Estimating the Chiasma Frequency in Diplotene-Diakinesis Stage

Introduction

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.

Origin of chiasmata and the Chiasmatype theory

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:

• At the site of connections, chromosomes penetrate one another.
• It is unlikely that dyads result from the simple coiling of two anatomically independent elements.
• The site where chromosomes penetrate each other is the site of inter-penetration and secondary fusion (where the segments of chromosomes break and recombine).
• The interaction between the chromosome is not simple. At chiasmata, the filaments of the chromosomes modify their organization by exchanging their segments. This creates a new segmental combination that affects the whole chromosome segment^[7].

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]

Crossover Distribution and concept of interference

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:

1. Single crossover: In this case, only one chiasmata form. It is the most common type of crossover.
2. Double crossover: Here, the formation of two chiasmata is observed, which may occur between two same or different chromatids.
3. Multiple crossovers: In this case, more than two chiasmata are observed, which rarely occurs.

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.

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]:

1. Differential Distance (denoted by “d”): It is the distance of the chiasma formation from a fixed point, like the centromere.
2. Interference Distance (denoted by “i”): It is the distance of the subsequent chiasmas from the first one, along the length of the chromosome.

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.

Mather’s Theory of Chiasma Distribution

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]:

• The subsequent formation of chiasmata occurs at “i” distance apart in all chromosome classes.
• If there is a linear relationship between chiasma frequency/chromosome length, then both “d” and “i” distance must be constant in all complements.
• If either “i” or “d” or both varies in the complements then there will be a non-linear relationship between chiasma frequency/chromosome length.
• If either “d” or “i” varies in an uncorrelated manner, a zig-zag relation will be observed.
• If the variation between the distances is correlated then a curvilinear relation is observed. The curve will be concave if the variation increases with the chromosome length, however, the curve is convex when the variation decreases with chromosome length^[5].

Factors affecting Chiasma Frequency

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].

Experiment to study the relation between chiasma frequency and Temperature

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

Procedure

1. Choose an insect (Locust, Schistocerca, and Stenobothrus are commonly used in labs) for your study.
2. Maintain and breed them in a laboratory condition and rear in a large glass tank filled with moist sand at the bottom.
3. Keep the bottle warm to a temperature of 30 ℃ using carbon filament lamps.
4. Transfer the animals to a constant temperature chamber between 24-72 hours, after the last ecdysis.
5. Fix the gonads in strong Flemming after 3-4 days of the animals transferred to the constant temperature chamber.
6. Choose the variations of the temperature at which you want to experiment and study the relation. For example, for the three mentioned insects,^[8] the following temperatures can be preferred:
   • Locust:   0, 13, 26, 42 ℃.
   • Schistocerca:   0, 15, 26, 45 ℃
   • Stenobothrus:   0, 10, 23, 37 ℃.
7.  Maintain the insects at the chosen temperature.
8. Section the tests of the insects at various thicknesses (20-26 µ), making sure the whole nuclei are present in the sections.
9. Stain the sections with gentian violet using Newton’s method and observe and analyze the results.
10. It is recommended to use more insects to eliminate all possible differences during the experiment^[8].

Calculation of Chiasma Frequency

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

f_c= 2 x f_r

Where f_c is the chiasma frequency and f_r is recombination frequency.

Recombination and the frequency of recombinants

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 (f_r) = (N x 100)/N_p

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

• The estimation of both, the frequency of recombination and the chiasmata frequency, is required in the construction of accurate genetic or linkage maps and to determine the whole length of the genome.

Method for the estimation of the chiasmata frequency in the arabidopsis

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

Procedure

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

1. Wash the fixed buds in a fixative consisting of ethanol and glacial acetic acid in the ratio of 3:1 followed by citrate buffer (pH4.5).
2. Incubate the buds in an enzyme mixture consisting of, 0.3% w/v pectolyase, 0.3% w/v cytohelicase, and 0.3% w/v cellulase (prepared in a citrate buffer) at 37 ℃ for 1.5 hours.
3. Put the buds in an ice-cold buffer to stop the reaction.
4. Transfer a single bud to a clean slide with a small volume of buffer.
5. Macerate the bud on the slide using a fine needle.
6. Add 10 µl of acetic acid to the slide.
7. Place the slide on a hot plate at 45 ℃ for 1 minute while continuously stirring with the needle.
8. Remove the slide from the hot plate, then add 10 µl of acetic acid to the slide followed by the addition of 200 µl of fixative prepared in the 3:1 ratio.
9. Rinse off the fixative from the slide and dry it using a hairdryer^[3].

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].

Method for the estimation of the chiasmata frequency in the Mice

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

Procedure

1. Prepare and characterize the somatic karyotypes of the organism on metaphases from bone marrow biopsy.
2. Take the testicular material from the adult mice for meiotic preparations.
3. Prepare diplotene-diakinesis chromosome spreads by treating the sample with a hypotonic solution.
4. After the treatment, fixation is done using the solution of methanol and acetic acid prepared in a 3:1 ratio.
5.  Process the slides with the C-banding staining technique.
6. Observe and analyze the spread of diplotene-diakinesis using light microscopy to locate chiasmata and determine the chiasma frequency by the chi-square method( ²)^[3].

What characteristics should you observe while noting down your results?

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]

1. Measure the relative length and centromere index of individual chromosomes.
2. Analyze the distribution of the chiasma between cells.
3. Study the correlation between chiasma frequency per cell and total autosomal length^[6].
4. Observe if there is competition for chiasmata between different chromosomes of the same cell.
5. Calculate the number of chiasmata formed on individual chromosomes and chromosome arms.
6. Analyze the effect of chiasma interference over the centromere.
7. Find the relation between the mean chiasma frequency and chromosomes as well as chromosome arm length and calculate the correlation coefficient (statistical term to determine the relationship between two variables)^[6].
8. Study and analyze the distribution of the chiasmata within chromosome arms. You can draw a histogram to analyze the results efficiently^[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!

Conclusion

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

References

1. Cano, M. I., Henriques-Gil, N., Arana, P., & Santos, J. L. (1986). The relationship between chiasma frequency and bivalent length: Effects of genotype and supernumerary chromosomes. Heredity, 56(3), 305–310. DOI:10.1038/hdy.1986.51.
2. E. López, M. Pradillo, C. Oliver, C. Romero, N. Cuñado, J. L. Santos (2012). Looking for natural variation in chiasma frequency in Arabidopsis thaliana. Journal of Experimental Botany, 63 (2), 887–894, https://doi.org/10.1093/jxb/err319.
3. E. Sanchez-Moran, S. J. Armstrong, J. L. Santos, F. C. H. Franklin, and G. H. Jones (2002).Variation in Chiasma Frequency Among Eight Accessions of Arabidopsis thaliana. Genetics 162 (3), 1415-1422.
4. Grieco, M. L., and Bidau, C. J. 1999. Chiasma frequency and distribution in males and females of Metaleptia brevicornis adspersa (Acridinae, Acrididae) with and without B chromosomes. Hereditus, 131, 101 – 107. Lund, Sweden. ISSN 0018-0661.
5. Henderson, S. A. (1963). Chiasma distribution at diplotene in a locust. Heredity, 18(2), 173–190. DOI:10.1038/hdy.1963.20.
6. Hulten M. (1974). Chiasma distribution at diakinesis in the normal human male.  Hereditas, 76, 55-78. Lund, Sweden. ISSN 0018-0661.
7. Koszul, R., Meselson, M., Van Doninck, K., Vandenhaute, J., & Zickler, D. (2012). The centenary of Janssens’s chiasmatype theory. Genetics, 191(2), 309–317. https://doi.org/10.1534/genetics.112.139733.
8. White, M. J. D. (1934). The influence of temperature on chiasma frequency. Journal of Genetics, 29(2), 203–215. DOI:10.1007/bf02982197.
9. https://www.chegg.com/homework-help/definitions/chiasma-frequency-14.