The following points highlight the top eight aspects of reciprocal translocation in genetics. The aspects are: 1. Chromosome Pairing 2. Orientation of the Quadruple at Ml 3. Sterility in Interchange Heterozygotes 4. Crossing Over in the Interchange Heterozygotes 5. Detection of Interchanges 6. Isolation of Homozygous Interchange Lines and a few others.
Aspects of Reciprocal Translocations
- Chromosome Pairing
- Orientation of the Quadruple at Ml
- Sterility in Interchange Heterozygotes
- Crossing Over in the Interchange Heterozygotes
- Detection of Interchanges
- Isolation of Homozygous Interchange Lines
- Identification of Chromosomes Involved in Translocations
- Location of Interchange Breakpoints
Reciprocal Translocations: Aspect # 1. Chromosome Pairing:
In 1930, McClintock for the first time showed that maize interchange heterozygotes involving two pairs of non-homologous chromosomes formed a “cross-shaped” configuration at pachytene, and a ring of four chromosomes at metaphase I, due to the pairing of homologous segments of the chromosomes involved in the translocation.
Such configurations should be called quadruple instead of quadrivalent; the latter term should be restricted to the pairing of four homologous chromosomes. However, the term “quadrivalent” is commonly used for the ring or chain of four chromosomes resulting from translocations. Some non-homologous pairing may also occur in cases as was observed in the translocation heterozygotes in maize by Burnham et al. in 1972.
In case of complete pairing, the following six paired segments can be distinguished in a quadruple (pachytene stage) within which chiasmata may be formed (Fig. 14.3):
(i) The two non-interchanged arms O and P,
(ii) The two interchanged segments R and S, and
(iii) The two interstitial segments (the regions between the centromeres and the breakpoints) T and U.
The MI configuration would depend on the chiasma formation in these segments.
The formation of chiasma (ta) in a particular segment depends on the following three factors:
(i) Length of segment,
(ii) The frequency of crossing over in the organism, and
(iii) The characteristic properties of the segments that are relevant to chiasma formation.
Considering all the above six segments, there are 26 (= 64) different possible combinations of chiasma (ta) formation. Each of these 64 possibilities leads to a particular configuration at MI, many of which are homomorphic, i.e., comparable in appearance. The most common configurations at MI are rings and chains of four chromosomes.
Occurrence of at least one chiasma in each of the terminal segments of the cross i.e., the two non-interchanged (O and P) and the two interchanged (R and S) segments, leads to the formation of a ring quadruple (ʘ 4) at MI (Fig. 14.4). But when any three of these segments have one chiasma each, a chain of four chromosomes results at MI.
Formation of chiasmata in only two adjacent segments produces a chain of three chromosomes plus a univalent (Fig. 14.4). Occurrence of chiasmata in all the four terminal segments (O, P, R and S) accompanied by chiasmata formation in both the two interstitial segments (T and U) yields a quadruple similar to ‘eight’ (8) in appearance at MI.
Chiasmata formation in the two trans-located arms (R and S) and a chiasma in one of the interstitial segments (either T or U) results in an open 8-shape configuration at MI. When only two opposite segments of the cross have chiasmata, two rod bivalents are formed.
Reciprocal Translocations:
Aspect # 2. Orientation of the Quadruple at Ml:
The orientation of interchange multiples at MI may be of the following two types:
(a) Alternate or zigzag and
(b) Adjacent orientation. In turn, the adjacent orientation may be of two types:
(i) Adjacent-I
(ii) Adjacent-II orientation.
Alternate orientation:
The homologous centromeres co-orient in such a manner at MI that the alternate chromosomes of the ring or chain lie toward one pole. As a result, in a ring of four chromosomes, the normal (unaltered) chromosomes move to one pole, whereas the other pole receives both the trans-located chromosomes.
In such a type of orientation at MI, a ring appears like the figure “8”, while a chain becomes zigzag (Fig. 14.4). Since both the trans-located chromosomes are included in the same gamete, the deficient segment of one chromosome is present in the other chromosome in the same gamete, therefore, all the gametes resulting from such orientations are functional (Fig. 14.5).
Adjacent orientation:
In this type of orientation, adjacent chromosomes of the ring move to the same pole. Ring or chain configuration occurs at MI.
This orientation is of the following two types:
(i) Adjacent-I Orientation:
The homologous centromeres co-orient, i.e., face the opposite poles, and the adjacent chromosomes go to the same pole. As a consequence, the homologous centromeres move to the opposite poles (Fig. 14.5), and each pole receives one normal and one trans-located chromosome. This results in the production of only deficiency-duplication gametes all of which are nonfunctional.
(ii) Adjacent-II orientation:
In such an orientation also, the adjacent chromosomes go to the same pole. But there is a co-orientation on non-homologous centromeres so that homologous centromeres move to same pole. As a result, each pole receives one normal and one trans-located chromosome so that all the gametes contain large deficiency-duplication and are nonfunctional.
If the two chromosomes involved in translocation differ considerably in length, the above three kinds of orientation can easily be recognized cytologically at MI. Interstitial chiasmata in the quadruple lead to a frying pan type orientation; such an orientation produces two balanced (functional) and two unbalanced (nonfunctional) gametes (Fig. 14.5).
Non-co-orientation:
This is an abnormal situation where two centromeres are co-oriented and are located equal distances from equator. The rest two centromeres are non-co-oriented and are stretched out between the two chromosomes. The two co-oriented chromosomes go to the opposite poles while the non-co-oriented chromosomes either go to opposite poles or move to the same pole.
If the non-co-oriented chromosomes go to the same pole, a 3 : 1 segregation occurs giving rise to n+1 and n – 1 gametes (Fig. 14.6). Non-co-orientation may be divided into the following two types.
(i) The normal chromosome co-orient, while the trans-located chromosomes non-co-orient. In such case, one pole receives one of the normal and the two trans-located chromosomes n + 1, whereas the other pole receives the remaining normal chromosome n – 1 [Fig. 14.6 (ii), (iii)], (ii) Alternatively, the trans-located chromosomes co-orient, while the two normal chromosomes non-co-orient.
As a result, one pole receives the two normal and one of the two trans-located chromosomes n + 1, whereas, the other pole receives the remaining trans-located chromosome n – 1 [Fig. 14.6 (iv), 14.6(v)].
Reorientation:
Reorientation of centromeres occurs for a controlled chromosome distribution because the initial orientation at the beginning of prometaphase is often inappropriate. This process involves the loss of chromosomal spindle fibre connections to one pole followed by formation of a new connection to the opposite pole. In rye, reorientation of chain quadrivalent is slower than those of ring quadrivalents.
Reciprocal Translocations:
Aspect # 3. Sterility in Interchange Heterozygotes:
Interchange heterozygotes show semi-sterility (SS) or partial sterility (PS) due to the production of deficiency-duplication gametes. The degree of sterility is dependent upon the frequencies of the different types of orientation at MI of the interchange multiples.
In the absence of crossing over in the interstitial region, only alternate segregation leads to functional gametes, while the adjacent segregations lead to nonfunctional gametes (Fig. 14.5).
If the alternate and adjacent segregations occur in equal frequency, 50% of the gametes will be sterile giving rise to semi-sterility observed in translocation heterozygotes of several plant species like maize. The frequency of alternate segregation above 50% will lead to a lower degree of sterility which may be called partial sterility (PS).
An excess of alternate orientation occurs in several plant species, such as, barley, rye, Datura, tomato, Triticum monococcum, T. aegilopoides and others. In barley (H. vulgare), alternate orientation has a frequency of more than 70%, and PS ranges between 25 and 30%.
Certain species show very low sterility or interchange heterozygotes. If the alternate orientation is more frequent and the interstitial region is short so that crossing over cannot occur sterility is greatly reduced. Yamashita in 1951 reported an average of 9% sterility in the interchange heterozygotes of T. monococcum and T. aegilopides. In Oenothera, interstitial segment is much short and the sterility is very low.
Several causes may be ascribed to the excess of alternate disjunction, such as, genetic variation in chromosome rigidity, capacity of the centromeres to re-orientate pre-metaphase stretch, chiasma terminalization and localization and the position of the break.
The tendency of alternate orientation may be different in ring and chain multiples. In certain species, e.g., Secale cereale and Campanula persicifolia, alternate segregation is greater in the case of rings than that in the case of chains.
Sterility increases with increase in the number of chromosomes involved in translocation. When the same number of chromosomes are involved, the degree of sterility is dependent upon the size of the interchange multivalent (Table 14.1).
In barley, translocation heterozygotes involving four pairs of chromosomes forming a ring or chain of 8 chromosomes showed 50-70% sterility which was greater than that (30-60%) exhibited by the translocation heterozygotes producing two rings of 4 chromosomes (two different translocations).
When all the 7 chromosomes of barley are involved in translocation, the sterility in the heterozygotes reaches 100% and, as a result, no seed is produced.
Reciprocal Translocations:
Aspect # 4. Crossing Over in the Interchange Heterozygotes:
Recombination values obtained in the interchange population may differ from those in standard normal populations. Crossing over is reduced in the region of translocation break position (interstitial region). Crossing over in the interstitial region followed by alternate disjunction leads to the production of nonfunctional gametes (Fig. 14.7), the crossover chromatids are lost in the aborted spores.
Contrary to this, the adjacent-I segregation leads to the production of functional gametes, and the crossover chromatids form the functional spores. Thus an excess of alternate segregation leads to a reduced recombination in the interstitial segment.
The patterns of reduction in recombination frequency in interchange heterozygotes furnishes a clue as to the position of centromere. In barley, the genetic marker K k (hooded vs. awned) is located in the short arm, while Bl bl (blue vs. non-blue aleurone) is located in the long arm of chromosome 4.
They show 40% recombination in normal populations. In a study of recombination in three barley interchange heterozygote lines, namely, T2-4a, T3-4a and T4-5a, (breakpoints located in the short arm) a reduction in the frequency of recombination between K and Bl was observed in the interchange populations by Hanson in 1952.
The reduction in recombination was 17% in the interchange T2-4a, 12% in T3-4a and 9% in T4-5a (Fig. 14.8).
The reduction in the frequency of recombination decreased as the breakpoint moved farther from K and closer to Bl indicating that the region in which crossing over was reduced was becoming progressively shorter; this region is the interstitial region.
Thus the genes K and Bl are located on the opposite arms of the chromosome and the breakpoints of the three translocations are between the gene K and the centromere. Utilizing this principle, genes can be located in the chromosomic in relation to its centromere.
Reciprocal Translocations:
Aspect # 5. Detection of Interchanges:
Interchanges can be detected by several ways, as given below:
(1) In natural or mutagen treated populations, plants showing partial (PS) or semi-sterility (SS) may be suspected to be interchange heterozygotes MI configuration of such plants are checked form the presence of ring or chain multiples, which confirms the presence of interchanges.
(2) In the species where interchange heterozygotes are not partially sterile, analysis of MI configurations are needed to detect interchange heterozygotes.
(3) Interchanges can be detected by studying the morphology of somatic chromosomes at mitotic metaphase and comparing it with that of known normal individual of the species. This method is used for white blood cells and other cells in human; it is considerably aided by chromosome banding techniques. Other methods such as genetic markers have also been used for detection of interchanges in different organisms.
Reciprocal Translocations:
Aspect # 6. Isolation of Homozygous Interchange Lines:
For this purpose, the interchange heterozygotes are selfed. The selfed progeny consists of normal, interchange heterozygotes and interchange homozygotes in the ratio of 1 : 2 : 1. The normal and interchange homozygote individuals will show normal fertility, while the interchange heterozygotes would show partial sterility.
Thus in the selfed progeny of interchange heterozygotes, 50% plants are fertile and 50% plants are partially sterile (Fig. 14.9). All the plants showing normal fertility are crossed with standard normal plants of the same variety, and the resulting F1 plants are scored for sterility and meiotic configurations.
The F1s from the cross “normal X standard normal” will show normal fertility and only bivalent configurations at MI. On the other hand, the F1s from the cross “interchange homozygote X standard normal” will show partial sterility and interchange multiples (ring or chain of 4 chromosomes) at MI. On this basis, the plants carrying homozygous translocation are identified. The scheme for the isolation of a homozygous interchange line is presented in Fig. 14.10.
Reciprocal Translocations:
Aspect # 7. Identification of Chromosomes Involved in Translocations:
The chromosomes involved in translocations are identified in the following two ways: (A) cytological method and (B) genetical method.
A. Cytological Method:
1. Karyotype studies:
Karyotypes of normal, translocation heterozygote and translocation homozygote individuals are compared. For such studies, root tip cells in plants and nerve ganglia or white blood cells in mammals are quite suitable. The different banding patterns are also used for identification of the chromosomes involved in the interchanges.
2. Study of pachytene chromosomes:
The cross-shaped configuration present at the pachytene of the interchange heterozygote is studied for identification of the chromosomes involved in translocation. The chromosomes can be identified easily if there are knobs or other distinctive markers and if the relative arm lengths and positions of these markers in the normal chromosomes are precisely known.
However, this method is useful only in those plants where pachytene analysis is easy.
3. Trisomic method:
In this method, trisomies for known chromosomes are crossed as females with the interchange homozygotes (used as males). The MI configuration of the resulting trisomic F1 plants are analysed. The presence of a chain of 5 chromosomes indicates that the chromosome for which the female parent used in the cross was trisomic is involved in the translocation.
On the other hand, the presence of a ring or chain of 4 chromosomes and a trivalent (ʘ 4+ lIII) configuration indicates that the chromosomes involved in the translocation are different from the chromosome for which the female parent of the F1 plant was trisomic.
4. Translocation tester method:
This method is easy and efficient and is free from errors. A translocation tester is a homozygous interchange line whose trans-located chromosomes are already known and produces a ʘ 4 configuration at MI in the heterozygous condition. Translocation tester sets have been developed in several crops, such as, barley, maize, Pennisetum, lentil etc.
The unknown translocation line is crossed with different translocation tester stocks and the MI chromosome configurations of the resulting F1 hybrids are studied.
(i) The presence of 2 rings of four chromosomes indicates that the chromosomes involved in translocation in the unknown line and those involved in the tester are quite different.
(ii) A ring of six chromosomes indicates that one chromosome is common to both the lines.
(iii) Only bivalents are expected when the trans-located chromosomes in the unknown interchange line and those in the tester stock are the same.
However, a ring of four chromosomes may also be observed even when the chromosomes involved are the same if the breakpoints are in the same arm of one of the chromosomes in both the interchange lines (unknown and tester lines), while they are located in different arms of the other chromosome.
An example is presented in the Table 14.2 in which chromosomes involved in 3 unknown translocation lines of barely have been identified using translocation testers. The F1s of the crosses of the unknownhomozygous translocation line 3047 with the translocation testers T3-5b, and T3-7a showed 2 ʘ 4 +3II at MI (Table 14.2) indicating that the chromosomes 3, 5 and 7 were not involved in translocation. The configuration ʘ6 + 4II was observed in crosses with testers T1-2a, T1-6a and T1-7d.
Since chromosome 1 was common to these three testers, this chromosome appeared to be involved in translocation. Again, a configuration ʘ6 + 4II was observed when the unknown line was crossed with T2-4a and T4-5a suggesting that chromosome 4, common to both testers was involved in translocation.
From these observations, conclusion was drawn that the line 3047 was T1-4. Similarly, the other lines 3103 and 3104 were T1-3 and T4-7, respectively.
Apart from the above methods, chromosomes involved in translocation can be identified by studying the interchange multiple associated with nucleolus. However, this method is useful for identifying the involvement of the nucleolar chromosome only.
B. Genetical method:
A very important method of identification of chromosomes involved in interchanges in the “linkage study” because not only the chromosomes are identified, the breakpoints can also be determined if suitable genetic markers are available. However, this method is a time consuming method.
The translocation line is crossed with the different linkage tester stocks (lines carrying genetic markers in the different chromosomes) and the independence of segregation for partial sterility (PS) from the various marker genes in F2 is investigated. The PS is considered as the “breakpoint”. The genetic marker present in the chromosome involved in translocation shows linkage with PS.
Reciprocal Translocations:
Aspect # 8. Location of Interchange Breakpoints:
Interchange breakpoints are located through various cytological and genetical techniques as described below.
A. Cytological location:
1. Karyotype study:
Karyotypes of somatic chromosomes of normal, interchange heterozygote and interchange homozygote individuals are analysed and compared to locate the breakpoint on the chromosomes. Study of banding patterns of normal and trans-located chromosomes is also useful in location of break-position.
2. Pachytene analysis:
Breakpoints can be located by analysis of the cross shaped configuration of chromosomes at pachytene. But this method is useful only where the pachytene chromosomes are easily analysed, e.g., in maize.
3. Intercross method:
This method is useful where pachytene analysis is difficult. It involves the study of types and frequencies of different chromosome configurations at diakinesis and MI in the hybrids between different interchanges involving the same two chromosomes.
B. Genetic location:
Genetic location of interchange breakpoints is made through linkage studies. It is the most accurate but time-consuming method. In this method, the breakpoint is considered as the partial sterility locus (PS) which segregates in F2 as PS and normal (N) in the ratio of 1 : 1. Recombination values between the PS and the different genetic markers are estimated from F2 data or from combined F2 and F3 data.
By using these recombination values, the breakpoint (PS locus) is located in relation to the genetic markers in the particular chromosome. A gene close to the centromere and another gene on the short arm are suitable markers for locating breakpoint in the short arm, while a gene close to the centromere and another gene on the long arm are suitable markers for locating breakpoint in the long arm of the chromosome.
Formulae and Tables to facilitate calculation of linkage intensity (recombination value) between the breakpoint (PS) and a qualitative character pair have been developed.
The homozygous translocation line is crossed to the stocks carrying genetic markers. In F2, the joint segregation of PS vs. normal (PS : N) and the marker genes (say R vs. r) is analysed. The segregants are divided into 4 classes namely, (i) PS with the dominant genetic marker (PS R) = a, (ii) normal with the dominant genetic marker (NR) = b, (iii) PS with the recessive genetic marker (PS r = c) and (iv) normal with the recessive genetic marker (N r) = d. Linkage between PS and genetic marker is detected by the Fisher’s X2 test for independence as follows:
where a, b, c and d are the observed frequencies in the four F2 classes described above.
A significant X2 value indicates the association of PS with the marker gene carried by the particular chromosome. The recombination value between the PS locus (breakpoint) and the genetic marker is then calculated by comparing the bc/ad ratio with the table value (presented by Joachim in 1947).
Estimation of recombination values using F2 data is presented in Table 14.3 for a barley interchange involving chromosomes 1 and 3. The recombination value between the breakpoint (PS locus) and the genetic marker n in the chromosome 1 is 19.41 ± 3.41%.
In chromosome 3 the breakpoint showed a recombination value of 5.64 ± 2.02% with the marker uz and 6.69 ± 2.67% with the marker zb. Recombination value between uz and zb was 9.26 ± 1.29% in normal population, while it was reduced to 4.35 ± 1.28% in the interchange (T1-3) population.
The marker zb is close to the centromere while uz is located in the short arm of chromosome 3. Utilizing the above data, a genetic map can be constructed showing the break position in the chromosome 3 (Fig. 14.11). The translocation break position is located on the short arm of this chromosome.
