The effect of stem growth habit on single seed weight and seed uniformity in soybean (Glycine max (L.) Merrill).

The timing of flower formation and length of the seed-filling period of indeterminate growth soybean varieties vary more than those of determinate varieties (Glycine max (L.) Merrill). These variations have been hypothesized to affect single seed weight and its uniformity which determine the processing quality of soybean used in foods. We derived near isogenic lines (NILs) with different growth characteristics from an indeterminate line (donor parent) and three determinate lines with heavy seeds (recurrent parents), and evaluated the effects of growth habit on seed weight and its uniformity. Each NIL population consisting of five indeterminate and five determinate BC4F4 lines tested at two locations in two different years with two replications. Split-plot analysis of variance, with main-plot and sub-plot being cross combination and growth habit, respectively, showed that indeterminate varieties had slightly heavier seeds than determinate varieties and that there was no significant difference in uniformity of single seed weights. The effects of growth habit on seed uniformity was related to genetic background, but differences between the two growth characteristics were less than the differences among genetic background. This indicates that indeterminate growth habit did not much influence seed weight or its uniformity.

Seed yield and its components of indeterminate and determinate lines in recombinant inbred lines of soybean.

The present study was conducted to evaluate the benefits of indeterminate growth habit in breeding to improve yield potential of Japanese soybean varieties, which exclusively have determinate growth habit. Two populations of recombinant inbred lines (RILs) derived from crosses between determinate Japanese cultivars and indeterminate US cultivars were grown in Akita and Kyoto, and seed weight per plant (SW) and its components were compared between indeterminate and determinate RILs. The difference of SW between the two growth habits in RILs varied depending on maturation time. The SW of early indeterminate lines was significantly higher than that of early determinate ones in Akita, but not in Kyoto. Among yield components, the number of seeds per pod was constantly larger in indeterminate lines than that in determinate ones irrespective of maturation time. The number of seeds per plant and the number of pods per plant of the indeterminate lines were greater than those of the determinate lines in early maturation in Akita. These results suggest that the indeterminate growth habit is an advantageous characteristic in breeding for high yield of early maturing soybean varieties in the Tohoku region.

A major and stable QTL associated with seed weight in soybean across multiple environments and genetic backgrounds.

KEY MESSAGE: We detected a QTL for single seed weight in soybean that was stable across multiple environments and genetic backgrounds with the use of two recombinant inbred line populations. Single seed weight (SSW) in soybean is a key determinant of both seed yield and the quality of soy food products, and it exhibits wide variation. SSW is under genetic control, but the molecular mechanisms of such control remain unclear. We have now investigated quantitative trait loci (QTLs) for SSW in soybean and have identified such a QTL that is stable across multiple environments and genetic backgrounds. Two populations of 225 and 250 recombinant inbred lines were developed from crosses between Japanese and US cultivars of soybean that differ in SSW by a factor of ~2, and these populations were grown in at least three different environments. A whole-genome panel comprising 304 simple sequence repeat (SSR) loci was applied to mapping in each population. We identified 15 significant QTLs for SSW dispersed among 11 chromosomes in the two populations. One QTL located between Sat_284 and Sat_292 on chromosome 17 was detected (3.6 < LOD < 14.1) in both populations grown in all environments. This QTL, tentatively designated qSw17-1, accounted for 9.4-20.9 % of phenotypic variation in SSW, with a dominant allele being associated with increased SSW. Given its substantial effect on SSW, qSw17-1 is an attractive target for positional cloning, and SSR markers closely associated with this locus may prove useful for marker-assisted selection for SSW control in soybean.

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.

Genetic relationships of soybean cyst nematode resistance originated in Gedenshirazu and PI84751 on Rhg1 and Rhg4 loci.

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is one of the most damaging pests of soybean (Glycine max (L.) Merr.). Host plant resistance has been the most effective control method. Because of the spread of multiple SCN races in Hokkaido, the Tokachi Agricultural Experiment Station has bred soybeans for SCN resistance since 1953 by using 2 main resistance resources PI84751 (resistant to races 1 and 3) and Gedenshirazu (resistant to race 3). In this study, we investigated the genetic relationships of SCN resistance originating from major SCN resistance genes in Gedenshirazu and PI84751 by using SSR markers. We confirmed that race 1 resistance in PI84751 was independently controlled by 4 genes, 2 of which were rhg1 and Rhg4. We classified the PI84751- type allele of Rhg1 as rhg1-s and the Gedenshirazu-type allele of Rhg1 as rhg1-g. In the cross of the Gedenshirazu-derived race 3-resistant lines and the PI84751-derived races 1- and 3-resistant lines, the presence of rhg1-s and Rhg4 was responsible for race 1-resistance. These results indicated that it was possible to select race 1 resistant plants by using marker-assisted selection for the rhg1-s and Rhg4 alleles through a PI84751 origin × Gedenshirazu origin cross.

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.

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.

Structural features of GmIRCHS, candidate of the I gene inhibiting seed coat pigmentation in soybean: implications for inducing endogenous RNA silencing of chalcone synthase genes.

Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The I gene inhibits pigmentation over the entire seed coat, resulting in a uniform yellow color of mature harvested seeds. We previously demonstrated that the inhibition of seed coat pigmentation by the I gene results from post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. Little is known about the structure of the I gene and the mechanism by which it induces PTGS of CHS genes. Here, we report a candidate of the I gene, GmIRCHS, which consists of a 5'-portion of a DnaJ-like gene containing a promoter region and a perfect inverted repeat (IR) of 1.1-kb truncated CHS3 sequences (5'-DeltaCHS3 and 3'-DeltaCHS3). RT-PCRs and RNase protection assay indicated the existence of the read-through product from 5'-DeltaCHS3 to 3'-DeltaCHS3 and the dsRNA region of DeltaCHS3, suggesting that dsRNA of DeltaCHS3 could be transcribed from GmIRCHS and could induce PTGS of CHS genes. Moreover, the IR structure of DeltaCHS3 in GmIRCHS was lost in the soybean mutants in which I was changed to i, supporting the conclusion that GmIRCHS is the I gene.