A phenylalanine in DGAT is a key determinant of oil content and composition in maize

Plant oil is an important renewable resource for biodiesel production and for dietary consumption by humans and livestock. Through genetic mapping of the oil trait in plants, studies have reported multiple quantitative trait loci (QTLs) with small effects, but the molecular basis of oil QTLs remains largely unknown. Here we show that a high-oil QTL (qHO6) affecting maize seed oil and oleic-acid contents encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT1-2), which catalyzes the final step of oil synthesis. We further show that a phenylalanine insertion in DGAT1-2 at position 469 (F469) is responsible for the increased oil and oleic-acid contents. The DGAT1-2 allele with F469 is ancestral, whereas the allele without F469 is a more recent mutant selected by domestication or breeding. Ectopic expression of the high-oil DGAT1-2 allele increases oil and oleic-acid contents by up to 41% and 107%, respectively. This work provides insights into the molecular basis of natural variation of oil and oleic-acid contents in plants and highlights DGAT as a promising target for increasing oil and oleic-acid contents in other crops.     

Identification of a functional transposon insertion in the maize domestication gene tb1

Genetic diversity created by transposable elements is an important source of functional variation upon which selection acts during evolution. Transposable elements are associated with adaptation to temperate climates in Drosophila, a SINE element is associated with the domestication of small dog breeds from the gray wolf and there is evidence that transposable elements were targets of selection during human evolution. Although the list of examples of transposable elements associated with host gene function continues to grow, proof that transposable elements are causative and not just correlated with functional variation is limited. Here we show that a transposable element (Hopscotch) inserted in a regulatory region of the maize domestication gene, teosinte branched1 (tb1), acts as an enhancer of gene expression and partially explains the increased apical dominance in maize compared to its progenitor, teosinte. Molecular dating indicates that the Hopscotch insertion predates maize domestication by at least 10,000 years, indicating that selection acted on standing variation rather than new mutation.     

Screening a large reference sample to identify very low frequency sequence variants: comparisons between two genes

Most human sequence variation is in the form of single-nucleotide polymorphisms (SNPs). It has been proposed that coding-region SNPs (cSNPs) be used for direct association studies to determine the genetic basis of complex traits. The success of such studies depends on the frequency of disease-associated alleles, and their distribution in different ethnic populations. If disease-associated alleles are frequent in most populations, then direct genotyping of candidate variants could show robust associations in manageable study samples. This approach is less feasible if the genetic risk from a given candidate gene is due to many infrequent alleles. Previous studies of several genes demonstrated that most variants are relatively infrequent (<0.05). These surveys genotyped small samples (n<75) and thus had limited ability to identify rare alleles. Here we evaluate the prevalence and distribution of such rare alleles by genotyping an ethnically diverse reference sample that is more than six times larger than those used in previous studies (n=450). We screened for variants in the complete coding sequence and intron-exon junctions of two candidate genes for neuropsychiatric phenotypes: SLC6A4, encoding the serotonin transporter; and SLC18A2, encoding the vesicular monoamine transporter. Both genes have unique roles in neuronal transmission, and variants in either gene might be associated with neurobehavioral phenotypes.     

Deletion in a gene associated with grain size increased yields during rice domestication

The domestication of crops involves a complex process of selection in plant evolution and is associated with changes in the DNA regulating agronomically important traits. Here we report the cloning of a newly identified QTL, qSW5 (QTL for seed width on chromosome 5), involved in the determination of grain width in rice. Through fine mapping, complementation testing and association analysis, we found that a deletion in qSW5 resulted in a significant increase in sink size owing to an increase in cell number in the outer glume of the rice flower; this trait might have been selected by ancient humans to increase yield of rice grains. In addition, we mapped two other defective functional nucleotide polymorphisms of rice domestication-related genes with genome-wide RFLP polymorphisms of various rice landraces. These analyses show that the qSW5 deletion had an important historical role in artificial selection, propagation of cultivation and natural crossings in rice domestication, and shed light on how the rice genome was domesticated.     

Exome sequencing of extreme phenotypes identifies DCTN4 as a modifier of chronic Pseudomonas aeruginosa infection in cystic fibrosis

Exome sequencing has become a powerful and effective strategy for the discovery of genes underlying Mendelian disorders. However, use of exome sequencing to identify variants associated with complex traits has been more challenging, partly because the sample sizes needed for adequate power may be very large. One strategy to increase efficiency is to sequence individuals who are at both ends of a phenotype distribution (those with extreme phenotypes). Because the frequency of alleles that contribute to the trait are enriched in one or both phenotype extremes, a modest sample size can potentially be used to identify novel candidate genes and/or alleles. As part of the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP), we used an extreme phenotype study design to discover that variants in DCTN4, encoding a dynactin protein, are associated with time to first P. aeruginosa airway infection, chronic P. aeruginosa infection and mucoid P. aeruginosa in individuals with cystic fibrosis.     

Control of rice grain-filling and yield by a gene with a potential signature of domestication

Grain-filling, an important trait that contributes greatly to grain weight, is regulated by quantitative trait loci and is associated with crop domestication syndrome. However, the genes and underlying molecular mechanisms controlling crop grain-filling remain elusive. Here we report the isolation and functional analysis of the rice GIF1 (GRAIN INCOMPLETE FILLING 1) gene that encodes a cell-wall invertase required for carbon partitioning during early grain-filling. The cultivated GIF1 gene shows a restricted expression pattern during grain-filling compared to the wild rice allele, probably a result of accumulated mutations in the gene's regulatory sequence through domestication. Fine mapping with introgression lines revealed that the wild rice GIF1 is responsible for grain weight reduction. Ectopic expression of the cultivated GIF1 gene with the 35S or rice Waxy promoter resulted in smaller grains, whereas overexpression of GIF1 driven by its native promoter increased grain production. These findings, together with the domestication signature that we identified by comparing nucleotide diversity of the GIF1 loci between cultivated and wild rice, strongly suggest that GIF1 is a potential domestication gene and that such a domestication-selected gene can be used for further crop improvement.     

Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles . Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics--from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.     

The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA

Retention of juvenile traits in the adult reproductive phase characterizes a process known as neoteny, and speculation exists over whether it has contributed to the evolution of new species. The dominant Corngrass1 (Cg1) mutant of maize is a neotenic mutation that results in phenotypes that may be present in the grass-like ancestors of maize. We cloned Cg1 and found that it encodes two tandem miR156 genes that are overexpressed in the meristem and lateral organs. Furthermore, a target of Cg1 is teosinte glume architecture1 (tga1), a gene known to have had a role in the domestication of maize from teosinte. Cg1 mutant plants overexpressing miR156 have lower levels of mir172, a microRNA that targets genes controlling juvenile development. By altering the relative levels of both microRNAs, it is possible to either prolong or shorten juvenile development in maize, thus providing a mechanism for how species-level heterochronic changes can occur in nature.     

The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.     

The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal Charcot-Marie-Tooth type 4A disease.

We identified three distinct mutations and six mutant alleles in GDAP1 in three families with axonal Charcot-Marie-Tooth (CMT) neuropathy and vocal cord paresis, which were previously linked to the CMT4A locus on chromosome 8q21.1. These results establish the molecular etiology of CMT4A (MIM 214400) and suggest that it may be associated with both axonal and demyelinating phenotypes.     

Conserved requirement for a plant host cell protein in powdery mildew pathogenesis

In the fungal phylum Ascomycota, the ability to cause disease in plants and animals has been gained and lost repeatedly during phylogenesis. In monocotyledonous barley, loss-of-function mlo alleles result in effective immunity against the Ascomycete Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. However, mlo-based disease resistance has been considered a barley-specific phenomenon to date. Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter. These findings imply that a common host cell entry mechanism of powdery mildew fungi evolved once and at least 200 million years ago, suggesting that within the Erysiphales (powdery mildews) the ability to cause disease has been a stable trait throughout phylogenesis.     

Identification of the gene (BBS1) most commonly involved in Bardet-Biedl syndrome,a complex human obesity syndrome

Bardet-Biedl syndrome (BBS, OMIM 209900) is a genetic disorder with the primary features of obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation and hypogenitalism. Individuals with BBS are also at increased risk for diabetes mellitus, hypertension and congenital heart disease. What was once thought to be a homogeneous autosomal recessive disorder is now known to map to at least six loci: 11q13 (BBS1), 16q21 (BBS2), 3p13 p12 (BBS3), 15q22.3 q23 (BBS4), 2q31 (BBS5) and 20p12 (BBS6). There has been considerable interest in identifying the genes that underlie BBS, because some components of the phenotype are common. Cases of BBS mapping ro BBS6 are caused by mutations in MKKS; mutations in this gene also cause McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly and congenital heart defects). In addition, we recently used positional cloning to identify the genes underlying BBS2 (ref. 16) and BBS4 (ref. 17). The BBS6 protein has similarity to a Thermoplasma acidophilum chaperonin, whereas BBS2 and BBS4 have no significant similarity to chaperonins. It has recently been suggested that three mutated alleles (two at one locus, and a third at a second locus) may be required for manifestation of BBS (triallelic inheritance). Here we report the identification of the gene BBS1 and show that a missense mutation of this gene is a frequent cause of BBS. In addition, we provide data showing that this common mutation is not involved in triallelic inheritance.     

Common variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular degeneration

Age-related macular degeneration (AMD) is a common, late-onset disease with seemingly typical complexity: recurrence ratios for siblings of an affected individual are three- to sixfold higher than in the general population, and family-based analysis has resulted in only modestly significant evidence for linkage. In a case-control study drawn from a US-based population of European descent, we have identified a previously unrecognized common, noncoding variant in CFH, the gene encoding complement factor H, that substantially increases the influence of this locus on AMD, and we have strongly replicated the associations of four other previously reported common alleles in three genes (P values ranging from 10(-6) to 10(-70)). Despite excellent power to detect epistasis, we observed purely additive accumulation of risk from alleles at these genes. We found no differences in association of these loci with major phenotypic categories of advanced AMD. Genotypes at these five common SNPs define a broad spectrum of interindividual disease risk and explain about half of the classical sibling risk of AMD in our study population.     

Control of a key transition from prostrate to erect growth in rice domestication

The transition from the prostrate growth of ancestral wild rice (O. rufipogon Griff.) to the erect growth of Oryza sativa cultivars was one of the most critical events in rice domestication. This evolutionary step importantly improved plant architecture and increased grain yield. Here we find that prostrate growth of wild rice from Yuanjiang County in China is controlled by a semi-dominant gene, PROG1 (PROSTRATE GROWTH 1), on chromosome 7 that encodes a single Cys(2)-His(2) zinc-finger protein. prog1 variants identified in O. sativa disrupt the prog1 function and inactivate prog1 expression, leading to erect growth, greater grain number and higher grain yield in cultivated rice. Sequence comparison shows that 182 varieties of cultivated rice, including 87 indica and 95 japonica cultivars from 17 countries, carry identical mutations in the prog1 coding region that may have become fixed during rice domestication.