A novel QTL associated with tolerance to cold-induced seed cracking in the soybean cultivar Toyomizuki.

Low temperatures after flowering cause seed cracking (SC) in soybean. Previously, we reported that proanthocyanidin accumulation on the dorsal side of the seed coat, controlled by the I locus, may lead to cracked seeds; and that homozygous IcIc alleles at the I locus confer SC tolerance in the line Toiku 248. To discover new genes related to SC tolerance, we evaluated the physical and genetic mechanisms of SC tolerance in the cultivar Toyomizuki (genotype II). Histological and texture analyses of the seed coat revealed that the ability to maintain hardness and flexibility under low temperature, regardless of proanthocyanidin accumulation in the dorsal seed coat, contributes to SC tolerance in Toyomizuki. This indicated that the SC tolerance mechanism differed between Toyomizuki and Toiku 248. A quantitative trait loci (QTL) analysis of recombinant inbred lines revealed a new, stable QTL related to SC tolerance. The relationship between this new QTL, designated as qCS8-2, and SC tolerance was confirmed in residual heterozygous lines. The distance between qCS8-2 and the previously identified QTL qCS8-1, which is likely the Ic allele, was estimated to be 2-3 Mb, so it will be possible to pyramid these regions to develop new cultivars with increased SC tolerance.

A pubescence color gene enhances tolerance to cold-induced seed cracking in yellow soybean.

In yellow soybean, severe cold weather causes seed cracking on the dorsal side. Yellow soybeans carry the I or ii allele of the I locus and have a yellow (I) or pigmented (ii ) hilum. We previously isolated an additional allele, designated as Ic, of the I locus, and reported that yellow soybeans with the IcIc genotype may be tolerant to cold-induced seed cracking. The Ic allele by itself, however, does not confer high tolerance. The association of a pubescence color gene (T) with suppression of low-temperature-induced seed coat deterioration has been previously reported. In the present study, we tested whether T is effective for the suppression of cold-induced seed cracking using two pairs of near-isogenic lines for the T locus in the iiii or IcIc background. In both backgrounds, the cracked seed rate of the near-isogenic line with the TT genotype was significantly lower than that with the tt genotype, which indicates that T has an inhibitory effect on cold-induced seed cracking. Furthermore, we also showed that gene pyramiding of Ic and T can improve tolerance to cold-induced seed cracking. Our findings should aid the development of highly SC-tolerant cultivars in soybean breeding programs.

Identification and validation of quantitative trait loci associated with seed yield in soybean.

In soybean [Glycine max (L.) Merrill], the genetic analysis of seed yield is important to aid in the breeding of high-yielding cultivars. Seed yield is a complex trait, and the number of quantitative trait loci (QTL) involved in seed yield is high. The aims of this study were to identify QTL associated with seed yield and validate their effects on seed yield using near-isogenic lines. The QTL analysis was conducted using a recombinant inbred line population derived from a cross between Japanese cultivars 'Toyoharuka' and 'Toyomusume', and eight seed yield-associated QTL were identified. There were significant positive correlations between seed yield and the number of favorable alleles at QTL associated with seed yield in the recombinant inbred lines for three years. The effects of qSY8-1, a QTL promoting greater seed yield, was validated in the Toyoharuka background. In a two-year yield trial, the 100-seed weight and seed yield of Toyoharuka-NIL, the near-isogenic line having the Toyomusume allele at qSY8-1, were significantly greater than those of Toyoharuka (106% and 107%, respectively) without any change for days to flowering and maturity. Our results suggest that qSY8-1 was not associated with maturity genes, and contributed to the 100-seed weight.

A major gene for tolerance to cold-induced seed coat discoloration relieves viral seed mottling in soybean.

In yellow soybeans, inhibition of seed coat pigmentation by RNA silencing of CHS genes is suppressed by low temperature and a viral suppressor, resulting in 'cold-induced seed coat discoloration' and 'seed mottling', respectively. Differences exist in the degree of cold-induced seed coat discoloration among Japanese yellow soybean cultivars; for example, Toyomusume is sensitive, Toyohomare has some tolerance, and Toyoharuka is highly tolerant. In this study, we compared the degree of seed mottling severity due to soybean mosaic virus (SMV) among these three soybean cultivars. Obvious differences were found, with the order of severity as follows: Toyohomare > Toyomusume > Toyoharuka. RNA gel blot analysis indicated that CHS transcript abundance in the seed coat, which was increased by SMV infection, was responsible for the severity of seed mottling. Quantitative reverse transcription PCR analysis revealed why mottling was most severe in SMV-infected Toyohomare: the SMV titer in its seed coat was higher than in the other two infected cultivars. We further suggest that a major gene (Ic) for tolerance to cold-induced seed coat discoloration can relieve the severity of seed mottling in SMV-infected Toyoharuka.

Occurrence and tolerance mechanisms of seed cracking under low temperatures in soybean (Glycine max).

MAIN CONCLUSION: In soybean, occurrence of, or tolerance to, seed cracking under low temperatures may be related to the presence or absence, respectively, of proanthocyanidin accumulation in the seed coat dorsal region. Soybean seeds sometimes undergo cracking during low temperatures in summer. In this study, we focused on the occurrence and tolerance mechanisms of low-temperature-induced seed cracking in the sensitive yellow soybean cultivar Yukihomare and the tolerant yellow soybean breeding line Toiku 248. Yukihomare exhibited seed cracking when subjected to a 21-day low-temperature treatment from 10 days after flowering. In yellow soybeans, seed coat pigmentation is inhibited, leading to low proanthocyanidin levels in the seed coat. Proanthocyanidins accumulated on the dorsal side of the seed coat in Yukihomare under the 21-day low-temperature treatment. In addition, a straight seed coat split occurred on the dorsal side at the full-sized seed stage, resulting in seed cracking in this cultivar. Conversely, proanthocyanidin accumulation was suppressed throughout the seed coat in low-temperature-treated Toiku 248. We propose the following mechanism of seed cracking: proanthocyanidin accumulation and subsequent lignin deposition under low temperatures affects the physical properties of the seed coat, making it more prone to splitting. Further analyses uncovered differences in the physical properties of the seed coat between Yukihomare and Toiku 248. In particular, seed coat hardness decreased in Yukihomare, but not in Toiku 248, under the low-temperature treatment. Seed coat flexibility was higher in Toiku 248 than in Yukihomare under the low-temperature treatment, suggesting that the seed coat of low-temperature-treated Toiku 248 is more flexible than that of low-temperature-treated Yukihomare. These physical properties of the Toiku 248 seed coat observed under low-temperature conditions may contribute to its seed-cracking tolerance.

Accumulation of proanthocyanidins and/or lignin deposition in buff-pigmented soybean seed coats may lead to frequent defective cracking.

MAIN CONCLUSION: Defective cracking frequently occurs in buff-pigmented soybean seed coats, where proanthocyanidins accumulate and lignin is deposited, suggesting that proanthocyanidins and/or lignin may change physical properties and lead to defective cracking. In the seed production of many yellow soybean (Glycine max) cultivars, very low percentages of self-pigmented seeds are commonly found. This phenomenon is derived from a recessive mutation of the I gene inhibiting seed coat pigmentation. In Japan, most of these self-pigmented seeds are buff-colored, and frequently show multiple defective cracks in the seed coat. However, it is not known why cracking occurs specifically in buff seed coats. In this study, quantitative analysis was performed between yellow and buff soybean seed coats. Compared with yellow soybeans, in which defective cracking rarely occurs, contents of proanthocyanidins (PAs) and lignin were significantly higher in buff seed coats. Histochemical data of PAs and lignin in the seed coats strongly supported this result. Measurements of the physical properties of seed coats using a texture analyzer showed that a hardness value was significantly decreased in the buff seed coats. These results suggest that PA accumulation and/or lignin deposition may affect the physical properties of buff seed coats and lead to the defective cracking. This work contributes to understanding of the mechanism of defective cracking, which decreases the seed quality of soybean and related legumes.

Method for selection of soybeans tolerant to seed cracking under chilling temperatures.

In Hokkaido, northern Japan, soybean [Glycine max (L.) Merr.] crops are damaged by cold weather. Chilling temperatures result in the appearance of cracking seeds (CS) in soybean crops, especially those grown in eastern and northern Hokkaido. Seed coats of CS are severely split on the dorsal side, and the cotyledons are exposed and frequently separated. CS occurrence causes unstable production because these seeds have no commodity value. However, little is known about the CS phenomenon. The aims of this study were to identify the cold-sensitive stage associated with CS occurrence and to develop a method to select CS-tolerant lines. First, we examined the relationship between chilling temperatures after flowering and CS occurrence in field tests. The average temperature 14 to 21 days after flowering was negatively correlated with the rate of CS. Second, we evaluated differences in CS tolerance among soybean cultivars and breeding lines in field tests. 'Toyohomare' and 'Toiku-238' were more CS-tolerant than 'Yukihomare' and 'Toyomusume'. Third, we developed a selection method in which plants were subjected to 21-day chilling-temperature treatment from 10 days after flowering in a phytotron. This enabled comparisons of CS tolerance among cultivars. This selection method will be useful for breeding CS-tolerant soybeans.

Pigmentation of soybean seed coats via a mutation that abolishes production of multiple-phased siRNAs of chalcone synthase genes.

Lack of pigmentation in seed coats of soybean is caused by natural RNA silencing of chalcone synthase (CHS) genes. This phenomenon is an evolutionary consequence of structural changes in DNA that resulted in the production of double-stranded RNAs (dsRNAs) that trigger RNA degradation. Here we determined that a mutant with pigmented seed coats derived from a cultivar that lacked the pigmentation had a deletion between DNA regions ICHS1 and a cytochrome P450 gene; the deletion included GmIRCHS, a candidate gene that triggers CHS RNA silencing via production of CHS dsRNAs. We also characterized CHS short interfering RNAs (siRNAs) produced in the wild-type seed coats that had CHS RNA silencing. Phased 21-nt CHS siRNAs were detected in all 21 phases and were widely distributed in exon 2 of CHS7, which indicates commonality in the pattern of RNA degradation in natural CHS RNA silencing between distantly related species. These results with the similarities in the rearrangements found in spontaneous mutants suggest that the structural organization that generates dsRNAs that trigger phased siRNA production is vulnerable to further structural changes, which eventually abolish the induction of RNA silencing.

A pair of transposons coordinately suppresses gene expression, independent of pathways mediated by siRNA in Antirrhinum.

Our knowledge is limited regarding mechanisms by which transposable elements control host gene expression. Two Antirrhinum lines, HAM2 and HAM5, show different petal colors, pale-red and white, respectively, although these lines contain the same insertion of transposon Tam3 in the promoter region of the nivea (niv) locus encoding chalcone synthase. Among 1000 progeny from HAM5 grown under the preferred conditions for the Tam3 transposition, a few showed an intermediate petal color between HAM2 and HAM5. Transposon tagging using these progeny identified a causative insertion of Tam3 for the HAM5 type (white) petal color, which was found 1.6 kb downstream of the niv gene. Insertion of Tam3 at the position 1.6 kb downstream of niv alone showed nearly wildtype petal pigmentation, and the niv expression reduced by only 50%. Severe suppression of niv observed in HAM5 required interaction of two Tam3 copies on either side of the niv coding sequence. DNA methylation and small interfering RNAs (siRNAs) were not associated with the suppression of niv expression in HAM5. Insertion of a pair of transposons in close proximity can interfere with the expression of gene located between the two copies, and also provide evidence that this interference is not directly associated with pathways mediated by siRNAs.

Seed coat pigmentation in transgenic soybean expressing the silencing suppressor 2b gene of Cucumber mosaic virus.

KEY MESSAGE: Soybean expressing the Cucumber mosaic virus 2b gene manifests seed coat pigmentation due to suppression of endogenous RNA silencing but no other morphological abnormality. This gene may help prevent transgene silencing. RNA silencing is an important mechanism for gene regulation and antiviral defense in plants. It is also responsible for transgene silencing, however, and thus hinders the establishment of transgenic plants. The 2b protein of Cucumber mosaic virus (CMV) functions as a suppressor of RNA silencing and therefore might prove beneficial for stabilization of transgene expression. We have now generated transgenic soybean that harbors the 2b gene of a CMV-soybean strain under the control of a constitutive promoter to investigate the effects of 2b expression. No growth abnormality was apparent in 2b transgenic plants, although the seed coat was pigmented in several of the transgenic lines. Genes for chalcone synthase (CHS), a key enzyme of the flavonoid pathway, are posttranscriptionally silenced by the inhibitor (I) locus in nonpigmented (yellow) soybean seeds. The levels of CHS mRNA and CHS small interfering RNA in strongly pigmented 2b transgenic seed coats were higher and lower, respectively, than those in the seed coat of a control transgenic line. The expression level of 2b also correlated with the extent of seed coat pigmentation. On the other hand, introduction of the 2b gene together with the DsRed2 gene into somatic embryos prevented the time-dependent decrease in transient DsRed2 expression. Our results indicate that the 2b gene alone is able to suppress RNA silencing of endogenous CHS genes regulated by the I locus, and that 2b is of potential utility for stabilization of transgene expression in soybean without detrimental effects other than seed coat pigmentation.

Comparative analysis of the inverted repeat of a chalcone synthase pseudogene between yellow soybean and seed coat pigmented mutants.

In soybean, the I gene inhibits pigmentation over the entire seed coat, resulting in yellow seeds. It is thought that this suppression of seed coat pigmentation is due to naturally occurring RNA silencing of chalcone synthase genes (CHS silencing). Fully pigmented seeds can be found among harvested yellow seeds at a very low percentage. These seed coat pigmented (scp) mutants are generated from yellow soybeans by spontaneous recessive mutation of the I gene. A candidate for the I gene, GmIRCHS, contains a perfect inverted repeat (IR) of a CHS pseudogene (pseudoCHS3) and transcripts of GmIRCHS form a double-stranded CHS RNA that potentially triggers CHS silencing. One CHS gene, ICHS1, is located 680 bp downstream of GmIRCHS. Here, the GmIRCHS-ICHS1 cluster was compared in scp mutants of various origins. In these mutants, sequence divergence in the cluster resulted in complete or partial loss of GmIRCHS in at least the pseudoCHS3 region. This result is consistent with the notion that the IR of pseudoCHS3 is sufficient to induce CHS silencing, and further supports that GmIRCHS is the I gene.

Suppressive mechanism of seed coat pigmentation in yellow soybean.

In soybean seeds, numerous variations in colors and pigmentation patterns exist, most of which are observed in the seed coat. Patterns of seed coat pigmentation are determined by four alleles (I, i(i), i(k) and i) of the classically defined I locus, which controls the spatial distribution of anthocyanins and proanthocyanidins in the seed coat. Most commercial soybean cultivars produce yellow seeds with yellow cotyledons and nonpigmented seed coats, which are important traits of high-quality seeds. Plants carrying the I or i(i) allele show complete inhibition of pigmentation in the seed coat or pigmentation only in the hilum, respectively, resulting in a yellow seed phenotype. Classical genetic analyses of the I locus were performed in the 1920s and 1930s but, until recently, the molecular mechanism by which the I locus regulated seed coat pigmentation remained unclear. In this review, we provide an overview of the molecular suppressive mechanism of seed coat pigmentation in yellow soybean, with the main focus on the effect of the I allele. In addition, we discuss seed coat pigmentation phenomena in yellow soybean and their relationship to inhibition of I allele action.

Variation of GmIRCHS (Glycine max inverted-repeat CHS pseudogene) is related to tolerance of low temperature-induced seed coat discoloration in yellow soybean.

In yellow soybean, seed coat pigmentation is inhibited by post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. A CHS cluster named GmIRCHS (Glycine max inverted-repeat CHS pseudogene) is suggested to cause PTGS in yellow-hilum cultivars. Cold-induced seed coat discoloration (CD), a commercially serious deterioration of seed appearance, is caused by an inhibition of this PTGS upon exposure to low temperatures. In the highly CD-tolerant cultivar Toyoharuka, the GmIRCHS structure differs from that of other cultivars. The aim of this study was to determine whether the variation of GmIRCHS structure among cultivars is related to variations in CD tolerance. Using two sets of recombinant inbred lines between Toyoharuka and CD-susceptible cultivars, we compared the GmIRCHS genotype and CD tolerance phenotype during low temperature treatment. The GmIRCHS genotype was related to the phenotype of CD tolerance. A QTL analysis around GmIRCHS showed that GmIRCHS itself or a region located very close to it was responsible for CD tolerance. The variation in GmIRCHS can serve as a useful DNA marker for marker-assisted selection for breeding CD tolerance. In addition, QTL analysis of the whole genome revealed a minor QTL that also affected CD tolerance.

Suppression of RNA-dependent RNA polymerase 6 in tomatoes allows potato spindle tuber viroid to invade basal part but not apical part including pluripotent stem cells of shoot apical meristem.

RNA-dependent RNA polymerase 6 (RDR6) is one of the key factors in plant defense responses and suppresses virus or viroid invasion into shoot apical meristem (SAM) in Nicotiana benthamiana. To evaluate the role of Solanum lycopersicum (Sl) RDR6 upon viroid infection, SlRDR6-suppressed (SlRDR6i) 'Moneymaker' tomatoes were generated by RNA interference and inoculated with intermediate or lethal strain of potato spindle tuber viroid (PSTVd). Suppression of SlRDR6 did not change disease symptoms of both PSTVd strains in 'Moneymaker' tomatoes. Analysis of PSTVd distribution in shoot apices by in situ hybridization revealed that both PSTVd strains similarly invade the basal part but not apical part including pluripotent stem cells of SAM in SlRDR6i plants at a low rate unlike a previous report in N. benthamiana. In addition, unexpectedly, amount of PSTVd accumulation was apparently lower in SlRDR6i plants than in control tomatoes transformed with empty cassette in early infection especially in the lethal strain. Meanwhile, SlRDR6 suppression did not affect the seed transmission rates of PSTVd. These results indicate that RDR6 generally suppresses PSTVd invasion into SAM in plants, while suppression of RDR6 does not necessarily elevate amount of PSTVd accumulation. Additionally, our results suggest that host factors such as RDR1 other than RDR6 may also be involved in the protection of SAM including pluripotent stem cells from PSTVd invasion and effective RNA silencing causing the decrease of PSTVd accumulation during early infection in tomato plants.

Molecular mechanism of seed coat discoloration induced by low temperature in yellow soybean.

Seed coat pigmentation is inhibited in yellow soybean. The I gene inhibits pigmentation over the entire seed coat. In yellow soybean, seed coat discoloration occurs when plants are exposed to low temperatures after the onset of flowering, a phenomenon named 'cold-induced discoloration (CD)'. Inhibition of seed coat pigmentation results from post-transcriptional gene silencing (PTGS) of the chalcone synthase (CHS) genes. PTGS is a sequence-specific RNA degradation mechanism in plants and occurs via short interfering RNAs (siRNAs). Similar post-transcriptional suppression is called RNAi (RNA interference) in animals. Recently, we identified a candidate of the I gene designated GmIRCHS. In this study, to elucidate the molecular mechanism of CD, CHS mRNA and siRNA levels in the seed coat were compared between CD-sensitive and CD-tolerant cultivars (Toyomusume and Toyoharuka, respectively). In Toyomusume, the CHS siRNA level was reduced markedly by low temperature treatment, and subsequently the CHS mRNA level increased rapidly after treatment. In contrast, low temperature treatment did not result in severe reduction of the CHS siRNA level in Toyoharuka, and the CHS mRNA level did not increase after the treatment. These results suggest that the rapid increase in CHS mRNA level after low temperature treatment may lead to enhanced pigmentation in some of the seed coat cells and finally in seed coat discoloration. Interestingly, we found a Toyoharuka-specific difference in the GmIRCHS region, which may be involved in CD tolerance.

A novel major quantitative trait locus controlling seed development at low temperature in soybean (Glycine max).

Low temperature is among the critical environmental factors that limit soybean production. To elucidate the genetic basis for chilling tolerance and identify useful markers, we conducted quantitative trait loci (QTL) analysis of seed-yielding ability at low temperature in soybean (Glycine max), using artificial climatic environments at usual and low temperatures and recombinant inbred lines derived from a cross between two contrasting cultivars in terms of chilling tolerance. We identified a QTL of a large effect (LOD > 15, r (2) > 0.3) associated with seed-yielding ability only at low temperature. The QTL was mapped near marker Sat_162 on linkage group A2, where no QTL for chilling tolerance has previously been identified. The tolerant genotype did not increase the pod number but maintained the seed number per pod and single seed weight, namely, the efficiency of seed development at low temperature. The effect of the QTL was confirmed in a segregating population of heterogeneous inbred families, which provided near-isogenic lines. The genomic region containing the QTL also influenced the node and pod numbers regardless of temperature condition, although this effect was not primarily associated with chilling tolerance. These results suggest the presence of a new major genetic factor that controls seed development specifically at low temperature. The findings will be useful for marker-assisted selection as well as for understanding of the mechanism underlying chilling tolerance in reproductive organs.

Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase (CHS) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase (F3'H) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.

Dose-dependent hypocholesterolemic actions of dietary apple polyphenol in rats fed cholesterol.

The dose-dependent hypocholesterolemic and antiatherogenic effects of dietary apple polyphenol (AP) from unripe apple, which contains approximately 85% catechin oligomers (procyanidins), were examined in male Sprague-Dawley rats (4 wk of age) given a purified diet containing 0.5% cholesterol. Dietary AP at 0.5 and 1.0% levels significantly decreased the liver cholesterol level compared with that in the control (AP-free diet-fed) group. Dietary AP also significantly lowered the serum cholesterol level compared with that in the control group. However, the HDL cholesterol level was significantly higher in the 1.0% AP-fed group than in the control group. Accordingly, the ratio of HDL-cholesterol/total cholesterol was significantly higher in the 0.5% AP-fed group and 1.0% AP-fed group than in the control group. Moreover, the atherogenic indices in the 0.5 and 1.0% AP-fed groups were significantly lower than those in the control group. The activity of hepatic cholesterol 7alpha-hydroxylase tended to be increased by dietary AP in a dose-dependent manner. In accord with this observation, dietary AP increased the excretion of acidic steroids in feces. Dietary AP also significantly promoted the fecal excretion of neutral steroids in a dose-dependent manner. These observations suggest that dietary AP at a 0.5 or 1.0% level exerts hypocholesterolemic and antiatherogenic effects through the promotion of cholesterol catabolism and inhibition of intestinal absorption of cholesterol.

Sequence divergence at chalcone synthase gene in pigmented seed coat soybean mutants of the Inhibitor locus.

Seed coat color in soybeans is determined by the I (Inhibitor) locus. The dominant I allele inhibits seed coat pigmentation, and it has been suggested that there is a correlation between the inhibition of pigmentation by the I allele and chalcone synthase (CHS) gene silencing in the seed coat. Analysis of spontaneous mutations from I to i has shown that these mutations are closely related to the deletion of one of the CHS genes (designated ICHS1). In soybeans with the I/I genotype (cv. Miyagi shirome), a truncated form of the CHS gene (CHS3) is located in an inverse orientation 680 bp upstream of ICHS1, and it was previously suggested that the truncated CHS3- ICHS1 cluster might be involved in CHS gene silencing in the seed coat. In the current study, the truncated CHS3- ICHS1 cluster was compared with the corresponding region of pigmented seed coat mutants in which I had changed to i in Miyagi shirome and in the strain Karikei 584. In the Karikei 584 mutant, the truncated CHS3-ICHS1 cluster was retained and the sequence diverged at a point immediately upstream (32 bp) of this cluster. The sequences upstream of the points of divergence in both mutants almost perfectly matched a part of the registered sequence in a soybean BAC clone containing the soybean cyst nematode resistance-associated gene, and inspection of the sequences suggested that the sequence divergence of the CHS gene in the Karikei 584 and Miyagi shirome mutants was due to an unequal crossing-over via 4-bp or 5-bp short repeats, respectively.

Endogenous RNA interference of chalcone synthase genes in soybean: formation of double-stranded RNA of GmIRCHS transcripts and structure of the 5' and 3' ends of short interfering RNAs.

In yellow soybean, seed coat pigmentation is inhibited via endogenous RNA interference (RNAi) of the chalcone synthase (CHS) genes. Genetic studies have shown that a single dominant gene, named the I gene, inhibits pigmentation over the entire seed coat in soybean. We previously isolated a candidate for the I gene from the yellow soybean genome with the I/I genotype, and designated it GmIRCHS. A structural feature of GmIRCHS is a perfect inverted repeat of the pseudoCHS gene lacking 5'-coding region. This suggests that the double-stranded RNA (dsRNA) structure of the pseudoCHS gene may be formed in the GmIRCHS transcript. RNAi is triggered by the dsRNA for a target gene, so the GmIRCHS transcript is likely to be a trigger for RNAi of CHS genes. In this study, we identified a 1087-bp dsRNA, including pseudoCHS region ranging from most of exon 2 to 3'-UTR, in the GmIRCHS transcript. Interestingly, this dsRNA was detected not only in the seed coat but also in the cotyledon and leaf tissues. Previously, CHS RNAi has been shown to be restricted to the seed coat, and we reported that endogenous short interfering RNAs of CHS genes (CHS siRNAs) are detected only in the seed coat and not in the cotyledon and leaf tissues. Taken together with these previous reports, our result suggests that seed-coat specificity of CHS RNAi may be determined in the amplification step of CHS siRNAs rather than dsRNA formation in the GmIRCHS transcript. Our studies further revealed that CHS siRNAs are modified at the 3' ends and bear 5' monophosphorylated ends, suggesting that CHS siRNA duplexes are generated by Dicer-like enzyme from CHS dsRNA and subsequently modified at the 3' ends for stabilizing CHS siRNAs.