The alleles at the E1 locus impact the expression pattern of two soybean FT-like genes shown to induce flowering in Arabidopsis

A small gene family of phosphatidyl ethanolamine-binding proteins (PEBP) has been shown to function as key regulators in flowering; in Arabidopsis thaliana the FT protein promotes flowering whilst the closely related TFL1 protein represses flowering. Control of flowering time in soybean [Glycine max (L.) Merrill] is important for geographic adaptation and maximizing yield. Soybean breeders have identified a series of loci, the E-genes, that control photoperiod-mediated flowering time, yet how these loci control flowering is poorly understood. The objectives of this study were to evaluate the expression of GmFT-like genes in the E1 near-isogenic line (NIL) background. Of the 20 closely related PEBP proteins in the soybean genome, ten are similar to the Arabidopsis FT protein. Expression analysis of these ten GmFT-like genes confirmed that only two are detectable in the conditions tested. Further analysis of these two genes in the E1 NILs grown under short-day (SD) and long-day (LD) conditions showed a diurnal expression and tissue specificity expression commensurate with soybean flowering time under SD and LD conditions, suggesting that these were good candidates for flowering induction in soybean. Arabidopsis ft mutant lines flowered early when transformed with the two soybean genes, suggesting that the soybean genes can complement the Arabidopsis FT function. Flowering time in E1 NILs is consistent with the differential expression of the two GmFT-like genes under SD and LD conditions, suggesting that the E1 locus, at least in part, impacts time to flowering through the regulation of soybean FT expression.     

Expression of flowering-time genes in soybean E1 near-isogenic lines under short and long day conditions

Control of soybean flowering time is important for geographic adaptation and maximizing yield. Plant breeders have identified a series of genes (E genes) that condition time to flowering; however, the molecular basis in the control of flowering by these E genes, in conjunction with canonical flowering-time genes, has not been studied. Time to flowering in near-isogenic lines (NILs) at the E1 locus was tested using a reciprocal transfer experiment under short day (SD) and long day (LD) conditions. Beginning 8 days after planting, three plant samples were harvested every 3 h for a 48-h period. RNA was isolated from these plants, and RNA samples were pooled for each line and each time period for cDNA synthesis. RT-PCR analysis was performed using primers synthesized for a number of putative flowering-time genes based on homology of soybean EST and genomic sequences to Arabidopsis genes. The results of the reciprocal transfer experiment suggest that the pre-inductive photoperiod-sensitive phase of the E1 NILs responsible for inducing flowering is perceived as early as 5–7-day post-planting. No gene expression differences were found between the E1 and e1 NILs, suggesting that the E1 gene does not directly affect the flowering-time genes during the time period tested; however, differences were observed in gene expression between SD and LD treatments for the putative soybean TOC1, CO, and FT genes. The gene expression results in this study were similar to those of flowering-time genes found in other SD species, suggesting that the selected genes correspond to the soybean flowering-time orthologs.     

An informative linkage map of soybean reveals QTLs for flowering time, leaflet morphology and regions of segregation distortion.

A genetic linkage map covering a large region of the genome with informative markers is essential for plant genome analysis, including identification of quantitative trait loci (QTLs), map-based cloning, and construction of a physical map. We constructed a soybean genetic linkage map using 190 F2 plants derived from a single cross between the soybean varieties Misuzudaizu and Moshidou Gong 503, based on restriction-fragment-length polymorphisms (RFLPs) and simple-sequence-repeat polymorphisms (SSRPs). This linkage map has 503 markers, including 189 RFLP markers derived from expressed sequence tag (EST) clones, and consists of 20 major linkage groups that may correspond to the 20 pairs of soybean chromosomes, covering 2908.7 cM of the soybean genome in the Kosambi function. Using this linkage map, we identified 4 QTLs--FT1, FT2, FT3, and FT4--for flowering time, the QTLs for the 5 largest principal components determining leaflet shape, 6 QTLs for single leaflet area, and 18 regions of segregation distortion. All 503 analyzed markers identified were located on the map, and almost all phenotypic variations in flowering time were explained by the detected QTLs. These results indicate that this map covers a large region of the soybean genome.     

Mapping and identification of a potential candidate gene for a novel maturity locus, <Emphasis Type="Italic">E10</Emphasis>, in soybean

Key message

E10 is a new maturity locus in soybean and FT4 is the predicted/potential functional gene underlying the locus.

Abstract

Flowering and maturity time traits play crucial roles in economic soybean production. Early maturity is critical for north and west expansion of soybean in Canada. To date, 11 genes/loci have been identified which control time to flowering and maturity; however, the molecular bases of almost half of them are not yet clear. We have identified a new maturity locus called “E10” located at the end of chromosome Gm08. The gene symbol E10e10 has been approved by the Soybean Genetics Committee. The e10e10 genotype results in 5–10 days earlier maturity than E10E10. A set of presumed E10E10 and e10e10 genotypes was used to identify contrasting SSR and SNP haplotypes. These haplotypes, and their association with maturity, were maintained through five backcross generations. A functional genomics approach using a predicted protein–protein interaction (PPI) approach (Protein–protein Interaction Prediction Engine, PIPE) was used to investigate approximately 75 genes located in the genomic region that SSR and SNP analyses identified as the location of the E10 locus. The PPI analysis identified FT4 as the most likely candidate gene underlying the E10 locus. Sequence analysis of the two FT4 alleles identified three SNPs, in the 5′UTR, 3′UTR and fourth exon in the coding region, which result in differential mRNA structures. Allele-specific markers were developed for this locus and are available for soybean breeders to efficiently develop earlier maturing cultivars using molecular marker assisted breeding.

     

Fine mapping of the FT1 locus for soybean flowering time using a residual heterozygous line derived from a recombinant inbred line

Fine-mapping of loci related to complex quantitative traits is essential for map-based cloning. A residual heterozygous line (RHL) of soybean (Glycine max) derived from a recombinant inbred line (RIL) was used for fine-mapping FT1, which is a major quantitative trait locus (QTL) responsible for soybean flowering time. The residual heterozygous line RHL1-156 was selected from the RILs that were derived from two distantly related varieties, Misuzudaizu and Moshidou Gong 503. The genome of RHL1-156 contains a heterozygous segment (approximately 17 cM) surrounding the FT1 locus but is homozygous in other regions, including three other loci affecting flowering time. A large segregating population of 1,006 individuals derived by selfing of RHL1-156 included two homozygous genotypes for the nearest marker of FT1 whose flowering time differed by 26 days. No continuous range of phenotypes was observed, in contrast to the F₂ population, suggesting that a single FT1 locus affected the flowering time in the RHL1-156 line. Linkage analysis revealed that the FT1 locus mapped as a single Mendelian factor between two tightly linked DNA markers, Satt365 and GM169, at distances of approximately 0.1 cM and 0.4 cM, respectively. Our results show that a RHL derived from RILs can be used to fine-map a QTL and that RHLs can be an efficient tool for a systematic fine-mapping of QTLs.     

Map-Based Cloning of the Gene Associated With the Soybean Maturity Locus E3

Photosensitivity plays an essential role in the response of plants to their changing environments throughout their life cycle. In soybean [Glycine max (L.) Merrill], several associations between photosensitivity and maturity loci are known, but only limited information at the molecular level is available. The FT3 locus is one of the quantitative trait loci (QTL) for flowering time that corresponds to the maturity locus E3. To identify the gene responsible for this QTL, a map-based cloning strategy was undertaken. One phytochrome A gene (GmPhyA3) was considered a strong candidate for the FT3 locus. Allelism tests and gene sequence comparisons showed that alleles of Misuzudaizu (FT3/FT3; JP28856) and Harosoy (E3/E3; PI548573) were identical. The GmPhyA3 alleles of Moshidou Gong 503 (ft3/ft3; JP27603) and L62-667 (e3/e3; PI547716) showed weak or complete loss of function, respectively. High red/far-red (R/FR) long-day conditions enhanced the effects of the E3/FT3 alleles in various genetic backgrounds. Moreover, a mutant line harboring the nonfunctional GmPhyA3 flowered earlier than the original Bay (E3/E3; PI553043) under similar conditions. These results suggest that the variation in phytochrome A may contribute to the complex systems of soybean flowering response and geographic adaptation.FLOWERING represents the transition from the vegetative to the reproductive phase in plants. Various external cues, such as photoperiod and temperature, are known to initiate plant flowering under the appropriate seasonal conditions. Of these cues, light is the most important, being received by several photoreceptors, including the red light (R) and the far-red light (FR)-absorbing phytochromes and the blue/UV-A absorbing cryptochromes and phototorpins (Chen et al. 2004).Phytochrome is the best characterized of these photoreceptors. All higher plant phytochromes are thought to exist as specific dimer combinations (Sharrock and Clack 2004), with each monomer being attached to a light-absorbing linear tetrapyrrole, phytochromobilin. The phytochrome apoproteins are synthesized within the cytosol and assemble autocatalytically with a chromophore to form the phytochrome holoproteins. The R-absorbing form (Pr) is thought to be inactive but is then converted to the active FR-absorbing form (Pfr) by R absorption. The absorption of light triggers the transfer of the phytochrome to the nucleus, where it regulates gene expression. In most plant species, the phytochrome apoproteins are encoded by a small gene family. Type I phytochrome is degraded in the light and is abundant in dark-grown seedlings, whereas type II phytochrome is relatively stable in the light (reviewed by Bae and Choi 2008). In Arabidopsis, five phytochromes (PhyA–E) have been characterized (Clack et al. 1994; Quail et al. 1995). PhyA is type I and is responsible for the very low fluence response and high irradiance response, whereas the other phytochromes are type II and are responsible for red-far/red reversible low fluence response (reviewed by Whitelam et al. 1998).It is well known that mutations in the phytochrome A gene affect the photoperiodic control of flowering. In Arabidopsis, a phyA mutant flowered later in either long-day or short-day conditions with a night break (Johnson et al. 1994; Reed et al. 1994). In rice, combinations of mutant alleles of phytochrome genes conferred various effects on the flowering phenotype. For example, the phyA phyB and phyA phyC double mutants grown under natural-day-length conditions showed earlier flowering phenotypes than wild-type plants (Takano et al. 2005). In pea, a long-day plant, loss- or gain-of-function phyA mutants displayed late or early flowering phenotypes, respectively (Weller et al. 1997, 2001). It is likely that photoperiodic response via phyA signaling is important for crop adaptation to a wide range of growing conditions.In soybean [Glycine max (L.) Merrill], several maturity loci, designated as E loci (Cober et al. 1996a), have been characterized by classical methods. These are E1 and E2 (Bernard 1971), E3 (Buzzell 1971), E4 (Buzzell and Voldeng 1980), E5 (McBlain and Bernard 1987), E6 (Bonato and Vello 1999), and E7 (Cober and Voldeng 2001). Of these, the E1, E3, and E4 loci have been suggested to be related to photoperiod sensitivity under various light conditions (Saidon et al. 1989; Cober et al. 1996b; Abe et al. 2003). In previous studies, using the same populations as in this study, three flowering-time quantitative trait loci (QTL)—FT1, FT2, and FT3 loci—were identified and considered to be identical with the maturity loci E1, E2, and E3, respectively (Yamanaka et al. 2001; Watanabe et al. 2004). Although many loci related to soybean flowering and maturity have been identified, and some candidate genes were recognized using near isogenic lines (NILs) (Tasma and Shoemaker 2003), most of the genes responsible for these loci have not yet been isolated except for the E4 gene. Liu et al. (2008) reported an association between phytochrome A and photoperiod sensitivity. A retrotransposon sequence inserted into the exon of the e4 allele conferred an early flowering phenotype under long-day conditions extended by incandescent lighting.A relationship between the E3 gene and some photoreceptor genes was suggested from different photosensitivity responses of various soybean NILs (Cober et al. 1996a). Cober and Voldeng (1996) also reported a linkage relationship between the E3 and Dt1 loci, which is related to a determinate or indeterminate growth habit phenotype. Additionally, Molnar et al. (2003) reported that Satt229, on linkage group (LG) L, was a proximal simple sequence repeat (SSR) marker to the E3 loci. According to the Soybean Genome Database (Shultz et al. 2006a,b, 2007; http://soybeangenome.siu.edu/) and the Legume Information System (LIS; http://www.comparative-legumes.org/), there are numerous QTL and >60 loci associated with various agronomic traits in the region between Dt1 and Satt373 (∼30–40 cM). This extremely large number of QTL may be the result of linkage between the Dt1 and E3 loci because both loci can affect many aspects of plant morphology. Among these QTL, several associations with the E3 gene have been reported (Mansur et al. 1996; Orf et al. 1999; Funatsuki et al. 2005; Kahn et al. 2008).To identify the genes responsible for the target QTL, fine mapping and map-based cloning strategies are necessary (Salvi and Tuberosa 2005). QTL analysis using intercross-derived populations, such as F₂ and recombinant inbred lines (RILs), have some limitations in genome resolution (10–30 cM) because of the simultaneous segregation of several loci affecting the same trait (Kearsey and Farquhar 1998). Additional strategies are therefore required to locate QTL more precisely. The use of NILs that differ at a single QTL is an effective approach for fine mapping and characterization of an individual locus (Salvi and Tuberosa 2005). However, the development of NILs through repeated backcrossing is time-consuming and laborious (Tuinstra et al. 1997). The use of a residual heterozygous line (RHL), as proposed by Yamanaka et al. (2004), and which is derived from RIL, is a powerful tool for precisely evaluating QTL (Haley et al. 1994). An RHL harbors a heterozygous region where the target QTL is located and a homozygous background in most other regions of the genome. Tuinstra et al. (1997) used a similar term, heterogeneous inbred family, for a selfed RHL population to identify the QTL associated with seed weight in sorghum.This RHL strategy has already been used to identify loci underlying resistance to pathogens in soybean (Njiti et al. 1998; Meksem et al. 1999; Triwitayakorn et al. 2005). After identification of the target loci, novel DNA markers tightly linked to the loci were developed using the amplified fragment length polymorphism (AFLP) method (Meksem et al. 2001a,b). Physical contigs, screened by sequence-characterized amplified region (SCAR) markers converted from these AFLP fragments, are ideal sources for identifying candidate genes for the target traits (Ruben et al. 2006).The aim of this study is to characterize the FT3 locus using a map-based cloning strategy and to confirm the gene responsible for the E3/FT3 locus by allelism tests through comparisons of gene sequences and photosensitivity of several alleles.     

A major QTL (<Emphasis Type="Italic">qFT12.1</Emphasis>) allele from wild soybean delays flowering time

Soybean is highly sensitive to photoperiod. To improve the adaptability and productivity of soybean, it is essential to understand the molecular mechanisms regulating flowering time. To identify new flowering time QTLs, we evaluated a BC₃F₅ population consisting of 120 chromosome segment substitution lines (CSSLs) over 2 years under field conditions. CSSLs were derived from a cross between the cultivated soybean cultivar Jackson and the wild soybean accession JWS156-1, followed by continuous backcrossing using Jackson as the recurrent parent. Four QTLs (qFT07.1, qFT12.1, qFT12.2, and qFT19.1) were detected on three chromosomes. Of these, qFT12.1 showed the highest effect, accounting for 36.37–38.27% of the total phenotypic variation over 2 years. This QTL was further confirmed in the F₇ recombinant inbred line population (n?=?94) derived from the same cross (Jackson × JWS156-1). Analysis of the qFT12.1 BC₃F₅ residual heterozygous line RHL509 validated the allele effect of qFT12.1 and revealed that the recessive allele of qFT12.1 resulted in delayed flowering. Evaluating the qFT12.1 near-isogenic lines (NILs) under different growth conditions showed that NILs with the wild soybean genotype always showed later flowering than those with the cultivated soybean genotype. qFT12.1 was delimited to a 2703-kb interval between the markers BARCSOYSSR_12_0220 and BARCSOYSSR_12_0368 on chromosome 12. qFT12.1 may be a new flowering time gene locus in soybean.     

Diurnal Expression Pattern,Allelic Variation,and Association Analysis Reveal Functional Features of the E1 Gene in Control of Photoperiodic Flowering in Soybean

Although four maturity genes, E1 to E4, in soybean have been successfully cloned, their functional mechanisms and the regulatory network of photoperiodic flowering remain to be elucidated. In this study, we investigated how the diurnal expression pattern of the E1 gene is related to photoperiodic length; and to what extent allelic variation in the B3-like domain of the E1 gene is associated with flowering time phenotype. The bimodal expression of the E1 gene peaked first at around 2 hours after dawn in long-day condition. The basal expression level of E1 was enhanced by the long light phase, and decreased by duration of dark. We identified a 5bp (3 SNP and 2-bp deletion) mutation, referred to an e1-b3a, which occurs in the middle of B3 domain of the E1 gene in the early flowering cultivar Yanhuang 3. Subcellular localization analysis showed that the putative truncated e1-b3a protein was predominately distributed in nuclei, indicating the distribution pattern of e1-b3a was similar to that of E1, but not to that of e1-as. Furthermore, genetic analysis demonstrated allelic variations at the E1 locus significantly underlay flowering time in three F₂ populations. Taken together, we can conclude the legume specific E1 gene confers some special features in photoperiodic control of flowering in soybean. Further characterization of the E1 gene will extend our understanding of the soybean flowering pathway in soybean.     

Simple sequence repeat (SSR) markers linked to E1, E3, E4, and E7 maturity genes in soybean.

Soybean near isogenic lines (NILs), contrasting for maturity and photoperiod sensitivity loci, were genotyped with approximately 430 mapped simple sequence repeats (SSRs), also known as microsatellite markers. By analysis of allele distributions across the NILs, it was possible to confirm the map location of the Dt1 indeterminate growth locus, to refine the SSR mapping of the T tawny pubescence locus, to map E1 and E3 maturity loci with molecular markers, and to map the E4 and E7 maturity loci for the first time. Molecular markers flanking these loci are now available for marker-assisted breeding for these traits. Analysis of map locations identified a putative homologous relationship among four chromosomal regions; one in the middle of linkage group (LG) C2 carrying E1 and E7, one on LG I carrying E4, one at the top of LG C2, at which there is a reproductive period quantitative trait locus (QTL), and the fourth on LG B1. Other evidence suggests that homology also exists between the E1 + E7 region on LG C2 and a region on LG L linked to a pod maturity QTL. Homology relationships predict possible locations in the soybean genome of additional maturity loci, as well as which maturity loci may share a common evolutionary origin and similar mechanism(s) of action.     

Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas9 contributes for expanding the regional adaptability of soybean

Flowering time is a key agronomic trait that directly influences the successful adaptation of soybean (Glycine max) to diverse latitudes and farming systems. GmFT2a and GmFT5a have been extensively identified as flowering activators and integrators in soybean. Here, we identified two quantitative trait loci (QTLs) regions harbouring GmFT2a and GmFT5a, respectively, associated with different genetic effects on flowering under different photoperiods. We analysed the flowering time of transgenic plants overexpressing GmFT2a or GmFT5a, ft2a mutants, ft5a mutants and ft2aft5a double mutants under long‐day (LD) and short‐day (SD) conditions. We confirmed that GmFT2a and GmFT5a are not redundant, they collectively regulate flowering time, and the effect of GmFT2a is more prominent than that of GmFT5a under SD conditions whereas GmFT5a has more significant effects than GmFT2a under LD conditions. GmFT5a, not GmFT2a, was essential for soybean to adapt to high latitude regions. The ft2aft5a double mutants showed late flowering by about 31.3 days under SD conditions and produced significantly increased numbers of pods and seeds per plant compared to the wild type. We speculate that these mutants may have enormous yield potential for the tropics. In addition, we examined the sequences of these two loci in 202 soybean accessions and investigated the flowering phenotypes, geographical distributions and maturity groups within major haplotypes. These results will contribute to soybean breeding and regional adaptability.     

Seed yield of near-isogenic soybean lines with introgressed quantitative trait loci conditioning resistance to corn earworm (Lepidoptera: Noctuidae) and soybean looper (Lepidoptera: Noctuidae) from PI 229358

The development of superior soybean, Glycine max (L.) Merr., cultivars exhibiting resistance to insects has been hindered due to linkage drag, a common phenomenon when introgressing alleles from exotic germplasm. Simple-sequence repeat (SSR) markers were used previously to map soybean insect resistance (SIR) quantitative trait loci (QTLs) in a'Cobb' X PI 229358 population, and subsequently used to create near-isogenic lines (NILs) with SIR QTL i n a 'Benning' genetic background. SIR QTLs were mapped on linkage groups (LGs) M (SIRQTL-M), G (SIRQTL-G), and H (SIRQTL-H). The objectives of this study were to 1) evaluate linkage drag for seed yield by using Benning-derived NILs selected for SIRQTL-M, SIRQTL-H, and SIRQTL-G; 2) assess the amount of PI 229358 genome surrounding the SIR QTL in each Benning NIL; and 3) evaluate the individual effects these three QTLs on antibiosis and antixenosis to corn earworm, Helicoverpa zea (Boddie), and soybean looper, Pseudoplusia includens (Walker). Yield data collected in five environments indicated that a significant yield reduction is associated with SIRQTL-G compared with NILs without SIR QTL. Overall, there was no yield reduction associated with SIRQTL-M or SIRQTL-H. A significant antixenosis and antibiosis effect was detected for SIRQTL-M in insect feeding assays, with no effect detected in antixenosis or antibiosis assays for SIRQTL-G or SIRQTL-H without the presence of PI 229358 alleles at SIRQTL-M. These results support recent findings concerning these loci.     

HUA2 is required for the expression of floral repressors in Arabidopsis thaliana

The HUA2 gene acts as a repressor of floral transition. Lesions in hua2 were identified through a study of natural variation and through two mutant screens. An allele of HUA2 from Landsberg erecta (Ler) contains a premature stop codon and acts as an enhancer of early flowering 4 (elf4) mutants. hua2 single mutants, in the absence of the elf4 lesion, flower earlier than wild type under short days. hua2 mutations partially suppress late flowering in FRIGIDA (FRI )-containing lines, autonomous pathway mutants, and a photoperiod pathway mutant. hua2 mutations suppress late flowering by reducing the expression of several MADS genes that act as floral repressors including FLOWERING LOCUS C (FLC ) and FLOWERING LOCUS M (FLM ).     

Identification of a new fragrance allele in soybean and development of its functional marker

We have previously reported an association between a single nucleotide polymorphism (SNP) in exon 10 of GmBADH2 gene and fragrance in vegetable soybean [Glycine max (L.) Merr.] cultivar Kaori. The SNP causes amino acid substitution in a highly conserved motif of GmBADH2 protein, which is necessary for functional activity of the protein. In this study, we sequenced GmBADH2 in another fragrant soybean cultivar Chamame and discovered a new fragrance allele, which has a 2-bp (TT) deletion in exon 10. The deletion causes a reading frame shift and introduces a premature stop codon, which could abolish protein function and result in fragrance. The old and new fragrance-promoting alleles were designated Gmbadh2-1 and Gmbadh2-2, respectively. A simple and co-dominant functional marker was developed for genotyping Gmbadh2-2. The marker can discriminate between fragrant and non-fragrant soybeans and distinguish the two different fragrant soybeans, and thus is useful for routine genotyping for the fragrance trait in breeding programs. Quantitative trait locus (QTL) mapping in an F₂ population using Chamame as the fragrance donor revealed that the location of the fragrance QTL nearly coincided with that of the functional marker, confirming the association between GmBADH2 and fragrance in Chamame.     

A loss-of-function mutant allele of a glycosyl hydrolase gene has been co-opted for seed weight control during soybean domestication

The resultant DNA from loss-of-function mutation can be recruited in biological evolution and development. Here, we present such a rare and potential case of “to gain by loss” as a neomorphic mutation during soybean domestication for increasing seed weight. Using a population derived from a chromosome segment substitution line of Glycine max(SN14) and Glycine soja(ZYD06), a quantitative trait locus(QTL) of 100-seed weight(q HSW) was mapped on chromosome 11, corresponding to a truncated β-1, 3-gl...     

Occurrence of a 2-bp (AT) deletion allele and a nonsense (G-to-T) mutant allele at the E2 (DBT) locus of six patients with maple syrup urine disease: multiple-exon skipping as a secondary effect of the mutations.

We have identified two novel mutant alleles in the transacylase (E2) gene of the human branched-chain alpha-keto acid dehydrogenase (BCKAD) complex in 6 of 38 patients with maple syrup urine disease (MSUD). One mutation, a 2-bp (AT) deletion in exon 2 of the E2 gene, causes a frameshift downstream of residue (-26) in the mitochondrial targeting presequence. The second mutation, a G-to-T transversion in exon 6 of the E2 gene, produces a premature stop codon at Glu-163 (E163*). Transfection of constructs harboring the E163* mutation into an E2-deficient MSUD cell line produced a truncated E2 subunit. However, this mutant E2 chain is unable to assemble into a 24-mer cubic structure and is degraded in the cell. The 2-bp (AT) deletion and the E163* mutant alleles occur in either the homozygous or compound-heterozygous state in the 6 of 38 unrelated MSUD patients studied. Moreover, an array of precise single- and multiple-exon deletions were observed in many amplified E2 mutant cDNAs. The latter results appear to represent secondary effects on RNA processing that are associated with the MSUD mutations at the E2 locus.