The question discussed here is related to the following question: How we can remove linkage drags? – Available from: https://www.researchgate.net/post/How_we_can_remove_linkage_drags [accessed Aug 11, 2016].
In relation to those, Micha Graciana Devi have raised some more questions and here are hers:
“Sore Pak, apa kabar? I’m sorry to message you informally via facebook since I don’t know your email address. I do have some questions regarding the post that you have responded on linkage drags. https://www.researchgate.net/post/How_we_can_remove_linkage_drags. Mr. Sidhu mentioned complete linkage drag, however, I’m not really sure to what extent do you define whether a linkage drag is near or complete? is it based on the distance between the markers in the map?”
Dear Micha: I may not answer your question directly but I will bring you to my way of thinking when someone asks me such questions.
What is linkage drag?
(1) When you have two block in a single chromosome, one chromosome block carrying good stuffs (i.e. genes controlling good or desirable characters) and other block carrying bad ones (i.e. genes controlling bad or undesirable characters) (Fig. 1.) then you will have both blocks inherited as one (i.e. the good block will be inherited to the progeny along with the bad one because they are both exist in a single chromosome [both blocks are linked]).
In this case, the genes controlling desirable characters from the good block and ones controlling undesirable characters from the bad block are always presence together in the progeny. In such case, any progeny carries desirable characters will always carry the linked undesirable ones while any progeny does not carry desirable characters neither will carry undesirable characters.
Fig. 1. Part of chromosome # 1 (blue line) with two blocks of chromosome parts having genes controlling desirable characters (green block) and those controlling undesirable characters (red block). Because the two block reside in a single chromosome (chr#1), they will be inherited as one entity (always together as if they are one block).
When you only want to have the good block (Fig. 1: the green) and do not want the bad block (Fig. 1: red) but you cannot get what you want because the two blocks are linked – than you are talking about linkage-drag (The linkage between the red- and the green block causes the red block always drags along with the green block in the progeny’s genome).
Is there different degree or level of linkage drag?
Although any two blocks in a single chromosome can always be called linkage drag; however, they are not the same in the sense of the percentage of dragging one block to the other. The reasons causing different levels of linkage drag are the occurrences of recombination among chromosomes and the distances between the two blocks (See Fig. 2 and Fig. 3 for illustrations). In Fig. 2. genetic recombination may actually remove the linkage drag. The recombination event that happen in chr#1 results in separation of the green and the red blocks and the formation of two new versions of Chr#1 (i.e. Chr#1* and Chr#1#). In the recombinant Chr#1*, the green block is separated from the red block, hence linkage drag between the green and the red block is broken. Ones can now obtain the desirable characters controlled by the green block without any undesirable characters controlled by the red block.
Fig. 2. A recombination event may results in breaking of the linkage drag between the green and the red blocks. Two recombinant chromosomes are obtained as the result of the recombination event.
The probability of occurrences of recombination event in between two blocks of the same chromosomes depends on the distance between blocks. Therefore, the ability to separate between two blocks of a chromosome depends on the distance between them. Recombination event separating two blocks of the same chromosome occurs more frequently if the distance between them is large (Fig. 2.A) while it is less frequent when the distance between them is close (Fig. 2.B). Recombination may not be possible if the two blocks are overlaps (Fig. 3.c). Therefore, if the linkage drag occurs in chr#1 as in Fig.3.A, it should be easy to break the linkage drag by recombination. However, the breaking of linkage drag will be more difficult if the configuration is as in Fig. 3.B. and event is impossible under the configuration as in Fig. 3.C. This Fig. 3.C. may be what Mr. Sidhu meant by “near or complete linkage drag” and you are correct, it is because of the close distance between chromosome block or markers.
Fig. 3. The frequency of recombination event that will separate two blocks (green and red blocks) of the same chromosomes is determined by the distance between the blocks (the green and the red blocks). The larger the distance between blocks, the more often the recombination event separating the blocks may take place and vice versa. Recombination event may not be possible if the two blocks overlap.
How to break linkage drag?
Linkage drag can be removed by conducting back cross (BC) breeding between the donor parents (usually wt=wild relatives) to the recurrent parents (usually commercial variety). Since the probability of removing linkage drag is affected by the distance between the two blocks of chromosomes (or we can regard them as between loci), therefore, the closer is the linkage between the blocks (loci), the more difficult to separate the blocks (loci). Ones can say that the closer is the linkage between blocks (loci); the bigger is the linkage drag. To be able to separate such blocks (loci), ones will require a large number of progeny arrays and or more generations of back cross breeding. However, with the advance of molecular marker technology, ones may have a better chance of removing linkage drag.
If you have access to marker technology, you can combine back crossing and background and foreground selection of molecular markers. However, firstly one will need to have marker loci closely associated with the desirable character in the donor parent genome (Fig. 4). Using molecular markers (if exist), introgression of B without C will easily be predicted by monitoring for the presence of the flanking markers (M1 and Mx) and the absence of the M2 marker in the Chr#1. This is an example of breaking the linkage drag assisted by molecular markers. The Chr#1* and Chr#1# are the recombinant chromosomes as a result of the recombination event.
Fig. 4. Donor parent (wt): with a good yield character, but a negative grain shattering one in a chromosome #1 (Chr#1). Transferring the “good yield character (B=Yield+)” from wt into commercial variety is always accompanied by the transfer of “bad grain shattering character (C=Grain Shattering+)” because they both are linked in one chromosome. This is as example of linkage drag and how recombination between the chromosome is able to break the drag.
In back-cross breeding, One will use marker loci linked to B=Yield+ for background selection to indirectly indicate the presence the desirable character (B=Yield+) in the segregated population. Secondly one will also need to have high density maps of the recurrent parent (the tester) to identify the back cross progeny individuals carrying the most genetic background of the recurrent parent (Fig. 5).
Fig. 5. Introgression of Chr#1 fragment from wt donor to Chr#1 of commercial variety and the use of high density molecular marker maps to monitor the introgressed fragments (Foreground selection).
Using molecular markers (if exist), the genome constitution of the commercial variety will be easily predicted by monitoring for the presence of marker loci in the high density maps (Chr#1: marker My to Mn). This is an example of the foreground selection. The recombinant Chr#1 is selected based on the presence of as many markers as those presence in the recurrent commercial variety genomes. The absence of foreground markers in the rec. chr#1 indicates the presence of wt Chr#1 fragment’s introgression into the commercial Chr#1 chromosome. The larger percentage of foreground markers are presence, the more portion of the commercial chr#1 fragments are presence in the selected BC progeny.
Once the BCing is done and segregated BC1 population arrays are obtained, one will screen individual progeny for markers associated with the trait/gene that one want to transfer from donor into the recurrent genetic background (foreground selection). One will also want to identify the progeny carrying the linked-markers (and eventually in it carries the trait/gene of interest). Subsequently, all progeny positively identified as carrying the linked-markers are subjected to foreground molecular marker analysis.
What one want to select is progeny carrying the back ground markers and as many foreground marker loci as possible. The more-the foreground markers are presence, the closer the selected progeny to the recurrent parent genetic background and the less linkage drag of the donor genome existing in the selected progeny. The final target of the molecular marker assisted BCing is to obtain BC individuals having ~ 100% of recurrent parent genetic background and carrying the introgression gene.
Usually it may require screening of a large number of the BC progeny to find such a desirable progeny. In case one BC generation fails to obtain such a desirable progeny, any progeny having the maximum number of foreground marker loci is further back-crossed to the recurrent parent to obtain BC2 progeny arrays and the same selection steps as in BC1 are conducted. Several generation of BCing may be necessary before one is able to identify such a desirable progeny. Once identified, self-fertilization of the desirable progeny is conducted to obtain pure lines.
Are there any other way to get ridge of linkage drag without back crossing?
With the advanced of plant molecular biology, it is possible to identify loci controlling many characters at the gene levels. Therefore, it should also be possible to identify the gene controlling undesirable characters. Once the genes controlling undesirable characters be identified, it should be possible to develop knock out transgenic lines to in-activate expression of the gene controlling undesirable characters. Hence, the undesirable characters are removed in the transgenic lines without relying on the genetic recombination. The knock out transgenic lines can be regenerated by introducing anti-sense construct or by introducing RNAi construct for the target gene.
Recently, the introduction of genome editing technology has opened up many possibilities in plant science research. The introduction of CRISPR/Cas9 systems for genome editing has made it possible to remove certain target genes from the genome. If the targeted genes are ones controlling undesirable characters which are linked with desirable characters, genome editing can be used to remove the undesirable genes without having to do back cross breeding. Hence, the linkage drag is eliminated by removing undesirable genes using genome editing. However, to be able to generate either knock out transgenic lines or conduct genome editing, ones need to identify the target genes associated with undesirable characters which cause linkage drags.
Example of linkage drag and back cross breeding?
- A marker linked (0.7 cM) to the Yd2 gene for resistance to barley yellow dwarf virus was successfully used to select for resistance in a barley back-cross breeding scheme (Jefferies et al., 2003). Field test data showed that BC2 F2-derived lines containing the linked marker had fewer leaf symptoms and higher grain yield when infected by the virus compared to lines lacking the marker.
(Jefferies, S.P., B.J. King, A.R. Barr, P. Warner, S.J. Logue, and P. Langridge. 2003. Marker-assisted backcross introgression of the Yd2 gene conferring resistance to barley yellow dwarf virus in barley. Plant Breeding 122:52-56.)
- Soybean yields were increased by using marker-assisted back-crossing to introgress a yield QTL from a wild accession into commercial genetic backgrounds (Concibido et al., 2003). Although the yield enhancement was observed in only two out of six commercial varieties, the study demonstrates the potential of incorporating wild alleles with the assistance of markers (MAB=marker assisted backcrossing).
(Concibido, V.C., B. La Vallee, P. Mclaird, N. Pineda, J. Meyer, L. Hummel, J. Yang, K. Wu, and X. Delannay. 2003. Introgression of a quantitative trait locus for yield from Glycine soja into commercial soybean cultivars. Theor. Appl. Genet. 106:575-582).
(The two examples above were copied exactly from http://passel.unl.edu/pages/informationmodule.php?idinformationmodule=1087488148&topicorder=10&maxto=10&minto=1)
- The third example was copied exactly from Integrated Breeding Platform website (https://www.integratedbreeding.net/courses/marker-assisted-breeding/index7567.html?id=137). A major advantage of using advanced backcross populations for QTL mapping is that each time a cross is made back to the recurrent parent (usually the cultivated variety, if the other parent is a wild relative), less of the donor genome remains. Once a locus in the segregating population becomes “fixed” (homozygous) for the recurrent parent allele (AA), you no longer need to check it with markers, because it can only stay homozygous in each subsequent back cross generation.
Therefore if your recurrent parent is AA, and in your segregating population Marker A and Marker D are AA, after a cross back to the recurrent parent, you only need to check the progeny with Markers B and C, and hopefully find a plant that is Aa only at B (linked to the yield QTL is) and now AA at Marker C (has lost the bad QTL due to recombination).
Before being used for MAB, a QTL needs to be validated (confirmed) to rule out the possibility of statistical anomalies or errors. The validation process tests whether the same QTL appears when the material is grown in other locations and/or years, and whether its effect can still be detected when introduced into a series of different genetic backgrounds. A clear and thorough example of this can be seen in Landi et al. (2005).
Landi P, Sanguineti MC, Salvi S, Giuliani S, Bellotti M, Maccaferri M, Conti S, Tuberosa R (2005). Validation and characterization of a major QTL affecting leaf ABA concentration in maize. Molecular Breeding 15: 291–303.