Hideyuki Funatsuki

High-density Integrated Linkage Map Based on SSR Markers in Soybean

A well-saturated molecular linkage map is a prerequisite for modern plant breeding. Several genetic maps have been developed for soybean with various types of molecular markers. Simple sequence repeats (SSRs) are single-locus markers with high allelic variation and are widely applicable to different genotypes. We have now mapped 1810 SSR or sequence-tagged site markers in one or more of three recombinant inbred populations of soybean (the US cultivar ‘Jack’ × the Japanese cultivar ‘Fukuyutaka’, the Chinese cultivar ‘Peking’ × the Japanese cultivar ‘Akita’, and the Japanese cultivar ‘Misuzudaizu’ × the Chinese breeding line ‘Moshidou Gong 503’) and have aligned these markers with the 20 consensus linkage groups (LGs). The total length of the integrated linkage map was 2442.9 cM, and the average number of molecular markers was 90.5 (range of 70–114) for the 20 LGs. We examined allelic diversity for 1238 of the SSR markers among 23 soybean cultivars or lines and a wild accession. The number of alleles per locus ranged from 2 to 7, with an average of 2.8. Our high-density linkage map should facilitate ongoing and future genomic research such as analysis of quantitative trait loci and positional cloning in addition to marker-assisted selection in soybean breeding.

Sequencing and analysis of approximately 40,000 soybean cDNA clones from a full-length-enriched cDNA library.

A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39 936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5′ and 3′ ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22 674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5′- and 3′-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.

Molecular basis of a shattering resistance boosting global dissemination of soybean

Significance Pod dehiscence is a critical step in the seed dispersal (shattering) of legume and crucifer crops and can cause significant yield losses. Upon drying, pod walls are dehisced by two factors: the reduction of pod-wall binding strength and the generation of dehiscing forces. Although the previously reported shattering-resistant mutants maintained binding strength, here, we show a gene regulating the dehiscing force. The gene, Pdh1, encodes a dirigent family protein, known to be involved in lignification, which increases dehiscing forces by promoting torsion of dried pod walls. The loss-of-function pdh1 gene has been widely used as a shattering-resistance gene in soybean breeding. This knowledge could be useful in improving other legume and crucifer crops, as well as soybean breeding. Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1. The gene for the shattering-resistant genotype, pdh1, was defective, having a premature stop codon. The functional gene, Pdh1, was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world’s leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1, or genes with the same role, will possibly be useful for crop improvement.

Development and Application of a Whole-Genome Simple Sequence Repeat Panel for High-Throughput Genotyping in Soybean

Among commonly applied molecular markers, simple sequence repeats (SSRs, or microsatellites) possess advantages such as a high level of polymorphism and codominant pattern of inheritance at individual loci. To facilitate systematic and rapid genetic mapping in soybean, we designed a genotyping panel comprised 304 SSR markers selected for allelic diversity and chromosomal location so as to provide wide coverage. Most primer pairs for the markers in the panel were redesigned to yield amplicons of 80–600 bp in multiplex polymerase chain reaction (PCR) and fluorescence-based sequencer analysis, and they were labelled with one of four different fluorescent dyes. Multiplex PCR with sets of six to eight primer pairs per reaction generated allelic data for 283 of the 304 SSR loci in three different mapping populations, with the loci mapping to the same positions as previously determined. Four SSRs on each chromosome were analysed for allelic diversity in 87 diverse soybean germplasms with four-plex PCR. These 80 loci showed an average allele number and polymorphic information content value of 14.8 and 0.78, respectively. The high level of polymorphism, ease of analysis, and high accuracy of the SSR genotyping panel should render it widely applicable to soybean genetics and breeding.

Fine mapping and development of DNA markers for the qPDH1 locus associated with pod dehiscence in soybean.

Pod dehiscence (shattering) is a major cause of yield loss in mechanical harvesting of soybeans. To develop useful selection markers, we conducted a high-resolution mapping of a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1. The progeny of a residual heterozygous line, which was a recombinant inbred line segregating only for the genomic region around qPDH1, was screened for flanking markers to obtain various recombinants in the vicinity of the QTL. Analysis of the relationship between degree of pod dehiscence and graphical genotype of these lines confined the location of qPDH1 to a 134-kb region on chromosome 16 (formerly linkage group J), where ten putative genes were predicted to be present. None of these genes showed significant sequence homology with the Arabidopsis genes that have previously been reported to be associated with pod dehiscence, suggesting the presence of a novel gene and mechanism underlying pod dehiscence in soybean. Sequencing analysis of the parental shattering-resistant and -susceptible cultivars for the candidate genes revealed a high-frequency nucleotide polymorphism in this genomic region between the cultivars. Three markers were developed using insertion/deletion variations in the region. Polymorphism at these marker loci was basically conserved between diverse shattering-resistant and -susceptible cultivars/lines, suggesting the versatility and usefulness of these markers for marker-assisted selection.

Identification of QTL controlling post-flowering period in soybean

The length of the reproductive period affects the grain yield of soybean (Glycine max [L.] Merr), and genetic control of the period might contribute to yield improvement. To detect genetic factor(s) controlling the reproductive period, a population of recombinant inbred lines (RILs) was developed from a cross between Japanese landrace ‘Ippon-Sangoh’ and, Japanese cultivar ‘Fukuyutaka’ which differ in their duration from flowering to maturation (DFM) relative to the difference in the duration from sowing to flowering (DSF). In the RIL population, the DFM correlated poorly (r = −0.16 to 0.34) with the DSF in all field trials over 3 years. Two stable QTLs for the DFM on chromosomes (Chr-) 10 and 11 as well as two stable QTLs for the DSF on Chr-10 and -16 were identified. The QTL on Chr-11 for the reproductive period (designated as qDfm1; quantitative trait locus for duration from flowering to maturation 1) affected all three trials, and the difference in the DFM between the Fukuyutaka and Ippon-Sangoh was mainly accounted for qDfm1, in which the Fukuyutaka allele promoted a longer period. qDfm1 affected predominantly the reproductive period, and thus it might be possible to alter the period with little influence on the vegetative period.

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.

A Major Soybean QTL, qPDH1, Controls Pod Dehiscence without Marked Morphological Change

Abstract Pod dehiscence (shattering) is a major source of yield loss in the mechanically harvested soybean. We examined near-isogenic lines (NILs) for a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1, to reveal the mechanism underlying the effect of this QTL on shattering resistance. The degree of shattering resistance differed among the NILs; as pod dehiscence percentage after 3 hr heat treatment was under 50% and over 90% for the genotypes resistant to shattering and those susceptible to shattering, respectively. On the other hand, there were no significant differences in the length, width and thickness of pods among the NILs. Anatomical analysis of the dorsal sutures of pods, at which pod dehiscence was found to commence most frequently, revealed no marked differences between the NILs. These results suggest that qPDH1 controls pod dehiscence without markedly changing the morphology of the pods.

Fertile plant regeneration from barley (Hordeum vulgare L.) protoplasts isolated from primary calluses

Abstract Barley (Hordeum vulgare L.) protoplasts were isolated from the immature embryo-derived primary calluses. These protoplasts were cultured with nurse cells, and they then divided to form colonies. After transfer of the colonies to regeneration medium, either green or albino shoots were regenerated from these colonies. A high agarose concentration (2.4% w/v) in the protoplast culture medium promoted protoplast division. The plantlets that developed strong root systems were transferred to the soil. These plants flowered and have set seeds.

Influence of primary callus induction conditions on the establishment of barley cell suspensions yielding regenerable protoplasts

SummaryWith the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.