Advances in Crop Science and Technology
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Linkage drag; Back cross; Undesirable gene; Molecular markers

Introduction

Wild relatives of crops are important sources of alleles to enhance crops, such as resistance to novel diseases and climate change. However, because of linkage drag, introgressions from wild relatives may negatively impact desired qualities, like as yield.

Usually, introgression or recurrent backcrossing are used to introduce wild genetic material into farmed lines (Tanksley and McCouch 1997). Reproductive obstacles that prevent crossings or lower the fitness of hybrid progeny can further impede this timeconsuming process (Moyle and Graham 2005; Tao et al. 2021).

Furthermore, the ensuing introgressions might negatively affect crop traits that are targeted (Chitwood-Brown et al. 2021). Although the target alleles’ negative pleiotropic effects may be to blame, linkage drag, the phenomenon whereby linked alleles that are detrimental in the crop genetic background seem to be the more common cause of adverse impacts (Von fels et al. 2017; Chitwood-Brown et al. 2021). In order to lessen the impact of the linkage drag, plant breeders tend to employ molecular markers to track the size and location of introgressions and/ or limit pre-breeding efforts to fully compatible wild relatives (Harlan and de Wet. 1971) [1].

It is thought that the factors that lead to species variations in natural populations also play a role in linkage drag. These comprise different types of hybrid incompatibilities and the genetic alterations that cause phenotypic divergences (Chitwood Brown et al. 2021; Tao et al. 2021). Strongly detrimental introductions are probably eliminated by selection during pre-breeding, thus those that are successfully added to the cultured gene pool ought to be less deleterious [2].

Backcross breeding programs introduce genes into cultivated plants, with segments of donor chromosomes inserted on each side of the introgression. Linkage drag, in which qualities other than those originally targeted are affected, is frequently observed in conjunction with this process (Young and Tanksley, 1989). Population structure, artificial selection, and genetic drift are all closely associated with linkage drag. In addition, the physical distance between linked genes strongly influences the degree of linkage drag (Liu et al. 2009).

According to Zhuang et al. (2001) breeding both high resistance and high yield progenies was challenging due to the negative correlation between the rice blast resistance gene pi25 (t) and yield-related quantitative trait loci (QTLs). Furthermore, throughout the long-term artificial selection and domestication process, certain characteristics exhibit linkage drag without recombination. For example, in tomatoes, selection for alleles linked to larger fruits changes metabolite profiles due to linkage with neighboring genes (Zhu et al. 2018) [3].

It is advantageous for a plant breeder when two or more loci governing different desired traits are connected. If there is a genetic relationship between two different desired qualities, then those two attributes can be improved simultaneously. However, reproductive obstacles like hybrid sterility and linkage drag have prevented undesired characteristics from transferring from cultivars to wild relatives. To overcome the linkage drag in plant breeding it is essential to break the linkage using one back crossing method and other advanced molecular techniques. So, the objective of this review article is to over view the linkage drag and how to overcome the challenges [4].

Descriptions of linkage drag in plant breeding

Linkage refers to the physical relationship of non-parental gene combinations. The linked genes are located in the same chromosome. They stay together in the gametes and the progeny rather than individually assorting. The selected gene’s chromosomal region shortens significantly more slowly. Linkage drag is the term used to describe the gradual segment degradation in backcross breeding (Kilian, B. et al. 2020). When trying to establish only the chosen allele in the intended background, this could be an issue. Linkage drag is one genetic characteristic of backcross breeding. This is the decrease in a cultivar’s fitness brought about by the introduction of harmful genes during backcrossing with favorable ones [5].

Backcrossing is a well-known important method of plant breeding that involves introducing alien material into crop cultivars from closely related or less adapted species (Reyes-Valdes MH., 2000). Progress can be rather sluggish when the goal is to transfer particular from a nonrecurrent source into the recurrent parent’s genetic background (Vikas, V.K. et al. 2020). It is anticipated that the remaining alien material, which segregates apart from the chosen gene, will drastically decrease by roughly 50% with every generation (Brown et al.1989) [6].

In rice breeding, interspecific hybridization is crucial because it increases access to desired traits like disease resistance and increases yields. However, skewed segregation, linkage drag, and hybrid sterility frequently impede interspecific hybridization. These findings suggest that the linking drag was caused by S20. We propose that hybrid sterility loci are the key components for the linkage drag in interspecific and sub specific hybridization of rice since a high number of these loci have been shown to be extensively dispersed on rice chromosomes (Figure 1) [7].

Figure 1: Diagram depiction of transferring undesirable genes with target gene. to loci that are not physically related. Thus, the greatest recombination Source: modified from IRRI, (2014) with permission (www.knowledgebank.irri.org).

Factors affecting linkage drag

Gene distance: The likelihood of linkage diminishes with increasing gene distance. Age: As people age, their odds of crossing over diminish, leading to an increase in linkage. Temperature: Chiasmata development is influenced by temperature increases. The linkage’s strength is diminished. X-rays: Genes exposed to X-rays lose some of their linking strength.

Types of linkage

Unlinked genes seem to exhibit autonomous assortment and segregation. The expected distribution of gamete types is even and random, with an RF of 0.50 (2011). There are two ways that this might happen: either the genes are on entirely distinct chromosomes, or the genes are spaced sufficiently apart on a single chromosome to cause so many crossings that the alleles are dispersed randomly (Figure 2). Either method, you should see an equal number of paternal and recombinant gametes with an RF close to ~0.50, as the alleles are sorting independently [8]. Due to real-life fluctuation, this figure may vary between approximately 0.40 and 0.60. https://opengenetics. pressbooks.tru.ca/?p=943#pb-interactive-content 2011

Figure 2: When Two Loci are on Non-Homologous Chromosomes Source: Plant Cell Rep (2011) 30:267-285.

Incomplete/Partial linkage

When two loci on the same chromosome are spaced sufficiently apart to allow crossovers to occur during some but not all meiosis, this is known as incomplete linkage. Incomplete linkage occurs when genes are spread out throughout a chromosome and there is a possibility of separation due to crossing over. They pass on to various gametes and progeny. (https://opengenetics.pressbooks.tru.ca/?p=943#pbinteractive- content 2011)

We refer to the state in which recombination frequencies fall between 0% and 50% as incomplete (or partial) linkage. When two loci on the same chromosome are spaced apart enough that crossovers happen between them during some but not all meioses, this is known as incomplete linkage (Figure 3). It makes no difference how fully or partially linked or unlinked genes are; they are all considered syntenic if they are located on the same chromosome [9]. As a result, while all syntenic genes are linked, not all linked genes are syntenic. https:// opengenetics.pressbooks.tru.ca/?p=943#pb-interactive-content 2011

Figure 3: Illustrated of partial/incomplete linkage Source: Plant Cell Rep (2011) 30:267-285.

The likelihood of a crossover between two loci increases with their distance from one another since crossover locations are essentially random for each base pair of the chromosome. Additionally, loci that are sufficiently isolated from one another but yet on the same chromosome will typically have many crossovers and function similarly to loci that are not physically related. Thus, the greatest recombination frequency that may be detected and a sign of either suitably separated loci on the same chromosome or loci on distinct chromosomes is 50% (Plant Cell Rep (2011).

Complete linkage

Complete linkage occurs when all of the genes are tightly packed together within a chromosome and cannot be separated by crossing over. They are usually passed on to the same gamete and progeny jointly. After discussing loci that are not related, let’s examine the opposite scenario, where two loci are positioned so closely on a chromosome that the parental allele combinations invariably segregate together. This is because crossover events become extremely rare due to the short physical distance between the two loci [10].

As a result, the two loci’s alleles are physically joined to the same chromatid and almost always segregate into the same gamete. In this instance, the frequency of recombination will be 0.00 and there won’t be any recombinants after meiosis. Because the loci must be so close to one another that, crossovers are practically impossible to detect (Figure 4).

Figure 4: When Two Loci Are Fully Connected.

Breaking linkage drag

Back cross (BC) mating between the donor parents, which are typically wild relatives, and the recurrent parents, that are typically commercial variety, can eliminate linkage drag. Since the distance between the two blocks of chromosomes (or, alternatively, between loci) influences the probability of reducing linkage drag, the closer the connection between the blocks (loci), the more challenging it is to separate the blocks (loci). One may claim that the linkage drag increases with the proximity of the linkage between blocks (loci). One will need a large number of progeny arrays and/or more generations of back cross breeding in order to be able to separate such blocks (loci). However, as molecular marker technology advances, there may be more possibilities of eliminating linkage drag [11].

Linkage drag reduction necessitates both background and foreground selection. Three markers are the minimum needed for linkage drag reduction: one for the target gene to confirm that it is still present in recombinants, and two flanking markers to look for recombinants. Feasible approaches to reduce the risk of losing the target allele through crossover events include flanking markers on both sides, but phenotypic validation is ultimately necessary to confirm that the target gene remains present. If the target gene sequence is known (e.g., a transgene), phenotypic validation may not be necessary, but it would still be done to ensure the gene is correctly expressed before a variety is released (https://pbea.agron.iastate.edu/).

If you have access to marker technology, you can select molecular markers for the foreground and background while also combining back crossing. Nevertheless, in the donor parent genome, one must first have marker loci that are strongly linked to the desired trait.

The term “linkage drag” describes the (often unwanted) consequences of genes connected to the genes or QTL we are attempting to introgressed. It is desirable to “break” the linkage drag, or separate the positive QTL from the negative QTL, if a favorable QTL for characteristic X is located near an undesirable gene impacting trait Y. In order to select a plant that has the desirable QTL but not the undesirable one, you must overcome linkage drag by passing the material through meiosis again and looking for recombinants which may be rare between the target QTL (which is tracked by the DNA markers used to identify it) and the undesirable gene/QTL. Here, a QTL that raises grain shattering is close to a QTL that raises yield [12].

Plant molecular biology is now advanced to the point that loci governing numerous traits at the gene level can be found. As a result, it ought to be feasible to pinpoint the gene governing undesirable traits as well. It should be able to create knockout transgenic lines to inactivate the expression of the gene causing unwanted traits after the genes causing them have been identified. Therefore, the transgenic lines eliminate the unwanted traits without the need for genetic recombination. Anti-sense or RNAi constructs for the target gene can be introduced to regenerate the knockout transgenic strains.

Apart from backcross, the advent of genome editing technologies has created a plethora of opportunities for plant science study. Target genes can now be deleted from the genome by the development of CRISPR/Cas9 genome editing tools. Genome editing can be used to eliminate undesired genes without resorting to backcross breeding if the targeted genes are those that regulate undesirable characters that are connected to desirable characters. Therefore, employing genome editing to eliminate unwanted genes, the linkage drag is removed. But in order to create knockout transgenic lines or do genome editing, one must first identify the target genes linked to the undesired traits that result in linkage drags [13].

Back cross breeding

Breeders employ backcrossing as a method to get rid of Linkage drag, however even after multiple backcrossing attempts, it might not work every time. A typical breeding technique used to transfer alleles at one or more loci from a donor to an elite variety is recurrent backcrossing (Allard RW. 1960, Reyes-Valdes MH. 2000). Six backcrosses would yield a 99.2% predicted recurrent parent (RP) genome recovery, which is most comparable to enhanced variety.

Essential elements for successful backcross

The selection of the recurrent parent, an efficient method for screening for the target trait or traits, and the quantity of backcrosses utilized to reconstitute the RP are the three primary components that make up a successful backcross breeding program (Allard RW. 1960, Acquaah G. 2007, Allard RW. 1953) [14].

Recurrent parent

Selecting the recurrent parent requires careful evaluation of various aspects, including agronomic performance, characteristics, target environment, and farmer preference (Figure 5 and Figure 6).

Figure 5: Markers.

Figure 6: Conventional (A) versus marker-assisted (B) backcrossing.

Screening for target trait

Because of the number of crossing cycles, phenotypic screening (i.e., a technique that distinctly distinguishes between segregating progeny) needs to be done effectively. Compared to qualities with low heritability, those with high heritability will be assimilated more successfully.

Number of backcrosses

Rapid development could lead to the creation of backcrossed lines with minimal foreign DNA paired with the desired gene. Following the initial backcross, an RFLP located one cM away might be used to screen all lines carrying the desired gene. The lines that showed a crossover at this point would be chosen. The recurrent parent would then have those lines crossed. Next, a second marker located 1 cM distant on the other side of the gene would be scored for the offspring of these crossings. Once more, progeny of crossings may be chosen. Thus, the amount of wild DNA might be lowered to 2 cM in just two generations. Having markers that are closely connected to the gene of interest and polymorphic between the two parents is essential for this process (Young and Tanklsley) (https://www.ndsu.edu/pubweb/~mcclean/ plsc731/analysis/analysis6.htm) [15].

The progeny that most closely resembles the recurrent parent can be visually selected by breeders in the early backcross generations; however, at later generations (i.e., after BC2), it may become impossible to distinguish backcross progeny from the RP based on individual plants; therefore, additional backcrosses must be made with the understanding that further backcrossing until at least BC6 will restore the recurrent parent as much as possible.

Source: https://www.integratedbreeding.net/courses/markerassisted- breeding/index7567.html?id=137

Less donor genome is left behind each time a cross is performed back to the recurrent parent, which is typically the cultivated variety if the other parent is a wild cousin. This is one of the key benefits of employing sophisticated backcross populations for QTL mapping. A locus in the segregating population can only remain homozygous in each future backcross generation, therefore once it is “fixed” (homozygous) for the recurrent parent allele (AA), markers are no longer necessary to verify it. You only need to check the progeny with Markers B and C after a cross back to the recurrent parent if, for example, your recurrent parent is AA and Markers A and D in your segregating population are also AA. Ideally, you will 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) [16].

The closer the linkage between loci, the more difficult to separate the loci (the higher the linkage drag). To be able to separate such loci will require a large number of progeny of more generation of back cross breeding. However, with the advance of molecular marker technology, you may have a better chance to remove linkage drag.

If you have access to marker technology, you can combine back crossing and background and foreground selection for molecular markers. However, (Allard RW. 1960) you will need to have marker loci closely associated with the desirable character in the donor parent and you will use these markers as background selection to indirectly indicate the presence the characters in segregated populations. (Reyes- Valdes MH. 2000) You will also need to have high density map of the recurrent parent (the tester) to identity the individual carrying the most genetic background of the recurrent parent among segregated back cross progeny.

Once you did your BC and obtain BC1 segregated population, you will screen among the progeny for marker associated with the trait/ gene that you want to introgres from the donor into the recurrent genetic background. You will want to identify the progeny carrying the linked-marker (and eventually in it carries the trait/gene) subsequently, all progeny positively identified as carrying the linked-markers, were subjected to foreground molecular marker analysis.

What you want to find are progeny that are positively carrying back ground markers, and as many as possible carrying the foreground marker loci. The more-the foreground markers are presence, the closer the selected progeny to the recurrent parent and the less genetic background of the donor existed in the genome of the selected progeny. Such condition means the less linkage drag will be seen in the selected progeny.

Backcrossing is a technique used by breeders to eliminate Linkage drag, however even after numerous backcrossing runs, it may not always be successful. The elite lines that were previously crossed may be negatively impacted by the residual “linkage drag.” One molecular technique known as “site-specific recombination” may be helpful in reducing linkage drag without requiring the time-consuming backcrossing procedure (Figure 7) [17].

Figure 7: Schematic representation of a recombinase mediated introgression

Conclusions and Prospective outcome

The physical association between non-parental gene combinations is referred to as linkage. On the same chromosome as the related genes. Rather of separately sorting, they remain together in the gametes and the progeny. The chromosomal region corresponding to the identified gene shortens noticeably more slowly. The phrase “linkage drag” refers to the progressive deterioration of segments in backcross breeding. This could be a problem when attempting to establish only the selected allele in the target background. One genetic feature of backcross breeding is linkage drag. This is the reduction in a cultivar’s fitness resulting from the backcrossing of unfavorable genes with favorable ones, which introduces detrimental alleles.

One genetic feature of backcross breeding is linkage drag. This is the reduction in a cultivar’s fitness resulting from the backcrossing of unfavorable genes with favorable ones, which introduces detrimental alleles.

Breeders employ backcrossing as a method to get rid of Linkage drag, however even after multiple backcrossing attempts, it might not work every time. The remaining “linkage drag” could have a negative effect on the elite lines that were previously crossed. “Site-specific recombination” is one molecular strategy that could be useful in lowering linkage drag without necessitating the difficult backcrossing process.

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