Characterization of the genomic sequence data around common cutworm resistance genes in soybean (Glycine max) using short- and long-read sequencing methods.

The common cutworm (CCW, Spodopteraab litura Fabricius) is one of the pests that most severely infect soybean (Glycine max L. Merr.). In a previous report, quantitative trait loci (QTL) analysis of CCW resistance using a recombinant inbred line derived from a cross between a susceptible cultivar 'Fukuyutaka' and a resistant cultivar 'Himeshirazu', identified two antixenosis resistance QTLs, CCW-1 and CCW-2. To reveal sequence variation between the aforementioned two cultivars, whole genome resequencing was performed using Illumina HiSeq2000 (75,632,747 and 91,540,849 reads). The generated datasets can be used for fine mapping and gene isolation of CCW-1 and CCW-2 as well as for revealing more detailed genetic differences between 'Fukuyutaka' and 'Himeshirazu' .

Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon.

KEY MESSAGE: A frame shift invoked by a single-base deletion in the gene encoding a cytochrome P450 hydroxylase, CYP81E22, causes the loss of bentazon detoxification function in soybean. Bentazon is an effective herbicide in soybean cultivation applied at post-emergence stages for control of several broadleaf weeds. However, some soybean cultivars are highly sensitive to bentazon and are killed upon application. In this study, the gene related to the high sensitivity of soybean cultivars to bentazon was mapped to chromosome 16, and its location was narrowed down to a 257-kb region where three cytochrome P450 genes were located. In these genes, a single-base deletion of cytosine was detected in the coding region of Glyma.16G149300, CYP81E22, at + 1465 bp downstream from the translation start codon, leading to a frame shift in the open reading frame and creating a premature stop codon. This stop codon resulted in the loss of more than half of the P450, and consequently, the remaining molecule failed to form a functioning protein. This single-base deletion was common among the highly sensitive cultivars screened from the soybean mini-core collection and other previously reported highly sensitive cultivars. Furthermore, we screened plant lines from the targeting-induced local lesions in genomes library of the soybean cultivar Enrei based on a modelled 3D structure of CYP81E22. The lines with mutations in Glyma.16G149300 were highly sensitive to bentazon, which provides strong evidence that Glyma.16G149300 is the gene responsible for high sensitivity to bentazon.

Evaluation of the resistance effect of QTLs derived from wild soybean (Glycine soja) to common cutworm (Spodoptera litura Fabricius).

Common cutworm (CCW) is a serious herbivorous insect pest of soybean. Previously, we conducted an antixenosis bioassay (measuring feeding preference) with CCW using recombinant inbred lines (RILs) derived from a cross between a wild soybean (Glycine soja) collected in Hiroshima prefecture (JP110755) and the leading cultivar, Fukuyutaka. The analysis revealed quantitative trait loci (QTLs) for antixenosis resistance, qRslx3 and qRslx4. In the present study we developed another RIL population using Fukuyutaka and a different G. soja, collected in Kumamoto prefecture (G406). An analysis revealed an antixenosis resistance QTL on chromosome 7, and the resistant allele of the QTL was derived from G406. The chromosomal position of the QTL was almost the same as that of CCW-2, a previously-reported antibiosis resistance QTL for CCW, detected in a F2 population derived from a cross between Fukuyutaka and a resistant cultivar Himeshirazu. These QTLs could be the same locus; however, G406 and Himeshirazu are likely to possess different alleles, because Himeshirazu allele exhibits no antixenosis effect. We expect that pyramiding of the resistance QTLs derived from G. soja will contribute to the development of CCW resistant cultivars.

Erratum: Quantitative trait loci associated with short inter-node length in soybean.

[This corrects the article on p. 554 in vol. 68, PMID: 30697116.].

Quantitative trait loci associated with short inter-node length in soybean.

Manipulating the genetic control of plant height is essential in soybean breeding to increase yield through the enlargement of the plant size while preventing lodging. A Japanese soybean germplasm, Y2, has distinctively shorter inter-node lengths than those of recently developed Japanese cultivars and is expected to provide new variation to prevent lodging. A quantitative trait loci (QTL) analysis for plant height-related traits was conducted using F2 individuals derived from a cross between the elite Japanese cultivar Fukuyutaka and Y2. A major QTL for average inter-node length (AIL) and plant height was identified on chromosome 13 and named qSI13-1 (QTL for short inter-node on chromosome 13). The Y2 allele of qSI13-1 was partially dominant for plant height. qSI13-1 exhibited no effect on either days to flowering or number of main stem nodes. The AILs and plant heights of the near-isogenic lines containing the Y2 allele of qSI13-1 in the genetic background of Fukuyutaka were significantly less than those of Fukuyutaka. No significant differences between the near-isogenic lines and Fukuyutaka were observed for seed yield and flowering date, indicating that qSI13-1 will be useful in developing cultivars with short plant heights without having negative effects on yield potential and days to flowering.

QTL mapping of antixenosis resistance to common cutworm (Spodoptera litura Fabricius) in wild soybean (Glycine soja).

The common cutworm (CCW; Spodoptera litura Fabricius) is a serious herbivorous insect pest of soybean (Glycine max) in Asia and Oceania. Previously, we identified quantitative trait loci (QTLs) for CCW-antibiosis-resistance, CCW-1 and CCW-2, and antixenosis-resistance, qRslx1 and qRslx2, in the cultivar 'Himeshirazu'. The effects of these QTLs are useful in the breeding of CCW-resistant cultivars. In this study, we conducted an antixenosis bioassay on CCW using recombinant inbred lines derived from a cross between a wild soybean (Glycine soja) and the leading cultivar 'Fukuyutaka' to identify CCW-resistance genes in G. soja. The QTL analysis revealed six and four novel antixenosis-resistance QTLs in 2012 and 2013, respectively. Among them, the QTLs on chromosomes 2 and 7, designated qRslx4 and qRslx3, respectively, were stably detected in both years. qRslx3 exhibited the largest effect in both years, suggesting that qRslx3 can be exploited in the breeding of CCW-resistant soybean. Furthermore, qRslx3 and qRslx4 can be used, along with previously reported QTLs from 'Himeshirazu', to enhance the CCW-resistance of soybean cultivars because their chromosomal positions are unique. These new CCW-resistance QTLs from G. soja should play important roles in the breeding of CCW-resistant soybean cultivars.

Genetic analysis of antixenosis resistance to the common cutworm (Spodoptera litura Fabricius) and its relationship with pubescence characteristics in soybean (Glycine max (L.) Merr.).

The common cutworm (CCW, Spodoptera litura Fabricius) is one of the most serious pests of soybean (Glycine max (L.) Merr.). Previously, two quantitative trait loci (QTLs) for antibiosis resistance to CCW, CCW-1 and CCW-2, were detected in the resistant cultivar Himeshirazu. In this study, we conducted an anti-xenosis bioassay using a recombinant inbred population derived from a cross between a susceptible cultivar Fukuyutaka and Himeshirazu to perform QTL analysis. Two QTLs for antixenosis resistance, qRslx1 and qRslx2, were identified on Chrs 7 and 12, and the resistant alleles of qRslx1 and qRslx2 were derived from Himeshirazu and Fukuyutaka, respectively. The position of qRslx1 is similar to that of CCW-1. We also analyzed pubescence characteristics because they have been reported to be associated with soybean insect resistance. Two QTLs for pubescence length (on Chrs 7 and 12) and two QTLs for pubescence density (on Chrs 1 and 12) were identified. The pubescence QTLs on Chrs 7 and 12 were located near qRslx1 and qRslx2, respectively. These results suggest that the antixenosis resistance could be controlled genetically by the identified QTLs and that the pubescence characteristics might contribute to the soybean antixenosis resistance to CCW.

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.

Effects on flowering and seed yield of dominant alleles at maturity loci E2 and E3 in a Japanese cultivar, Enrei.

'Enrei' is the second leading variety of soybean (Glycine max (L.) Merr.) in Japan. Its cultivation area is mainly restricted to the Hokuriku region. In order to expand the adaptability of 'Enrei', we developed two near-isogenic lines (NILs) of 'Enrei' for the dominant alleles controlling late flowering at the maturity loci, E2 and E3, by backcrossing with marker-assisted selection. The resultant NILs and the original variety were evaluated for flowering, maturity, seed productivity and other agronomic traits in five different locations. Expectedly, NILs with E2 or E3 alleles flowered later than the original variety in most locations. These NILs produced comparatively larger plants in all locations. Seed yields were improved by E2 and E3 in the southern location or in late-sowing conditions, whereas the NIL for E2 exhibited almost the same or lower productivity in the northern locations due to higher degrees of lodging. Seed quality-related traits, such as 100-seed weight and protein content, were not significantly different between the original variety and its NILs. These results suggest that the modification of genotypes at maturity loci provides new varieties that are adaptive to environments of different latitudes while retaining almost the same seed quality as that of the original.

OryzaExpress: an integrated database of gene expression networks and omics annotations in rice.

Similarity of gene expression profiles provides important clues for understanding the biological functions of genes, biological processes and metabolic pathways related to genes. A gene expression network (GEN) is an ideal choice to grasp such expression profile similarities among genes simultaneously. For GEN construction, the Pearson correlation coefficient (PCC) has been widely used as an index to evaluate the similarities of expression profiles for gene pairs. However, calculation of PCCs for all gene pairs requires large amounts of both time and computer resources. Based on correspondence analysis, we developed a new method for GEN construction, which takes minimal time even for large-scale expression data with general computational circumstances. Moreover, our method requires no prior parameters to remove sample redundancies in the data set. Using the new method, we constructed rice GENs from large-scale microarray data stored in a public database. We then collected and integrated various principal rice omics annotations in public and distinct databases. The integrated information contains annotations of genome, transcriptome and metabolic pathways. We thus developed the integrated database OryzaExpress for browsing GENs with an interactive and graphical viewer and principal omics annotations (http://riceball.lab.nig.ac.jp/oryzaexpress/). With integration of Arabidopsis GEN data from ATTED-II, OryzaExpress also allows us to compare GENs between rice and Arabidopsis. Thus, OryzaExpress is a comprehensive rice database that exploits powerful omics approaches from all perspectives in plant science and leads to systems biology.

High potential of a transposon mPing as a marker system in japonica x japonica cross in rice.

Although quantitative traits loci (QTL) analysis has been widely performed to isolate agronomically important genes, it has been difficult to obtain molecular markers between individuals with similar phenotypes (assortative mating). Recently, the miniature inverted-repeat transposable element mPing was shown to be active in the japonica strain Gimbozu EG4 where it had accumulated more than 1000 copies. In contrast, most other japonicas, including Nipponbare, have 50 or fewer mPing insertions in their genome. In this study we have exploited the polymorphism of mPing insertion sites to generate 150 PCR markers in a cross between the closely related japonicas, Nipponbare x Gimbozu (EG4). These new markers were distributed in genic regions of the whole genome and showed significantly higher polymorphism (150 of 183) than all other molecular markers tested including short sequence repeat markers (46 of 661). In addition, we performed QTL analysis with these markers using recombinant inbred lines derived from Nipponbare x Gimbozu EG4, and successfully mapped a locus involved in heading date on the short arm of chromosome 6. Moreover, we could easily map two novel loci involved in the culm length on the short arms of chromosomes 3 and 10.

A genome-wide view of miniature inverted-repeat transposable elements (MITEs) in rice, Oryza sativa ssp. japonica.

Transposable elements (TEs) have played important roles in the evolution of genes and genomes of higher eukaryotes. Among the TEs in the rice genome, miniature inverted-repeat transposable elements (MITEs) exist at the highest copy number. Some of MITEs in the rice genome contain poly(A) signals and putative cis-acting regulatory domains. Insertion events of such MITEs may have caused many structural and functional changes of genomes. The genome-wide examination of MITE-derived sequences could elucidate the contribution of MITEs to gene evolution. Here we report on the MITEs in the rice genome that have contributed to the emergence of novel genes and the expansion of the sequence diversity of the genome and mRNAs. Of the MITE-derived sequences, approximately 6000 were found in gene regions (exons and introns) and 67,000 in intergenic regions. In gene regions, most MITEs are located in introns rather than exons. For over 300 protein-coding genes, coding sequences, poly(A) sites, transcription start sites, and splicing sites overlap with MITEs. These sequence alterations via MITE insertions potentially affect the biological functions of gene products. Many MITE insertions also exist in 5'-untranslated regions (UTRs), 3'-UTRs, and in the proximity of genes. Although mutations in these non-protein coding regions do not alter protein sequences, these regions have key roles for gene regulation. Moreover, MITE family sequences (Tourist, Stowaway, and others) are unevenly distributed in introns. Our findings suggest that MITEs may have contributed to expansion of genome diversity by causing alterations not only in gene functions but also in regulation of many genes.