs have 78 chromosomes total, so 2n=78, meaning n, the number of pairs, is 39. Humans have n=23 pairs of chromosomes.

Every cell with a nucleus has a 2n complement of chromosomes. These cells group themselves into specialised populations to form tissues and organs. Muscle cells make up a muscle, liver cells make up a liver, kidney cells make up a kidney, and so on. Cells in these tissues and organs continually replicate themselves to replace others that die off: new muscle cells replace old muscle cells, liver cells replace liver cells, etc.

Cells replicate themselves by dividing into two identical copies with a full set of chromosomes each: for example, a 2n kidney cell produces two 2n daughter kidney cells, each of which will in turn produce two 2n daughter kidney cells of their own.This process is called mitosis, and I’ll elaborate more on this in the next post.

I said above that each cell in an animal has a 2n complement of chromosomes. This is true of all cells that make up the tissues and organs, with one sole exception: the sex cells produced by the sex organs. The eggs produced by the ovaries in females and the sperm produced by the testes in males have an n complement of chromosomes. In other words, a  sex cell (also known as a gamete) contains just one copy of each chromosome pair, and thus one copy of each gene pair.

When a sperm fertilises an egg, the n complement of the sperm joins with the n complement of the egg, conferring a full 2n complement to the developing embryo. This is how a parent donates just one gene copy and why offspring inherit two.

This process — the production of gametes — is called meiosis, and I’ll go into far more detail on this after covering mitosis.

But how does this apply to Mendel’s first two Laws?

Gametes (eggs and sperm) have an n complement, but they actually arise from 2n precursors. But instead of producing two 2n cells, these precursors divide into four n cells. Unlike mitosis, where two 2n cells arise from one, in meiosis four n gametes arise from one 2n cell. Each gamete has just one copy of every original chromosome pair.

This is Mendel’s First Law, the law of segregation (of genes). One 2n cell produces four n gametes, each of which contains just one of each chromosome pair, which in turn contains one copy of each gene associated with that chromosome. The chromosome pairs — and thus the genes on those chromosomes — separate out into four distinct gametes when a 2n cell splits into four n cells.

 Any one of those chromosome pairs in the original 2n cell could end up in a gamete with any other one of the other chromosome pairs. Imagine a very simple 2n cell with just three chromosome pairs, named 1a and 1b, 2a and 2b, and 3a and 3b. After meiosis, we could end up with a gamete containing chromosome 1a, chromosome 2b and chromosome 3b. Or 1b, 2b and 3a. Or some other combination. (There would be eight possible combinations just from these three pairs alone — I’ll explain the maths in a later post!)

This is Mendel’s Second Law, the law of independent assortment (of genes). There is nothing determining which chromosome of any pair (and its genes) ends up with any other chromosome from any other pair in a gamete. And thus nothing determining which copy of a gene pair ends up with any other copy of a different gene pair.

This admittedly was a very brief coverage of a rather involved subject: please regard it as an introduction to set the scene. The next few weeks will be far more in depth, and with diagrams and a bit of maths to boot!

Last week I touched on mitosis, the process by which regular body cells replicate themselves, and meiosis, the process specialist cells undertake to produce gametes (the sex cells, sperm and eggs).

Today I shall elaborate on mitosis and leave meiosis for the next post. Mitosis isn’t as relevant to our long-term discussion of breeding as meiosis is, but it is well worth covering here, as understanding meiosis is far easier once you understand mitosis!

Normally when something is divided two halves form, not two entire copies, so what exactly goes on during mitosis to cause a cell to produce two identical versions of itself? What causes a 2n cell to produce two 2n cells, and not two n cells?

What happens is the chromosomes actually replicate themselves before the cell divides, such that there are two complete 2n sets within. Each set goes to either end of the cell, which then splits down the middle to form two daughter cells, each with a single 2n set again.

Mitosis is broken down into five stages to make it easier to study, though the whole process is actually seamless. The stages are:

• Interphase
• Prophase
• Metaphase
• Anaphase
• Telophase

The diagrams below are of an imaginary 2n = 4 cell, ie a cell with two pairs of chromosomes: a pair of long ones and a pair of short ones. I colour-coded each one to make the steps easier to follow, so please think of the dark grey and dark blue chromosomes as a pair, as are the light grey and light blue ones.

Interphase
The resting stage: not quite true, as the cell is actually growing and preparing for division. The nuclear membrane is visible. Chromosomes can’t be seen, but they are duplicating during this stage.

Prophase
(From the Greek pro, “before” and phasis, “stage")
The chromosomes shorten and thicken and become visible strands if stained. Each duplicated chromosome (called a chromatid) is joined to its pair at the centromere. In animal cells a structure called the centriole splits in two, each part going to opposite ends of the cell. The nuclear membrane disappears at the end of this stage.

Metaphase
(From the Greek meta, “adjacent” and phasis, “stage")
Long protein fibres called the spindle form from one end of the cell to the other. The chromatid pairs move to the middle of the cell and the centromeres attach to the spindle fibres.

Anaphase
(From the Greek ana, “up” and phasis, “stage")
The centromere splits in two, separating the chromatids into two distinct chromosomes. The spindle fibres contract and pull each chromosome to either end of the cell. The cell membrane begins to indent.

Telophase
(From the Greek telos, “end” and phasis, “stage")
A nuclear membrane forms around each chromosome set, such that there are now two nuclei per cell. The chromosomes lengthen and are no longer visible. The cytoplasm divides in two, the indenting cell membrane joins up, and two new, identical, cells arise.

Now that you’ve read about it, why not see it in real life?! This video is a close-up of the chromatids separating and moving to opposite ends of the cell, while this video better shows the spindle fibres in action. Just amazing.