Chiasma is the point of crossing over or site where the exchange of genetic material takes place between two homologous, non-sister chromatids. The crossover occurs in the pachytene stage, however, it is observed in the diplotene stage of meiosis-I[2].

The cross-over between the two homologs also creates a new combination of parental genes, forming recombinants. The recombination of the genes causes variation in the population and exert a profound effect on genomic diversity and evolution.

Meiotic recombination and variation in the population have been a concern for scientists to understand the impact and significance of crossing over in a population. Over time, various techniques, such as immunolocalization and electron microscopy of recombination nodules[2], were discovered for the analysis of meiotic recombination and quantification of crossing over.

However, estimation of chiasma frequency is the traditional method followed widely to understand the phenomenon.

Chiasma Frequency is defined as the estimation of the level of genetic recombination in a population. It is especially very effective to estimate the genetic recombination in organisms in which genetic analysis is impossible/difficult to perform[2].

So, this article is a layout of the origin of the concept of chiasmata, the factors affecting chiasma frequency, and its distribution in chromosomes. Also discussed, is the procedure for estimating chiasma frequency in plants as well as animals.

The concept of chiasmata was first introduced by the scientist Frans Alfons Janssens in 1909. He explained, that when the tetrads (two pairs of sister chromatids) separate, they only show one point of connection at chiasmata, where they exchange their segments.

The prophase of meiosis-I consists of five stages: leptotene, zygotene, pachytene, diplotene, and diakinesis. The actual crossover occurs at the pachytene stage but they can be visualized only in the diplotene stage of the division[7].

Mendel observed, the random assortment and segregation of characters is the result of meiotic behavior, however, how it occurs was shown by Janssens.

He explained that out of four chromatids, two cross each other, and two don’t. At the end of the division, four gametes are formed, out of which two are parental type and two recombinant. His observation and studies were included in the “Chiasmatype theory”[7].

Janssens studies were a breakthrough in the understanding of meiotic behavior and chiasmata formation.The highlights of the theory[7] are:

Figure: Drawing from Jasson’s 1909 article showing illustrations of chiasmata.

(A) Shema XXI: drawing of a single chiasma; Schema XXII: drawing of chiasma in dyads at diplotene stage; Schema XXIII: anaphase I; Schema XXIV: Result of crossover and recombination.

(B) Arrows showing two chiasmata formation in diplotene dyad.

(C) Micrograph of a diplotene bivalent from the salamander Oedipina poelzi.

(D) Jasson’s drawing showing multiple chiasmata in dyad which was used by many cytologists to criticize Jasson’s theory.

Source https://doi.org/10.1534/genetics.112.139733[7]

Chiasmata form at the point where crossover occurs and based on the number of chiasmata forms between the chromosomes, the crossover is of three types:

The distribution of cross-over is not random over the length of the chromosome. However, it is measured and affected by interference[5].

Interference is the phenomenon in which the occurrence of one cross-over between the chromosomes reduces the chance of the occurrence of the second cross-over.

The interference, in the second cross-over, decreases as the distance from the chiasma or cross-over (already examined) increases. Interference is more in the adjacent region of the cross-over.

Calculation of Interference

The phenomenon of interference results in the occurrence of fewer double crossover types.

                   Interference= 1 – Coefficient of Coincidence

The interference varies in different regions of the chromosomes which are measured by the coefficient of coincidence. It is defined as the ratio between the number of observed double crossovers and the number of expected double crossovers.

The frequency of the formation of chiasma has also regional dependencies, such as, in some regions, there are higher chiasma frequencies while lower in some other regions[5].

So, a scientist named Mather defined the chiasma distribution over two parameters[5]:

He explained that the consideration of these two parameters will help to determine the frequency and position of the chiasmata formation in the bivalents[5]. The points of the study are discussed in the next section.

He explained that the position of chiasma frequency is bivalent specific. Then he described the relationship between chiasmata frequency and length of the chromosome by using differential (d) and interference distance (i). Some points of this interesting study are mentioned below[5]:

Various factors affect the chiasma frequency, like the length of the chromosome, maternal age, the distance between the subsequent chiasmata, and even temperature.

It has been observed that the frequency of crossover is maximum at temperatures 41℃ and 31 ℃.

A study by Nolte et al and Shaw (1971 & 1972) showed that the chiasmata frequency depends on both genetic and environmental components[1]. It also includes the presence of supernumerary chromosomes.

Supernumerary chromosomes are extra chromosomes that are only found in some species of the organisms. These chromosomes are not important for the survival of the species[1]. So, apart from the primary set of chromosomes, the organism may have a “B or supernumerary chromosome”.

The chiasmata formation is also affected by the nature of chromosomal regions. If a heterochromatic region is present, it restricts the chiasmata in adjacent euchromatic segments. So, a terminal presence of heterochromatic regions leads to redistribution of chiasmata away from the segments to proximal sites[1].

Some studies have shown that the value of chiasma frequency is dependent on the sex of the organisms. For example, a study by Darlington (1973) showed that sometimes both the sexes (male and female) of the organism display differences in recombination by divergent patterns of chiasma frequency[4]. He called it “two-track heredity”. However, in some situations, both sexes have similar chiasma frequency and distribution[4].

Here’s a study by White M. J. D. (1934) that demonstrates the process of chiasma frequency against temperature[8].

Chiasma frequency is calculated by using the formula[9]:

fc= 2 x fr

Where fc is the chiasma frequency and fr is recombination frequency.

The recombination is a process in which the DNA strands break and recombine to form a new combination of alleles, called recombinant. The recombinants are produced by two processes: independent assortment and crossing over.

Crossing over results in the formation of recombinants which brings variation in the population. The impact of crossing over compelled scientists to study the relationship between crossing over frequency and the formation of recombinants.

Calculation of the frequency of recombination[9]:

Frequency of recombination (fr) = (N x 100)/Np

Where N is the number of recombinants and Np is the total number of progeny[9].

A study by Moran et. al. is presented here to demonstrate variation in the chiasma frequency in Arabidopsis[3].

Plant material: In this study, the plant material was grown on soilless compost and grown to flower in a constant environmental condition at a temperature of 18℃ for 16 hours[3].

Fixation: Detach the flower buds and fix them in a fixative consisting of ethanol, chloroform, and acetic acid in the ratio of 6:3:1. Store the fixed buds at -20 °C until required[3].

Slide Preparation

Fluorescence in situ hybridization

Counterstain the slides using 4’,6’-diamidino-2-phenylindole (DAPI- 4 µg/ml) and mount it in the antifade mounting medium[3].

To learn more about the principle and procedure of the FISH technique, you can refer to the article “Molecular Cytogenetics: in situ Hybridization-based technology”.

Statistical analyses

Analyze the chiasmata data using the Minitab software and note down your results[3].

This section presents a study by Gorlov et al. The sample used in the study was the testicles of the mice[3].

What leads to a complete understanding of the chiasma distribution in the cell? What should you observe and analyze while studying the distribution of diplotene-diakinesis in normal human males or any organisms?

Here are a few points:[6]

Keeping the points in your mind while evaluating and analyzing your results, will help you to draw a discrete conclusion about your experiment without missing anything!

Genetic recombination is the main source of variability in a population, which is necessary for its continued evolution. One of the causes of genetic recombination is crossing over. The crossover counts or estimation of chiasmata frequency at the diplotene-diakinesis phase of meiosis-I is used as a cytological measure for the estimation of the total length of the genome. This method is an efficient approach to other available techniques because it can be easily scored for a large sample of meiocytes.

The chiasma frequency is a non-random phenomenon and depends on the length and type of chromosomes (acrocentric and non-acrocentric), the type of organisms, and the age of the organisms.

The decades of meticulous studies have still left some loose ends, as the actual mechanism of the control of crossing over or chiasma formation, over genetic recombination, is still not well understood. Furthermore, there is much to explore in the findings of the relation between the variation in chiasma frequency and chromosome length