Complete genetic linkage maps from an interspecific cross between Fusarium circinatum and Fusarium subglutinans

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CHAPTER 3 Transmission ratio distortion in an interspecific cross between Fusarium circinatum and Fusarium subglutinans

Abstract

, an interspecific cross between Fusarium circinatum and Fusarium subglutinans was used to generate a genetic linkage map. About 55% of the genotyped markers in this cross displayed genome-wide transmission ratio distortion (TRD). The working hypothesis for this study was that TRD would be nonrandomly distributed throughout the genome due to distorting loci. Using a genome-wide threshold of α = 0.01, 79 markers displaying TRD were distributed on all twelve linkage groups. Eleven putative TRDLs (transmission ratio distortion loci), on eight linkage groups, were identified in regions containing three or more adjacent markers displaying distortion. No epistatic interactions were observed between these TRDLs. Thus, it is uncertain whether the genome-wide TRD was due to linkage between markers and genomic regions causing distortion. The parental origins of markers followed a non-random distribution throughout the linkage map, with linkage groups contained stretches of markers originating from only one parent. Thus, due to the nature of the interspecific cross, the current hypothesis to explain these observations is that the observed genome-wide segregation was caused by the high level of genomic divergence between the parental isolates. Thus, homologous chromosomes would not align properly during meiosis, resulting in aberrant transmission of markers. This would explain previous observations of the preferential transmission of F. subglutinans alleles to the F₁ progeny.

Introduction

The Gibberella fujikuroi species complex accommodates the sexual stage of Fusarium spp. collectively treated in the section Liseola (Leslie & Summerell, 2006). This complex includes some of the most ubiquitous and economically important fungal pathogens of plants. The biological species concept had been used to formally classify species in this complex into ten mating populations or biological species (Nirenberg & O‟Donnell, 1988; Samuels et al., 2001; Zeller et al., 2003; Lepoint et al., 2005). Species delineation, when applying the biological species concept, implies that individual species are reproductively isolated (Mayr, 1940; Dobzhansky, 1951). This is somewhat complicated in fungi where interspecific crosses can occur, such as those found between some taxa in the G. fujikuroi species complex (Desjardins et al., 1997; Desjardins et al., 2000; Leslie et al., 2004b).
Several examples exist for interspecific crosses within this species complex. Certain isolates from Fusarium fujikuroi (mating population C) and Fusarium proliferatum (mating population are interfertile and produce viable progeny (Desjardins et al., 1997; Desjardins et al., 2000; Leslie et al., 2004b), and a naturally occurring hybrid has been identified (Leslie et al., 2004a). The current hypothesis is that genetic isolation between these two biological species is not complete, allowing reproductive barriers to be overcome (Leslie et al., 2004b). Another example of a laboratory generated interspecific cross is one between Fusarium subglutinans and Fusarium circinatum, residing in mating populations E and H, respectively (Desjardins et al., 2000; De Vos et al., 2007; Friel et al., 2007).
De Vos et al. (2007) constructed a genetic map for the interspecific cross between F. circinatum and F. subglutinans. These authors found that ca. 55% of the markers exhibited significant TRD (transmission ratio distortion) from the expected ratio of 1:1 of a haploid cross (P < 0.05). Ninety-six percent of the TRD markers were skewed towards the F. subglutinans male parent. There was also preferential transmission of alleles, as well as complete chromosomes, from the genome of F. subglutinans. The clear bias towards the transmission of F. subglutinans alleles led to the conclusion that the F₁ progeny that inherited F. subglutinans alleles exhibited a general fitness benefit (De Vos et al., 2007).
Mendel‟s postulate of segregation dictates that during the formation of gametes, the paired unit factors segregate randomly such that each gamete receives one or the other with equal likelihood (Klug & Cummings, 1994). When deviations from the expected Mendelian ratio of segregation (TRD) occur, they are frequently observed in interspecific crosses (Zamir & Tadmor, 1986). It has been demonstrated that the larger the genetic divergence between the parental lines, the higher the levels of TRD (Grandillo & Tanksley, 1996; Lee et al., 2009). Interspecific crosses in Fusarium display the same tendency. Thus, interspecific crosses display higher levels of segregation (Jurgenson et al., 2002; Leslie et al., 2004b), than intraspecific crosses (Gale et al., 2005).
TRD has been attributed to linkage between markers and genetic factor(s) that affect the fitness of gametes leading to unbalanced transmission of parental alleles to the next generation (Zamir Tadmor, 1986). This functions during the pre- and postzygotic stages of reproduction and can also affect the zygotic viability. The presence of non-random marker loci exhibiting TRD throughout the genome, would suggest the presence of a distorting genetic factor in that region of the genome (transmission ratio distortion loci; TRDL) (Jiang et al., 2000; Myburg et al., 2004). These loci form barriers that prevent recombination from occurring in these parts of the genome, leading to unbalanced transmission of parental alleles to the zygotes.
With the advent of high-throughput molecular markers such as AFLPs (Vos et al., 1995), the construction of genetic linkage maps with high levels of map coverage has been possible. This has allowed for detailed examination of the transmission of these markers to the next generation, including markers displaying TRD. The working hypothesis for this study was that TRD in an interspecific cross between F. circinatum and F. subglutinans would be non-randomly distributed throughout the genome. The aim was, therefore, to determine the positions and effects of possible TRDLs, by means of a previously compiled genetic linkage map derived from an interspecific cross between F. circinatum and F. subglutinans.

Materials & Methods

Identification of TRD and putative TRDL

The direction and percentage of distortion of each marker from the genetic linkage map (De Vos et al., 2007), was determined by employing the formula (allele frequency – 0.5) x 100% (Myburg et al., 2004). Markers displaying TRD could have occurred by chance or by displaying linkage to genetic factor(s) that affect the fitness of gametes (Zamir & Tador, 1986). To distinguish between a “chance” TRDL or a “true” TRDL, a genome-wide significance threshold is needed (Myburg et al., 2004). Using this method with results of De Vos et al. (2007), it was assumed that the 12 linkage groups identified correspond to the 12 chromosomes (n = 12 for F. subglutinans, Xu et al., 1995). Assuming that each chromosome contains at least two independent segregating regions, there was an expectation of a minimum of 24 independently segregating regions. To acquire a genome-wide significance level of P = 0.05, a significance threshold of 0.05/24 » 0.002 would be necessary. However, in order to include weak TRDLs in this study, a significance threshold of α = 0.01 (χ² = 6.64) was employed. All regions that displayed three or more distorted markers were noted. The most skewed marker in this region was considered the most likely position of the distorting factor (Lu et al., 2002).

Epistatic interactions between the TRDLs

To determine whether epistatic interactions occur between pairs of TRDLs, the most distorted marker in each TRDL was used (i.e. the most likely position of the distorting factor). Marker scores were evaluated for each pair of markers using Fisher‟s exact test in Statistica (v. 10, StatSoft, Inc. 2011, www.statsoft.com).

Results

Identification of TRD and putative TRDL

Of the 252 markers that were placed on the genetic linkage map, 138 (55%) showed distortion at the 5% level of significance, 79 (31%) at the 1% level of significance and 37 (15%) at the 0.1% level of significance. Markers displaying TRD were distributed throughout the genome; all linkage groups had markers that deviated from the expected 1:1 ratio (P < 0.05, Figure 2). Sampling error was excluded as a possible reason for distorted frequencies, as at a 5% level of significance, only 29 markers would be expected to show TRD.
When comparing the transmission of markers on the linkage map, the distribution of the F. subglutinans genetic composition amongst the F₁ progeny showed a mean of 59.8% (Figure 1). This was significantly different (P = 0.049996) to the predicted value of 50%. Also, the distribution of the F. subglutinans genome in the progeny did not follow a normal distribution as expected (Shapiro-Wilk W-test; SW-W = 0.87, P = 0.00). The distribution showed two „tails‟, with the second “tail” at the 90-100% genomic constitution of F. subglutinans (Figure 1). This is an indication that some (thirteen of ninety-four) F₁ progeny closely resembled the F. subglutinans parental isolate in their genetic constitution, compared to F. circinatum (De Vos et al., 2011). In contrast, only one of the F₁ progeny showed a F. circinatum genomic constitution of >90%.
In determining the direction and percentage of distortion of each marker placed on the genetic linkage map, only 12 markers (4%) were skewed towards the F. circinatum parent (Figure 2). This directional distortion extended over whole linkage groups, except in LG 2, 5, 6, 8, 9 and 11, where isolated markers were skewed towards F. circinatum (one marker on LG 5, 8, 9 and 11 and two markers each on LG 2 and 6). The exception was LG 7, where four of six markers at the beginning of the linkage group were skewed towards F. circinatum.
Using the genome-wide significance threshold of P = 0.01, 79 markers displayed TRD (Figure 2). Eleven regions were identified that displayed three or more distorted markers (P < 0.01), with the marker displaying the highest distortion as the most probable area for the TRDL (Figure 2, indicated with arrows). TRD regions that displayed three or more distorted markers were all unidirectionally distorted towards the F. subglutinans parent. These were located on LG 1 (markers GA/CC-353be and GA/AC-523bh), on LG 2 (marker CA/TC-463fh), LG 4 (markers AA/AC-337bh and CA/TC-263be), LG 5 (between markers CA/TC-149fh and GA/CC-111be), LG 6 (markers GA/AC-213bh and AA/AA-142fh), LG 8 (marker AG/AC-315bh), LG 10 (marker CA/TC-189be) and LG 11 (marker AG/AC-751fh) (Table 1). Thus, the putative TRDLs were not evenly distributed across the linkage groups, with LG 1, 4 and 6 having two TRDLs, LG 2, 5, 8, 10 and 11 having one TRDL and LG 3, 7, 9 and 12 not containing any. These TRDLs covered a total of 396.5 cM which accounts for 14.29 % of the observed map length.
A haploid population has allelic frequencies that equal genotypic frequencies, so it was not possible to establish whether TRD was caused by gametic or zygotic selection. However, the TRDL effects can also be expressed as the differential viability, t (0 < t < 1), of gametes or zygotes with alternate genotypes to that of normal gametes or zygotes (Cheng et al., 1998). The relative viability or fertilization ability of gametes or viability of zygotes affected by the TRDL ranged from 0.34 to 0.53 (Table 1), indicating the unidirectional skewing towards the F. subglutinans parent.

CHAPTER 1 Fusarium genomics and underlying opportunities to understand the genetics of the pitch canker fungus
Introduction
The Gibberella fujikuroi species complex
Fusarium circinatum
Classification
The pitch canker disease
Genetic linkage mapping
Genetic linkage mapping in fungi
Genetic linkage mapping in Fusarium
The genome sequence of Fusarium graminearum
Interspecific crosses in the G. fujikuroi species complex
Conclusion
References
CHAPTER 2 Complete genetic linkage maps from an interspecific cross between Fusarium circinatum and Fusarium subglutinans
Abstrac
Introduction
Materials & Methods
Fungal isolates
DNA isolation
AFLP analysis
Additional marker analysis
Framework linkage map construction
Bin mapping of accessory markers
Estimated genome coverage and length
DNA isolation and AFLP analysis.
Framework linkage map construction
Discussion
References
CHAPTER 3 Transmission ratio distortion in an interspecific cross between Fusarium circinatum and Fusarium subglutinans
Abstract
Introduction
Materials & Methods
Identification of TRD and putative TRDL
Epistatic interactions between the TRDLs
Results
Identification of TRD and putative TRDLs
Epistatic interactions between the TRDLs
Discussion
References
CHAPTER 4 Genetic analysis of growth, morphology and pathogenicity in the F1 progeny of an interspecific cross between Fusarium circinatum and Fusarium subglutinans
Abstract
Introduction
Materials & Method
Results
Discussion
References
CHAPTER 5 Macrosynteny within the Gibberella fujikuroi species complex allows for the identification of a putative reciprocal translocation
Abstract
Introduction
Materials & Methods
AFLP generation
PCR addition of sequencing adapters
Pyrosequencing
Results
Discussion
References
Summary
Conclusions and Future Prospects
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