Unintentional selection during the process of population development

The process by which genetic and plant breeding populations are developed may include various unintended selection pres-sures which result in the deviation of geno-typic and allelic frequencies from Mendelian expectation. Segregation distortion has been documented in a wide range of organisms including plants. Distorted segregation can be detected with almost any type of genetic marker, including morphological mutants, isozymes and DNA markers (Table 3 in Xu et al., 1997). The same allele at a given locus can be distorted in either of two directions, with an allele frequency higher or lower than the expected. For example, waxy and non-waxy rice kernels on a segregating plant could show ratios of equal to or larger or smaller than the expected ratio (3:1), with the proportion of waxy rice kernels ranging from 8.9 to 95.6% depending on the crosses (Xu and Shen, 1992d).

As reviewed by Xu et al. (1997), aber-rant segregation ratios in plants may arise from a variety of physiological or genetic causes and may be manifested as differ-ential transmission in either the male or female germ line or may result from post-zygotic selection prior to genotypic evalu-ation. Most commonly, however, skewed segregation appears to arise from male gametophytic selection, through the selec-tive influences of the gynoecium, includ-ing genetic incompatibility, environmental effects and the differential competitive abil-ity of genetically-variable pollen.

Selection pressure associated with DH development

The representativeness of DH lines can be severely affected by the process involved in DH development. For the DH populations

derived from anther culture (male gameto-phytes), the observed distortion in segrega-tion can be attributed to differential viability or lethality of pollen or to selective regen-eration in in vitro culture and clearly not to the selective influences of the gynoecium or the differential competitive ability of pollen.

The distortion in three chromosomal regions (two on chromosome 2 and one on chromo-some 10) detected in the DH populations by Xu et al. (1997) indicated an overrepresenta-tion of alleles from the japonica parent that has been proven to be easily regenerated by anther culture. The other parent is indica which belongs to a subspecies that is more recalcitrant to anther culture (Shen et al., 1982; Yang et al., 1983). It has been suggested that these regions may be associated with the preferential regeneration of japonica geno-types during anther culture. Yamagishi et al.

(1996) also identified markers in several chromosomal regions that showed aberrant segregation ratios favouring japonica alleles in a DH population, although these markers segregated normally in the corresponding F₂ population. They concluded that these regions contained genetic factors which con-ferred a selective advantage on the japonica genotypes during anther culture.

Selective regeneration of genotypes has also been reported in other plants. Very strong distortions of single locus segrega-tions were observed in an anther culture-derived barley population (Devaux et al., 1995). Devaux and Zivy (1994) demon-strated that some markers showing distorted segregation are linked to genes involved in the anther culture response. In another bar-ley DH population, a significant proportion (44%) of the mapped markers showed dis-torted segregation which was caused mainly by the prevalence of alleles from the parent that responded better to in vitro culture (Graner et al., 1991). Although segregation distortion may arise from genetic, physio-logical and/or environmental causes and the relative contribution of each of these factors may differ in specific populations, much of the reported segregation distortion in anther culture-derived populations is likely to be the result of using parental genotypes that differ in their response to anther culture.

Selection pressure involved in RIL development

Deviation from randomness due to selec-tion pressure in the producselec-tion of RILs is a potential problem that needs more atten-tion. In contrast to the populations derived from a one-step homogenization, RILs are produced by many generations of inbreed-ing durinbreed-ing which plants are subjected to selection pressures generated by various environmental disturbances and competi-tion among plants that may well occur for many years and seasons and in many loca-tions. The distortions resulting from selec-tion pressures involved in RIL development can be understood by comparison of mul-tiple populations of different genetic struc-tures derived from the same crosses and by comparison of populations produced by dif-ferent approaches.

He et al. (2001) compared molecular marker segregations between DH and RIL populations derived by anther culture and SSD respectively from the same rice cross, ZYQ8 (indica) × JX17 (japonica). In the RIL population, 27.3% of the markers showed distorted segregation at the P < 0.01 level, of which 90% of the markers favoured indica alleles while in the DH population, 18.2%

of the markers were skewed almost equally towards indica and japonica alleles. This might reflect the different types of selection pressures to which the DH and RIL popula-tions were subjected. Eight commonly dis-torted regions on chromosomes 1, 3, 4, 7, 8, 10, 11 and 12 were detected in both RIL and DH populations of which seven skewed towards indica alleles and one towards a japonica allele. Five of them were located near gametophytic gene loci (ga) and/or ste-rility gene loci (S).

To compare the frequency and location of loci showing distorted allele frequencies between different population types (F₂, DHs and RILs), information from 53 populations with a known number of distorted markers was summarized and analysed (Xu et al., 1997). In summary, RIL populations had significantly higher frequencies of distorted markers (39.4 ± 2.5%) than other population structures (DH: 29.4 ± 3.5%; BC: 28.6 ± 2.8%;

F₂: 19.3 ± 11.2%), which may indicate the cumulative effects of selection pressures dur-ing the process of RIL development. Distorted segregation in RIL populations derived via SSD represents the cumulative effect of both genetic (G) and environmental (E) factors on multiple generations and the G × E interac-tion becomes more pronounced with the progress of selfing. Thus, it is difficult to dis-tinguish genetic from environmental causes of distortion in RIL populations. However, an over-representation of indica alleles in two chromosomal regions on chromosomes 3 and 6 was specific to one RIL population. These chromosomal regions may be associated with a selective advantage in the indica growth environment in which the RIL population was developed.

In contrast to DH and RIL populations where genotypic frequencies are a perfect reflection of the allele frequencies due to lack of heterozygotes, F₂ populations offer the potential to detect an advantage or dis-advantage associated with the heterozygote class at specific loci, even when the paren-tal allele frequency is normal.

Expression of distorters with low her-itability will be influenced by the environ-ment and therefore these will be detected only in experiments carried out under well-controlled conditions. Because the segrega-tion distorsegrega-tion occurs either during, or just before or after meiosis, the experimental environment must be controlled during the reproductive phase of the parental lines, although the effect will only be detected in the offspring.

Genetics of selection associated segregation distortion

The genetic control of distorted segregation has been studied in rice (as summarized by Xu et al., 1997) and barley (Konishi et al., 1990, 1992) using morphological and iso-zyme markers. The genetic basis of seg-regation distortion may be the abortion of male or female gametes or the selective fer-tilization of particular gametic genotypes.

Distortion at a marker locus in rice may be caused by linkage between the marker and the gene conferring lower pollinating

abil-ity, the gametophyte gene (ga) (Nakagahra, 1972) also referred to as a gamete eliminator or pollen killer, causing abortion of gametes (Sano, 1990). A large number of ga loci and sterility gene loci (S) have been identified using morphological markers.

If segregation distorters have high herit-ability, they will be detected in almost any population if the parents differ at the genetic locus in question and in almost any environ-ment in which the population is grown. For a specific chromosomal region, the prob-ability of a distortion locus being falsely assigned will decrease with the number of populations sharing the same distortion and with the number of markers in a cluster of distorted markers. Use of multiple popula-tions developed in multiple environments would facilitate the detection of highly heritable genetic segregation distortion fac-tors. Chromosomal regions associated with marker segregation distortion in rice were compared using six molecular linkage maps (Xu et al., 1997). Mapping populations were derived from one interspecfic backcross and five inter-subspecfic (indica/japonica) crosses including two F₂ populations, two DH populations and one RIL population.

Marker loci associated with skewed allele frequencies were distributed on all 12 chro-mosomes. Distortion in eight chromosomal regions showed the grouping of previ-ously identified gametophyte (ga) or steril-ity genes (S). Three additional clusters of skewed markers were observed in more than one population in regions where no game-tophytic or sterility loci had been reported previously. A total of 17 segregation distor-tion loci were postulated and their locadistor-tions in the rice genome were estimated. Using a single F₂ cross, Harushima et al. (1996) iden-tified 11 major segregation distortions at ten positions on chromosomes 1, 3, 6, 8, 9 and 10 and at least two of these segregation dis-tortion regions (on chromosomes 1 and 3) were also detected by Xu et al. (1997).

A similar comparison was undertaken among four maize mapping populations using 1820 co-dominant markers (Lu et al., 2002). On a given chromosome nearly all of the markers showing segregation distortion favoured the allele from the same parent.

A total of 18 chromosomal regions on the ten maize chromosomes were associated with segregation distortion. The consistent location of these chromosomal regions in four populations suggested the presence of segregation distortion regions. Three known gametophytic factors are possible genetic causes for the presence of these regions.

In Populus most markers exhibiting segregation distortion generally occurred in large contiguous blocks on two linkage groups and it has been hypothesized that divergent selection had occurred on the chromosomal scale among the parental spe-cies (Yin, T.M. et al., 2004).

Segregation distortion loci were map-ped to chromosomal regions including three regions on chromosome 5D in Aegilops tauschii using 194 molecular markers for an F₂ population (Faris et al., 1998). Two sets of reciprocal BC populations were used to further analyse the effect of sex and cyto-plasm on segregation distortion. Extreme distortion of marker segregation ratios in the chromosome 5D regions was observed in populations in which the F₁ was used as the male parent and ratios were skewed in favour of one parent. There was some evi-dence of differential transmission caused by nucleo-cytoplasmic interactions. This result, along with other studies, indicated that loci affecting gametophyte competition in male gametes are located on 5DL.

To map segregation-distorting loci using molecular markers, both a maximum likeli-hood (ML) method and a Bayesian method were developed (Vogl and Xu, 2000). ML mapping was implemented by use of an expectation-maximization algorithm and the Bayesian method was developed using the Markov chain Monte Carlo (MCMC) approach. Bayesian mapping is computa-tionally more intensive than ML mapping but can handle more complicated models such as multiple segregation-distorting loci.

Implications for genetics and plant breeding The phenomenon of segregation distortion is intimately linked to the probability of producing specific recombinants of inter-est in genetics and breeding populations. In

genetics, construction of genetic maps and identification of linkage among markers and between markers and genes depends on the segregation patterns of all the mark-ers and genes involved. In breeding, the success of obtaining specific genes, geno-types and gene combinations depends on the probability of the target genes and gene combinations occurring at a ratio expected by Mendelian segregation. To broaden the genetic base of cultivated species, breed-ers often undertake wide hybridization but they frequently fail to recover recombinants of interest, in part as a result of non-random survival or generation of offspring. On the other hand however, phenotypic selec-tion during inbreeding including the back-crossing process, which breeders of course utilize, can significantly improve the prob-ability of recovering the desired alleles.

Identification of genetic factors asso-ciated with segregation distortion will contribute to our understanding of where these genetic factors are located and how they might be managed in a breeding pro-gramme. If a target locus is known to be linked to a segregation distortion locus and is underrepresented in a desired pop-ulation, the frequency of the favourable allele can be increased by using molecular markers to select for recombinants in the region of interest. To reduce the negative influence of segregation distortion in plant breeding, it is reasonable to decrease the number of generations required for stabiliz-ing breedstabiliz-ing lines. The production of DH populations from F₁ hybrids minimizes the number of generations required to reach homozygosity and therefore maximizes the chance of retaining desirable alleles in a population unless they are linked to segre-gation distortion factors that affect DH pro-duction. In wide crosses where wild alleles tend to be disproportionately lost, the fre-quency of rare alleles can be enhanced by adjusting the type of selection and popu-lation structure used in accordance with genetic information relating to segregation distortion, thus providing further opportu-nities for favourable recombination in later generations (Xu et al., 1997). To under-stand the underlying mechanism(s) that

is responsible for segregation distortion, it would be useful to develop NILs contain-ing individual segregation distortion loci so that the effect of these factors could be evaluated systematically in different

genetic backgrounds and environments.

NILs would also provide material for clon-ing these genetic factors to permit a more in-depth characterization of their molecu-lar structure and function.

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