A stepwise route to domesticate rice by controlling seed shattering and panicle shape.

Asian rice (Oryza sativa L.) is consumed by more than half of the world's population. Despite its global importance, the process of early rice domestication remains unclear. During domestication, wild rice (Oryza rufipogon Griff.) acquired non-seed-shattering behavior, allowing humans to increase grain yield. Previous studies argued that a reduction in seed shattering triggered by the sh4 mutation led to increased yield during rice domestication, but our experiments using wild introgression lines show that the domesticated sh4 allele alone is insufficient for shattering loss in O. rufipogon. The interruption of abscission layer formation requires both sh4 and qSH3 mutations, demonstrating that the selection of shattering loss in wild rice was not as simple as previously suggested. Here we identified a causal single-nucleotide polymorphism at qSH3 within the seed-shattering gene OsSh1, which is conserved in indica and japonica subspecies but absent in the circum-aus group of rice. Through harvest experiments, we further demonstrated that seed shattering alone did not significantly impact yield; rather, yield increases were observed with closed panicle formation controlled by SPR3 and further augmented by nonshattering, conferred by integration of sh4 and qSH3 alleles. Complementary manipulation of panicle shape and seed shattering results in a mechanically stable panicle structure. We propose a stepwise route for the earliest phase of rice domestication, wherein selection of visible SPR3-controlled closed panicle morphology was instrumental in the sequential recruitment of sh4 and qSH3, which together led to the loss of shattering.

Detection of novel loci involved in non-seed-shattering behaviour of an indica rice cultivar, Oryza sativa IR36.

Asian rice (Oryza sativa) was domesticated from O. rufipogon, and reduced seed-shattering behaviour was selected to increase yields. Two seed-shattering loci, qSH3 and sh4, are involved in reducing seed shattering in both japonica and indica rice cultivars, while qSH1 and qCSS3 are likely specific to japonica cultivars. In indica cultivars, qSH3 and sh4 fail to explain the degree of seed shattering, as an introgression line (IL) of O. rufipogon W630 carrying domesticated alleles at qSH3 and sh4 still showed seed shattering. Here we analysed differences in seed-shattering degree between the IL and the indica cultivar IR36. The values for grain detachment in the segregating population between the IL and IR36 were continuous. Based on QTL-seq analysis using the BC1F2 population between the IL and IR36, we detected two novel loci, qCSS2 and qCSS7 (QTLs for the Control of Seed Shattering in rice on chromosomes 2 and 7), which contributed to the reduced seed shattering in IR36. We further investigated the genetic interaction of qCSS2 and qCSS7 under the presence of qSH3 and sh4 mutations in O. rufipogon W630 and found that IL carrying IR36 chromosomal segments covering all four loci are required to explain seed-shattering degree in IR36. Since qCSS2 and qCSS7 were not detected in previous studies on seed shattering in japonica, their control may be specific to indica cultivars. Therefore, they are important to understanding the history of rice domestication as well as to adjusting the seed-shattering degree of indica cultivars to maximise their yield.

Different contributions of PROG1 and TAC1 to the angular kinematics of the main culm and tillers of wild rice (Oryza rufipogon).

MAIN CONCLUSION: PROG1 is necessary but insufficient for the main culm inclination while TAC1 partially takes part in it, and both genes promote tiller inclination in Asian wild rice. Asian wild rice (Oryza rufipogon), the ancestor of cultivated rice (O. sativa), has a prostrate architecture, with tillers branching from near the ground. The main culm of each plant grows upward and then tilts during the vegetative stage. Genes controlling tiller angle have been reported; however, their genetic contributions to the culm movement have not been quantified. Here, we quantified their genetic contributions to angular kinematics in the main culm and tillers. For the main culm inclination, one major QTL surrounding the PROG1 region was found. In cultivated rice, tillers firstly inclined and lately rose, while it kept inclining in wild rice. It was suggested that PROG1 affected the tiller elevation angle in the later kinematics, whereas TAC1 was weakly associated with the tiller angle in the whole vegetative stage. Micro-computed tomography (micro-CT) suggested that these angular changes are produced by the bending of culm bases. Because near-isogenic lines (NILs) of wild rice-type Prog1 and Tac1 alleles in the genetic background of cultivated rice did not show the prostrate architecture, the involvement of another gene(s) for inclination of the main culm was suggested. Our findings will not only contribute to the understanding of the morphological transition during domestication but also be used in plant breeding to precisely reproduce the ideal plant architecture by combining the effects of multiple genes.

Wild and cultivated allele effects on rice phenotypic traits in reciprocal backcross populations between Oryza rufipogon and two cultivars, O. sativa Nipponbare and IR36.

A total of four populations of reciprocal backcross recombinant inbred lines were produced from a cross between a wild accession of Oryza rufipogon W630 and two major cultivars, O. sativa Japonica Nipponbare and Indica IR36. Using these populations, quantitative trait locus (QTL) analysis for eight morphological traits (culm length, panicle length, days to heading, panicle shape, pericarp color, hull color, seed shattering and seed awning) was carried out, and the putative QTL regions were compared among the populations. The QTLs with strong allele effects were commonly detected for culm length, panicle shape, pericarp color and hull color in all four populations, and their peak locations were close to the major genes of sd1, Spr3, Rc and Bh4, respectively. For panicle length and days to heading, some QTL regions overlapped between two or three populations. In the case of seed shattering and seed awning, strong wild allele effects at major loci were observed only in the populations with cultivated backgrounds. Since the wild and cultivated alleles have never been evaluated in the reciprocal genetic backgrounds, the present results provide new information on gene effects in breeding and domestication studies.

Assessment of skin inflammation using near-infrared Raman spectroscopy combined with artificial intelligence analysis in an animal model.

Raman spectroscopy is a powerful method for estimating the molecular structure of a target that can be adapted for biomedical analysis given its non-destructive nature. Inflammatory skin diseases impair the skin's barrier function and interfere with the patient's quality of life. There are limited methods for non-invasive and objective assessment of skin inflammation. We examined whether Raman spectroscopy can be used to predict skin inflammation with high sensitivity and specificity when combined with artificial intelligence (AI) analysis. Inflammation was chemically induced in mouse ears, and Raman spectra induced by a 785 nm laser were recorded. A principal component (PC) analysis of the Raman spectra was performed to extract PCs with the highest percentage of variance and to estimate the statistical score. The accuracy in predicting inflammation based on the Raman spectra with or without AI analysis was assessed using receiver operating characteristic (ROC) curves. We observed some typical changes in the Raman spectra upon skin inflammation, which may have resulted from vasodilation and interstitial oedema. The estimated statistical scores based on spectral changes correlated with the histopathological changes in the skin. The ROC curve based on PC2, which appeared to include some spectral features, revealed a maximum accuracy rate of 80.0% with an area under the curve (AUC) of 0.864. The AI analysis improved the accuracy rate to 93.1% with an AUC of 0.972. The current findings demonstrate that the combination of Raman spectroscopy with near-infrared excitation and AI analysis can provide highly accurate information on the pathology of skin inflammation.

Interspecific introgression and natural selection in the evolution of Japanese apricot (Prunus mume).

Domestication and population differentiation in crops involve considerable phenotypic changes. The logs of these evolutionary paths, including natural/artificial selection, can be found in the genomes of the current populations. However, these profiles have been little studied in tree crops, which have specific characters, such as long generation time and clonal propagation, maintaining high levels of heterozygosity. We conducted exon-targeted resequencing of 129 genomes in the genus Prunus, mainly Japanese apricot (Prunus mume), and apricot (Prunus armeniaca), plum (Prunus salicina), and peach (Prunus persica). Based on their genome-wide single-nucleotide polymorphisms merged with published resequencing data of 79 Chinese P. mume cultivars, we inferred complete and ongoing population differentiation in P. mume. Sliding window characterization of the indexes for genetic differentiation identified interspecific fragment introgressions between P. mume and related species (plum and apricot). These regions often exhibited strong selective sweeps formed in the paths of establishment or formation of substructures of P. mume, suggesting that P. mume has frequently imported advantageous genes from other species in the subgenus Prunus as adaptive evolution. These findings shed light on the complicated nature of adaptive evolution in a tree crop that has undergone interspecific exchange of genome fragments with natural/artificial selections.

Role of qGZn9a in controlling grain zinc concentration in rice, Oryza sativa L.

KEY MESSAGE: A candidate gene responsible for higher grain zinc accumulation in rice was identified, which was probably associated with a partial defect in anther dehiscence. Zinc (Zn) is an essential mineral element in many organisms. Zn deficiency in humans causes various health problems; therefore, an adequate dietary Zn intake is required daily. Rice, Oryza sativa, is one of the main crops cultivated in Asian countries, and one of the breeding scopes of rice is to increase the grain Zn levels. Previously, we found that an Australian wild rice strain, O. meridionalis W1627, exhibits higher grain Zn levels than cultivated rice, O. sativa Nipponbare, and identified responsible genomic loci. An increase in grain Zn levels caused by one of the loci, qGZn9a, is associated with fertility reduction, but how this negative effect on grain productivity is regulated remains unknown. In this study, we artificially trimmed spikelets on the flowering day and found that a reduction in number of seeds was associated with an increase in the grain Zn levels. We also found that a partial defect in anther dehiscence correlated with the increase in grain Zn levels in plants carrying the W1627 chromosomal segment at qGZn9a in a Nipponbare genetic background. Among eight candidate genes in the qGZn9a region, three were absent from the corresponding region of W1627; one of these, Os09g0384900, encoding a DUF295 protein with an unknown function, was found to be specifically expressed in the developing anther, thereby suggesting that the gene may be involved in the regulation of anther dehiscence. As fertility and grain Zn levels are essential agronomic traits in rice, our results highlight the importance of balancing these two traits.

Detection of a novel locus involved in non-seed-shattering behaviour of Japonica rice cultivar, Oryzasativa 'Nipponbare'.

KEY MESSAGE: A novel locus, qCSS3, involved in the non-seed-shattering behaviour of Japonica rice cultivar, 'Nipponbare', was detected by QTL-seq analysis using the segregating population with the fixed known seed-shattering loci. Asian cultivated rice, Oryzasativa, was domesticated from its wild ancestor, O.rufipogon. Loss of seed shattering is one of the most recognisable traits selected during rice domestication. Three quantitative trait loci (QTLs), qSH1, qSH3, and sh4, were previously reported to be involved in the loss of seed shattering of Japonica cultivated rice, O.sativa 'Nipponbare'. However, the introgression line (IL) carrying 'Nipponbare' alleles at these three loci in the genetic background of wild rice, O.rufipogon W630, showed a lower value for detaching a grain from the pedicel than 'Nipponbare'. Here, we investigated abscission layer formation in the IL and found a partially formed abscission layer in the central region between the epidermis and vascular bundles. Based on QTL-seq analysis using the F2 population obtained from a cross between 'Nipponbare' and the IL, we detected two novel loci qCSS3 and qCSS9 (QTL for the Control of Seed Shattering in rice on chromosomes 3 and 9), which were found to be involved in the difference in seed-shattering degree between 'Nipponbare' and W630. Then, we further focused on qCSS3 in order to understand its potential role on the loss of seed shattering. The candidate region of qCSS3 was found to be located within a 526-kb region using substitution mapping analysis. Interestingly, the qCSS3 candidate region partially overlaps the selective sweep detected for Japonica but not for Indica rice cultivars, suggesting that this region harbours the mutation at a novel seed-shattering locus specifically selected for non-seed-shattering behaviour in Japonica cultivars.

Assessment of genetic diversity and genetic structure of wild rice populations in Myanmar.

To evaluate genetic diversity and genetic structure of wild rice (Oryza rufipogon) populations in Myanmar, seven research sites were selected based on various ecological conditions. A large number of samples under natural growth conditions were collected and studied using six simple sequence repeats (SSRs) and two chloroplast DNA markers. A total of 77 alleles were detected from 1559 samples over six SSR loci. The mean number of alleles per population ranged from 3.167 to 8.667, and the mean expected heterozygosity ranged from 0.140 to 0.701. Wild rice populations survived under various environmental conditions and retained different levels of genetic diversity. The large number of samples was effective to confirm the spatial genetic structure of wild rice populations in a relatively small area. Regarding chloroplast DNA polymorphisms, four populations possessed only one pattern, while the other three showed two or five combinations of haplotypes, even within the same population. Additionally, the existence of a new genotype was revealed. Considerable variations in chloroplast DNA exist in the wild rice populations of Myanmar. A high proportion of genetic variation was detected within, rather than among, populations. To ensure maintenance of allelic diversity, it is advisable to preserve many individuals from a large population.

Estimation of loci involved in non-shattering of seeds in early rice domestication.

Rice (Oryza sativa L.) is widely cultivated around the world and is known to be domesticated from its wild form, O. rufipogon. A loss of seed shattering is one of the most obvious phenotypic changes selected for during rice domestication. Previously, three seed-shattering loci, qSH1, sh4, and qSH3 were reported to be involved in non-shattering of seeds of Japonica-type cultivated rice, O. sativa cv. Nipponbare. In this study, we focused on non-shattering characteristics of O. sativa Indica cv. IR36 having functional allele at qSH1. We produced backcross recombinant inbred lines having chromosomal segments from IR36 in the genetic background of wild rice, O. rufipogon W630. Histological and quantitative trait loci analyses of abscission layer formation were conducted. In the analysis of quantitative trait loci, a strong peak was observed close to sh4. We, nevertheless, found that some lines showed complete abscission layer formation despite carrying the IR36 allele at sh4, implying that non-shattering of seeds of IR36 could be regulated by the combination of mutations at sh4 and other seed-shattering loci. We also genotyped qSH3, a recently identified seed-shattering locus. Lines that have the IR36 alleles at sh4 and qSH3 showed inhibition of abscission layer formation but the degree of seed shattering was different from that of IR36. On the basis of these results, we estimated that non-shattering of seeds in early rice domestication involved mutations in at least three loci, and these genetic materials produced in this study may help to identify novel seed-shattering loci.

Evaluation of genetic variation among wild rice populations in Cambodia.

A total of 448 samples in five natural populations of wild rice (Oryza rufipogon) were collected in Cambodia. They were examined using 12 SSR and two chloroplast markers to evaluate the degree of variation among populations and the genetic structure within populations. In the two annual populations, the number of plants with homozygous alleles at all 12 SSR loci were high (66.3% and 79.5%), suggesting that these plants propagate mainly through self-pollination. In the three perennial populations, no individuals had all homozygous genotypes, but redundant genotypes resulted from clonal propagation were observed. Percentages of the redundant genotypes were highly varied (3.6%, 29.2% and 86.0%). This may be due to the different stable levels of environmental conditions. As for chloroplast genome, most of the wild plants showed the same chloroplast types as most Indica-type cultivars have. However, plants with different chloroplast types were maintained, even in the same population. In tropical Asian countries, many wild rice populations were observed under similar ecological conditions examined in this study. Therefore, the present results concerning population structure will be important to further elucidate genetic features of wild rice, and will also give strong clues to utilize and conserve wild natural genetic resources.

Effect of quantitative trait loci for seed shattering on abscission layer formation in Asian wild rice Oryza rufipogon.

Asian cultivated rice Oryza sativa L. was domesticated from its wild ancestor, O. rufipogon. During domestication, the cultivated rice lost its seed-shattering behaviour. Previous studies have shown that two major quantitative trait loci (QTLs; qSH1 and sh4) are responsible for the seed-shattering degree. Here, we produced introgression lines carrying non-functional alleles from O. sativa 'Nipponbare' at the two major QTLs in the genetic background of wild rice O. rufipogon W630, and examined the effects of the two QTLs on seed shattering and abscission layer formation. The introgression lines, with Nipponbare alleles at either or both loci, showed complete or partial abscission layer formation, respectively, indicating that other unknown loci might be involved in enhancing seed shattering in wild rice. We detected a single QTL named qSH3 regulating seed-shattering degree using an F2 population between Nipponbare and the introgression line carrying Nipponbare alleles at the two QTLs. Although we generated an introgression line for qSH3 alone, no effects on seed shattering were observed. However, a significant effect on seed-shattering degree was observed for the introgression line carrying Nipponbare alleles at qSH3 and the two QTLs, suggesting an important role of qSH3 on seed shattering in coordination with the two QTLs.

Estimation of the outcrossing rate for annual Asian wild rice under field conditions.

The Asian wild rice, Oryza rufipogon, has partial outcrossing behavior and shows high levels of genetic variation. To estimate an accurate outcrossing rate of annual form of O. rufipogon, two backcross lines (Lines R1 and R2) between Oryza sativa Nipponbare and O. rufipogon W630 were examined under field conditions. A chromosome survey confirmed that these lines had wild chromosomal segments at more than 92% of marker loci. As for the traits of glume, stamen and pistil, Line R1 showed similar floral morphology as that of O. rufipogon W630, whereas Line R2 had larger glumes. In 2005, 2006 and 2008, a total of 22 backcross plants were planted in the middle of wild rice plots. The successive progenies of each plant were examined using microsatellite markers that could clearly detect self-pollination and outcrossing. The outcrossing rates of Line R1 plants ranged from 4.04% to 25.50% with an average of 10.20%. This indicates that cross-pollination of wild rice is a chance event affected by many environmental factors. The outcrossing rates of Line R2 plants also varied, however, no significant difference was observed between the averages of Lines R1 and R2, suggesting that the glume sizes are not critical for outcrossing ability.

Variations in Grain Traits among Local Rice Varieties Collected More Than Half-Century Ago in Indochinese Countries.

More than half-century ago, local rice varieties were collected from Indochinese countries (Cambodia, Thailand, Laos, and Vietnam). Of these, 162 local varieties were examined for 7 grain-size traits: seed length/width/thickness, brown rice length/width/thickness, and 100-seed weight. Since these traits varied considerably, a survey of functional mutations was performed in the genes related to these traits. In total, 19 markers (12 InDel and 7 dCAPS markers) were used to investigate the mutations at 14 grain-size loci of GW2, GS2, qLGY3, GS3, GL3.1, TGW3, GS5, GW5, GS6, TGW6, GW6a, GLW7, GL7, and GW8. Significant allele effects were observed with six markers detecting base substitution mutations at GW2 and GS3 and insertion/deletion mutations at GS5, GW5, and GW6a, suggesting that these mutations might have affected the grain trait and caused variation among local varieties in the Indochinese countries. In addition to grain size, the hull color, grain color, and glutinosity were also examined using a survey of loss-of-function mutations at major responsible loci. Most phenotypes were reflected based on functional mutations at these loci. Since the local varieties have wide genetic variation, they are important genetic resources for future rice breeding.

Evaluation of Domestication Loci Associated with Awnlessness in Cultivated Rice, Oryza sativa.

BACKGROUND: Awns are bristle-like organs at the tips of the glumes. Wild rice has maintained long awns for successful seed propagation through seed dispersal. Seed awning is an interesting trait in rice domestication. Long awns might have been beneficial for seed gatherers in the initial phase of domestication; however, awnless phenotypes were preferably selected in a later phase with non-seed-shattering plants. Investigation of domestication loci associated with awnlessness in cultivated rice will be useful in clarifying the process and history of rice domestication. RESULTS: Quantitative trait locus (QTL) analysis for seed awning was carried out using a BC3F2 population between Oryza sativa IR36 (a cultivated donor parent with awnless phenotype) and O. rufipogon W630 (a wild recurrent parent with awns). As a result, two QTLs on chromosome 4 (corresponding to An-1 and LABA1) and one on chromosome 2 (designated as qAWNL2) were detected. Gene interaction among three seed-awning QTLs were further examined with the plants having eight different combinations of homozygous genotypes. Their awn length variation indicated that the IR36 alleles at these loci had the additive awnlessness effects in the genetic background of wild rice. The shortest awn length was observed for the plants having IR36 homozygous alleles at all loci, giving about 75% reduction in awn length. By the fine mapping, the candidate region of the novel qAWNL2 locus was delimited in a 157.4-kb region containing 22 predicted genes in Nipponbare genome. CONCLUSIONS: QTL analysis revealed that three loci, An-1, LABA1 and qAWNL2, were mainly responsible for the awnlessness of O. sativa IR36. In the wild genetic background, loss-of-function alleles at three awning loci showed additive effects on length reduction. In rice domestication, awnless forms may be gradually generated through the accumulation of mutations at awning loci.

Gene interaction at seed-awning loci in the genetic background of wild rice.

Seed awning is one of the important traits for successful propagation in wild rice. During the domestication of rice by ancient humans, plants with awnless seeds may have been selected because long awns hindered collection and handling activities. To investigate domestication of awnless rice, QTL analysis for seed awning was first carried out using backcross recombinant inbred lines between Oryza sativa Nipponbare (recurrent parent) and O. rufipogon W630 (donor parent). Two strong QTLs were detected in the same regions as known major seed-awning loci, An-1 and RAE2. Subsequent causal mutation surveying and fine mapping confirmed that O. rufipogon W630 has functional alleles at both loci. The gene effects and interactions at these loci were examined using two backcross populations with reciprocal genetic backgrounds of O. sativa Nipponbare and O. rufipogon W630. As awn length in wild rice varied among seeds even in the same plant, awn length was measured based on spikelet position. In the genetic background of cultivated rice, the wild alleles at An-1 and RAE2 had awning effects, and plants having both wild homozygous alleles produced awns whose length was about 70% of those of the wild parent. On the other hand, in the genetic background of wild rice, the substitution of cultivated alleles at An-1 and RAE2 contributed little to awn length reduction. These results indicate that the domestication process of awnless seeds was complicated because many genes are involved in awn formation in wild rice.

Detection of quantitative trait loci controlling grain zinc concentration using Australian wild rice, Oryza meridionalis, a potential genetic resource for biofortification of rice.

Zinc (Zn) is one of the essential mineral elements for both plants and humans. Zn deficiency in human is one of the major causes of hidden hunger, a serious health problem observed in many developing countries. Therefore, increasing Zn concentration in edible part is an important issue for improving human Zn nutrition. Here, we found that an Australian wild rice O. meridionalis showed higher grain Zn concentrations compared with cultivated and other wild rice species. The quantitative trait loci (QTL) analysis was then performed to identify the genomic regions controlling grain Zn levels using backcross recombinant inbred lines derived from O. sativa 'Nipponbare' and O. meridionalis W1627. Four QTLs responsible for high grain Zn were detected on chromosomes 2, 9, and 10. The QTL on the chromosome 9 (named qGZn9), which showed the largest effect on grain Zn concentration was confirmed with the introgression line, which had a W1627 chromosomal segment covering the qGZn9 region in the genetic background of O. sativa 'Nipponbare'. Fine mapping of this QTL resulted in identification of two tightly linked loci, qGZn9a and qGZn9b. The candidate regions of qGZn9a and qGZn9b were estimated to be 190 and 950 kb, respectively. Furthermore, we also found that plants having a wild chromosomal segment covering qGZn9a, but not qGZn9b, is associated with fertility reduction. qGZn9b, therefore, provides a valuable allele for breeding rice with high Zn in the grains.

Mitochondrial microsatellite variability in common wheat and its ancestral species.

On the basis of the entire mitochondrial DNA sequence of common wheat, Triticum aestivum, 21 mitochondrial microsatellite loci having more than ten mononucleotide repeats were identified. The mitochondrial microsatellite variability at all loci was examined with 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploidy evolution. Polymorphic banding patterns were obtained at 15 out of 21 mitochondrial microsatellite loci. The number of alleles per polymorphic microsatellite ranged from 2 to 5 with an average of 3.07, and the diversity values (H) ranged from 0.09 to 0.50 with an average of 0.29. These values are almost two third of wheat chloroplast microsatellite values, indicating that variability of mitochondrial microsatellite is much less than that of chloroplast microsatellite. Based on the allele variation at all loci, a total of seven mitochondrial haplotypes were identified among common wheat and its ancestral species. Three diploid species showed their own specific haplotypes and timopheevi group (11 accessions) had three types, whereas 29 accessions of emmer and common wheat groups shared the same haplotype. These results indicate that a single mitochondrial haplotype determined by microsatellite analysis has conservatively been maintained in the evolutionary lineage from wild tetraploid to cultivated hexaploid species.

DNA variation in the metallothionein genes in wild rice Oryza rufipogon: relationship between DNA sequence polymorphism, codon bias and gene expression.

This study examines the relationship between DNA sequence variation and level of gene expression in four metallothionein genes from wild rice Oryza rufipogon. The nucleotide diversity was 0.0028 to 0.0117 over the entire coding and non-coding region, and it was negatively correlated with gene expression for three type 2 metallothionein genes. In contrast, codon bias and percent of preferred codons correlated positively with gene expression. These results indicate that the intensity of natural selection depends on the level of gene expression, which in turn shapes the level of nucleotide polymorphism. In addition, significant linkage disequilibria were frequent between the metallothionein genes, although significance was not confirmed after multiple test correction. This result suggests that metallothionein genes expressed at different levels are epistatic with respect to fitness, and that gene expression is an important factor determining level of DNA polymorphism.