The Genetic Of Hybrid Incompatibility

Published Date: 02 Nov 2017

Research on the genetic basis of evolution of reproductive barrier has been great interest for understanding the process of speciation. There are two major type of reproductive isolation(RI) preventing gene flow between species including pre-zygotic RI and post-zygotic RI. For pre-zygotic RI, it restrict the potential of copulation or mating between different species which examples include flowering at different season in plants and courtship behavior fail to stimulate individuals of other species in animals. For post-zygotic RI, it reduces the fitness of the hybrid offspring so the hybrid is usually inviable or sterile. Hybrid incompatibility is regarded as the main cause of post-zygotic RI and thus plays an important role in species diversification and species maintenance which are key topics in evolutionary biology. However, pre-zygotic RI can be much more simply recognized as a byproduct of differential adaptation to varying environment or selection against the production of unfit hybrid while the evolution of post-zygotic RI have posed a question to scientists because such a maladaptive inviability and infertility could not evolve by natural selection.

Mode of hybrid incompatibility

One of the theories trying to explain how incompatibilities between closely related species develop without either of the them going through an adaptive valley is the Dobzhansky-Muller Model. It posits that hybrid incompatibility is unlikely to be arisen from only a single change at one locus due to underdominance (viable AA or aa but inviable Aa) that such hybrid being heterozygotes have lower fitness should an allelic substitution not take place by natural selection. In contrast, Dobzhansky-Muller incompatibilities result from negative epistatic interaction between alleles located at least at two loci.Those incompatibility casuing gene, called ‘complementary gene’ show no deleterious effects within either population but dysfunction with genes from other species when brought in hybrid. (Fig.1)

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The basic model is that two allopatric populations starting with common ancestral genetic background, while accumulation of genetic change continues along their divergence such that allele substitution at different loci occur in the separated populations. The derived alleles are neutral or even beneficial in their own genetic background but incompatible with the later derived allele at different loci in another population. When considering many loci, the accumulation of complementary gen can be shown by the following diagram:

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The two lines that forming a V shape represent the two lineage from a common ancestor and both population have all their allele lowercase fixed at initial. Time proceed upward and the first allele substitution takes place at locus a, then locus b followed by locus c and so on. The arrows show possible incompatibilities due to epistatic interaction between loci form the two populations. Some principle can be concluded from the figure. Firstly, according to the graph, since the arrows never point upwards, it indicate incompatibility must occur between two loci that both have allele substitution, which in term alleles at undiverged loci must be compatible with those diverged loci as the two population share the common genetic background. Secondly, later substitutions are more potent in causing incompatibilities than that of the earlier (D may be incompatible with c,B,a but B could only be incompatible with A)which suggest the rate of increase of intensity of reproductive isolation may be faster than first order (Snowball effect). Thirdly, all the incompatibilities are initially asymmetric that for example C could be incompatible with a but b cannot. This further deduce that a evolutionarily derived allele(uppercase) tend to be involved in incompatibility more than a ancestral allele(lowercase).The reason is that when considering all the possible types of incompatibility, there are derived allele-derived allele and derived allele-ancestral allele interaction but not including a ancestral allele-ancestral allele type.

As a result, there can be transitional genotypes adaptive or neutral in ancestral populations so can escape the elimination by natural selection. However, even if the DM model can explain the above problem, it provides no answer to how the genetic change can lead to HI at molecular level and what force drive the divergence of those HI genes. How is the molecular interaction cause hybrid malfunction? Do the speciation gene diverge by positive selection, sexual selection or genetic drift? Fortunately, with the recent identification of some gene underlying reproductive isolation, we may find some clues to the above questions. Also, molecular studies may tell us the relative importance of allopatric and parapatric modes of speciation.

Hybrid incompatibility in animals:

A ‘Dobzhansky-Muller" type incompatibility was identified in a platyfish species. Hybrid between platyfish Xiphophorus maculatus and a related species the swordtail Xiphophorus helleri show inviability. Also, backcross hybrid of those species often develop malignant tumor and eventually die. With the help of molecular genetic analysis, a X-linked hybrid incompatibility gene Xmrk-2 which code for a novel receptor tyrosine kinase was found misexpressed in hybrid, leading to cancer formation. Xmrk-2 gene functions as a spot producing gene in platyfish with a repressor gene locating at an autosome. The autosomal repressor is missing in hybrid resulting in improper regulation of the Xmrk2 gene expression. The Xmrk2 gene has received great attention due to its role in cancer formation. However, more important was found in a frequently used and well-studied genetic tool –Drosophila. Those identified incompatibility genes are shown in the following table:

Gene

Hybrid phenotype

Normal Gene function

Possibly genetic cause

Odysseus-Homeobox (OdsH)

Male sterility

transcription factor

Misexpressed in hybrid

Lethal hybrid rescue (Lhr)

inviability

Heterochromatin protein

Failure in mediating chromosome condensation in mitosis

Hybrid male rescue(Hmr)

inviability

transcription factor

Disruption in gene regulation

Nup96

inviability

Nucleoporin

Negative interaction with a unknown loci on X chromosome of D. melanogaster

Research found loss of function mutation of Hmr gene in D. melanogaster and Lhr in D. simulans suppress hybrid male lethality. Both gene demonstrate asymmetry in causing hybrid lethality which is stated in Dobzhansky-Muller model by the result that deletion mutation of those alleles of another species cannot rescue the hybrid. Among those incompatibility genes, Nup96 seem to be more informative in nowadays study. Nup96 gene codes for a protein that stably bound to the nuclear pore complex which is the largest macromolecular complex in eukaryotes. The Nup96 nucleoporins play a structural roles in nuclear pore compex mediating nucleocytoplasmic trafficking of RNAs and proteins. In hybrid cross between D. melanogaster and D. simulans , Nup96 allele cause lethality in hemizygous for the D. melanogaster X chromosome but not in hemizygous for the D. simulans X chromosome. The observation match the Dobzhansky-Muller model predication that a D. simulans allele of Nup96 would not be incompatible with loci on its own X chromosome.Nup96 protein is found interacting with others nucleoporins such as Nup75, Nup107, Nup133, Nup160, Seh1, Nup37, and Nup43 to form a Nup 107 subcomplex. Sequencing analyses of polymorphism of those genes suggest a trend of adaptive coevolution between Nup96 and some of its interacting proteins. Such coevolution event may provide a new model different from the assumption of population genetic that substitution take place independently. Comparing with independent substitutions model, non-independent evolution may speed up divergence of hybrid incompatibilities since substitutions at 1-locus increase the probability of substitutions at interacting loci within a lineage. Also, Coevolution of interacting genes may concentrate substitutions in one lineage which imply a increase in possibility of derived–ancestral allele hybrid incompatibilities, opposing to the predication of Dobzhansky-Muller Model.

Hybrid incompatibility in plants:

Past studies have shown that postzygotic RI is often evolved through mechanisms consistent with the classic DM model involving two or more locus incompatibility but there are some exceptional cases that yield the similar result with incompatibility involving only one locus through a multiple step in allele substitution. The empirical mechanism is shown in the fig.3

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According to the graph, two allopatrically isolated subpopulations diverged from a common ancestor have different allelic fixation (red or blue) at the same locus. The two derived alleles are incompatible when brought in hybrid. It is thus possible to avoid initial fitness cost within species.

Example includes HPA1 and HPA2 loci found in Arabidopsis thaliana population which divergent duplication of those allele cause some of the F2 hybrid offspring inviable; and ancestral polymorphism of SaF and SaM loci in domesticated Oryza sativa subspecies causing hybrid infertility between cross of O.sativa japonica and O.sativa indica.

For classic DM model, hybrid necrosis was found usually result from negative epistatic interaction of complementary gene. Also, there are examples that hybrid necrosis preventing gene flow between species in the plant. Hybrids between different wild strains of Arabidopsis thaliana strains sometimes show hybrid necrosis due to a negative epistatic interaction between 2 to 4 loci. A highly polymorphic NB-LRR genes, which is most common resistant gene in plant was mapped and found responsible for the improper regulation of immune system in hybrid. The NB-LRR expression is normally regulated by a trans-acting element encoded at second loci which a failure of coevolution of the two interacting loci may disturb the balance of the interaction, leading to ectopic activation. Hybrid show lower threshold for activation of active immune response and some hybrid may have enhanced resistance against pathogen when comparing to their parents. This type of hybrid necrosis is experimentally found temperature sensitive which hybrid suffer from lethality at its typical habitat temperature but the autoimmune response is greatly suppressed at a higher temperature. The temperature-dependent effect suggests a quantitative incompatibility which the deleterious phenotype is subjected to the dosage of the incompatible alleles.

Second example of autoimmune response in rice was found in cross between japonica variety, Koshihikari, and indica variety, Habataki. Two unlinked recessive gene hbd2 and hbd3 interaction was found responsible for the hybrid breakdown. By fine mapping, hbd2 was found code for a causal gene as casein kinase I which they differ in one amino acid in coding sequence and over expression of the ‘Habataki’ alleles in ‘Koshihikari’ background lead to necrosis. While hbd3 was mapped within a cluster of NBS-LRR gene region which is the most common class of resistance gene, and a high degree of structural divergence was revealed.

There are also hybrid necrosis cases found in tomato. In cross between a domestic species S. lycopersicon and wild species S. pimpinellifolium, hybrid show necrotic phenotype when a fungal resistant gene-cf2 from S. pimpinellifolium is introgressed into S. lycopersicon that lack the cf2 gene. However, the autonecrosis is suppressed when an additional gene RCR3 which code for a secreted cysteine protease from S. pimpinellifolium is also introgressed into the hybrid. The tomato resistance loci cf2 is found containing two almost identical genes which confer resistance to the tomato leaf mould C. fulvum expressing the corresponding Avr2 gene product. The cf-2 gene encode a extracellular protein with leucine rich repeat motif that form complex with the Avr-2 protein which then bind to the RCR3 protease, causing its change of conformation to elicit a hypersensitive response. The molecular mechanism for S. lycopersicon Rcr3 –dependent autonecrosis is that S. lycopersicon Rcr3 protein have one amino acid deleted and six substitution when comparing with that of S. pimpinellifoliu, which those change resemble the cf-avr2 binding. Hence autoimmune response resulted even without infection.

Above disease resistance loci usually involve several tightly-related gene which are often subjected to diversifying selection that are believed as a driving force of sympatric speciation.

Cytoplasmic male sterility

Cytoplasmic male sterility (CMS) was found take a important part in causing hybrid sterility as most of the loci underlying sterility are mapped to be within plant mitochondria genome.CMS usually result from rearrangements of the mitochondrial genome through the formation and expression of chimeric open reading frames.CMS consist of two part, as like the DM model, one is the CMS-associated mitochondrial genes and the other is the non-functioning nuclear restorer of fertility (RF) genes.There are different mitochondrial genotypes triggering CMS in different ways while each of them have a specific set of matching nuclear genes that restore normal function. The RF genes achieve restoration usually by regulating the transcript profile or protein accumulation of the CMS locus while in some cases they lessen the destructive metabolic effects or act to reduce the abundance of mitochondrial segments carrying the CMS locus. As a result, the fertility is regulated by the balance of disruptive effects of mitochondria and the defensive effect of the nuclear genes but this delicate mitochondrial–nuclear balance is disturbed in hybrid. Since mitochondrial gene is maternally inherited while cytoplasmic gene is byparentally transmitted, difference in inheritance pattern creates a genetic conflict between nuclear and cytoplasmic genes which may contribute to the driving force of CMS. CMS loci are regarded as selfish genetic elements as they spread in population by mean of slightly increasing female fitness. Some CMS mutant show higher female fitness when comparing with hermaphrodites as they save the cost of producing pollen. The improved female fitness provides chances for natural selection to act on.

There are some interspecific CMS-related loci identified showing in table 2.

Table 2

CMS loci

species

Normal function

Possibly genetic cause

Brassica tour cytoplasm (ORF263), (ORF193)

Brassica juncea/B. tournefortii

Disruption of pollen development

Recombination created chimeric gene

Moricandia arvensis cytoplasm (ORF108)

Moricandia arvensis/Brassica napus

Disruption of pollen development

Recombination created chimeric gene

Wheat As or Tt cytoplasm (ORF256)

Triticum aestivum/T. timopheevi

Disruption of pollen development

Recombination created chimeric gene

Rice Boro II cytoplasm(ORF79)

Oryza sativa

Disruption of pollen development

Recombination created chimeric gene

Also, a table concluding some of the nuclear restorer loci is shown below:

Table 3

RF loci

species

function

Non-restoring cause

Maize RF2

maize

aldehyde dehydrogenase which restore URF13 CMS with the presence of Zm-RF1 gene

Loss of function mutation resulting from amino acid substitution in active site

RF-PPR 592

Petunia

Restore PCF CMS by down-regulate PCF CMS loci and thus reduce PCF protein production

Loss of function mutation resulting from base pair deletion in promoter region

SH-RF 1

Sorghum bicolor L.

Restore fertility of sorghum A1 cytoplasm

19 sequence polymorphisms in the third gene which code for a PPR protein

Rice RF1A

Oryza sativa

Restore BT CMS by endonucleolytic cleavage of ORF79 protein

Loss of function mutation result from frame shift mutation

Rice RF1B

Oryza sativa

Restore BT CMS by degrading ORF79 transcripts

Loss of function mutation result from amino acid substitution

Discussion:

Interspecific hybrid incompatibility is one of the causes of reproductive isolation between species which is a defining feature of the biological concept of species. Knowing the genetic basis of hybrid incompatibility is therefore a crucial procedure of discovering the origin of speciation. As genetic drift and selection continues after speciation, the split species continue to diverge and the intensity of incompatibility will only keep on increasing. Therefore, finding a incompatibility gene not necessarily mean finding a speciation gene. So, the candidate speciation genes discussed above should be termed RI genes as in strictly speaking speciation genes should be defined as evolved to cause incompatibility only at the time of speciation but it poses a great difficulty to isolate a speciation gene after speciation have taken place over millions years. However, hints about the phylogenetic relation among species may be obtained by observing the incompatibility pattern of hybrid crossed from different related species. For example, a Nup96-dependent lethality show in hybrid between D. melanogaster-D. simulans and D. melanogaster -D. sechellia, but not in D. melanogaster- D. mauritiana hybrid suggesting D. mauritiana may have speciated before D. simulans and D. sechellia. Apart from this, more evolutionary event could be predicted with the incidents that all non-synonymous substitution of Nup96 allele of D. simulans are also found in D. mauritiana ,which indicate Nup96 allele may had diverged prior to the divergence of D. simulans and D. mauritiana. Also, a gene locating on X chromosome incompatible with Nup96 alleles to cause lethality should have diverged on the D. melanogaster lineage due to the occurrence of lethality only on hybrids with the D. melanogaster X chromosome, neither with D. simulans nor D. sechellia .

Effect of incompatibility on fertility and viability:

Another part concerning HI I want to discuss is the differential effect of hybrid inviabilty and hybrid sterility in affecting hybrid fitness. Increasing evidence show that hybrid sterility evolves faster than hybrid inviability in many organism such as fruit fly Drosophila and yeast. A past experiment partially proved this using two yeast species S. cerevisiae and S. paradoxs. Their relative fertility and viability were measured by the sporulation frequency and the clonal growth rate respectively . The heterozygous F1 hybrids were not used in the test as recessive incompatibility may be masked. Instead, homozygous F2 hybrids formed by autodiploidization of the F1 gamete were examined. Result show that for each F2 individual, the growth-based was much higher than sporulation-based fitness, indicating hybrid sterility is more pronounced than hybrid viability. A possible molecular mechanisms is that a S. cerevisiae nuclear gene MRS1 was found incompatible with a S. paradoxs mitochondrial gene COX1. This cytonuclear incompatibility disturbs cellular metabolism on non-fermentable medium where yeast sporulates and therefore cause hybrid sterility. There are three main incompatibility-causing sites on the Sc-MRS1 gene while one of those is found polymorphic, suggesting that MRS1 alleles are evolutionarily young. As clonal growth proceeds with mitosis while sporulation are meiosis-required, the two different process involved different sets of genes with different molecular features .Normally viability-related incompatibility should evolve faster than that of fertility-related incompatibility due to more genes involved in clonal growth than in sporulation. But a new breakthrough in protein evolution suggest a different answer which there are a rapid evolutionary trend of the meiosis-related genes resulted from a alleviated deleterious effect of protein mis-folding caused by a non-synonymous mutation on a lowly expressed gene. While In major, wild yeast reproduces with asexual clonal growth which meiosis-related genes merely expressed, constructing a possible cause why hybrid infertility is more common than hybrid inviability in yeast.

Evolutionary forces that drive the divergence of speciation genes

Additional to the identity and the characteristic of the speciation genes, biologists are also interested in understanding the driving force to the divergence of speciation genes. Increasing evidence show that hybrid incompatibility gene evolves as a by-product of adaptive evolution and is rapidly evolving. For example, by comparative genetic and DNA sequencing, Nup96 gene was found diverged among Drosophila species with significant excessive non-synonymous substitution relative to synonymous substitution, demonstrating Nup96 allele is under positive natural selection rather than genetic drift. OdsH gene was also found rapidly evolving between D. simulans and D. mauritiana with non-synonymous substitution greatly outnumber synonymous substitution. A much higher than normal value of non-synonymous divergence for Hmr and Lhr gene between D. melanogaster and its sibling species is also recorded. All of the above cases strongly suggest a divergence through adaptive evolution. In addition, new hypotheses propose intragenomic conflict may take a part in which meiotic drive may contribute to the evolution of postmating reproductive isolation. Mutations causing meiotic drive are usually suppressed within species due to fixation of suppressor mutations soon after the distorters arise but can re-express in hybrids. A autosomal repressor allele of sex-ratio distortion was found to associate with hybrid sterility in Drosophila hybrid. A small fragment of D. mauritiana genome causes meiotic drive in an otherwise D. simulans genetic background while that region of chromosome is also found causing hybrid male sterility which suggesting a likely candidate gene tmy which is held responsible for both hybrid segregation distortion and hybrid sterility.

Conclusion

From the evidence shown by the HI genes discovered, it is convincing that HI is resulted from negative epistatic interaction of 2 or more loci involving multiple allelic substitution as predicted by the DM model which provide a path for HI to evolve without compromised fitness with species.

When comparing RI between plant and animals, there are much less evidence that diseases resistance gene are involved in RI of animals species. Also, mitochondria-associated gene seem rare in animals RI. There are evidence showing sympatric speciation make some contribution to plant speciation while in animal are much more subjected to allopatric evolution.

Future direction

To understand speciation, first we should know the origin of reproductive isolation. Although the majority of the truth of hybrid incompatibility remains uncertain, more and more HI genes is being discovered and the new innovation of searching HI gene is keep on developing. For example, a rapid and cost-effective method for mapping HI loci is invented using a nematode species, Caenorhabditis briggsae and its recently identified close relative C. sp.9.Before there is no sister species capable of mating and producing viable hybrid progeny with the well-studied model species C. elegan which lead to little contribution of C. elegan to this field. However, a newly discovered species C. sp.9 can mate with C. briggsae to produce viable offspring, offering an exceptional chance for studying the genetic and molecular mechanisms of HI between nematode species which in the past nematode species received very little attention in isolating HI loci except only one intra-species HI gene was identified in C. elegans using SNP based mapping. The method consist of systematic introgression of genomic fragments between the two species and the help of genetic map using a dominant and visible C. briggsae specific primer. If the genomic fragments reduce hybrid fitness in genetic background of otherwise species, then the fragment should be related to the HI. A lot of problem preventing effective mapping is tackled suck as the lack of a dominant and visible maker that evenly flagging of the C. briggsae chromosome which is done by repeated selection of suitable primers using PCR and GFP with the help of comparative genomics tools provided in UCSC genome browser, to ensure specific amplification of the genomic sequences of C. briggsae but not that from C. sp.9.Also this method helps generate introgression line as a side-product which aids in the subsequent mapping of HI loci. This can open up a new study field on HI alternative to those relatively well-established cases in rice and fruit fly and hopefully bring us closer to the mystery of the process of the speciation.