On the cover

Job’s tears growing in a field (background). Job’s tears flowers have protruding stamens and pistils (bottom of right). This indicates that they can easily cross-pollinate between cultivars (middle of right) and wild types (top of right). The wild type has several traits that make it undesirable for medicinal use, such as a hard seed coat and non-glutinous endosperm. Therefore, when growing Job’s tears, it is important to avoid crossing with wild species (This issue, p. 408–414).

(K. Matsui: NARO, Institute of Crop Science)

In this study, the mutagenic effects of different doses and exposure times of oryzalin and Nitrogen Protoxide (N₂O) were tested for stimulating polyploid on 41 B and Fercal grapevine rootstocks seedlings. Ploidy changes were examined by morphological, cytological, macroscopic, and microscopic methods. Leaf thickness, chlorophyll contents, stomatal sizes, and chloroplast numbers of polyploid seedlings stimulated with mutagens increased but their stomatal densities decreased. Flow cytometry (FC) analyses were performed on 50 samples selected by morphological and microscopic preliminary determinations. In FC analyses, 1 tetraploid seedling and 4 mixoploid seedlings from Fercal offspring and 1 mixoploid seedling from 41 B offspring were verified. The nuclear DNA content of tetraploid and mixoploid seedlings were increased by 2.00 and 1.34-fold, respectively, when compared to their diploid parents. Chromosome counts in root tip samples propagated in vitro from the tetraploid Fercal offspring confirmed a 2-fold increase compared to the diploid parent. In polyploidy induction studies, it was deemed appropriate to use FC analysis and chromosome count together to confirm the ploidy levels of mutants. Oryzalin and N₂O applications at different doses and exposure times were found to be effective for inducing polyploidy in 41 B and Fercal grapevine rootstocks.

A large vascular bundle number (VBN) in the panicle neck in rice (Oryza sativa L.) is related to the ability to transport assimilates from stem and leaf to reproductive organs during seed maturation. Several quantitative trait loci (QTLs) for VBN have been identified by using segregating populations derived from a cross between indica and japonica rice cultivars. However, the detailed location, effect, and interaction of QTLs for VBN were not understood well. Here, to elucidate the genetic basis of VBN, we identified three stable QTLs for VBN—qVBN5, qVBN6 and qVBN11—by using 71 recombinant inbred lines derived from a cross between indica ‘IR24’ and japonica ‘Asominori’. We confirmed their positions and characterized their effects by using chromosome segment substitution lines (CSSLs) with an ‘IR24’ genetic background. qVBN6 had the most substantial effect on VBN, followed by qVBN11 and qVBN5. We developed pyramided lines carrying two QTLs for VBN to estimate their interaction. The combination of qVBN6 and qVBN11 accumulated VBN negatively in the pyramided lines owing to the independent actions of each QTL. The QTLs detected for VBN will enhance our understanding of genetic mechanisms of VBN and can be used in rice breeding.

A total of four populations of reciprocal backcross recombinant inbred lines were produced from a cross between a wild accession of Oryza rufipogon W630 and two major cultivars, O. sativa Japonica Nipponbare and Indica IR36. Using these populations, quantitative trait locus (QTL) analysis for eight morphological traits (culm length, panicle length, days to heading, panicle shape, pericarp color, hull color, seed shattering and seed awning) was carried out, and the putative QTL regions were compared among the populations. The QTLs with strong allele effects were commonly detected for culm length, panicle shape, pericarp color and hull color in all four populations, and their peak locations were close to the major genes of sd1, Spr3, Rc and Bh4, respectively. For panicle length and days to heading, some QTL regions overlapped between two or three populations. In the case of seed shattering and seed awning, strong wild allele effects at major loci were observed only in the populations with cultivated backgrounds. Since the wild and cultivated alleles have never been evaluated in the reciprocal genetic backgrounds, the present results provide new information on gene effects in breeding and domestication studies.

The brown planthopper (BPH: Nilaparvata lugens Stål) is one of the most destructive insects in rice production. The use of host plant resistance has potential to reduce damage caused by BPH. The heat tolerance japonica rice ‘Sagabiyori’, with superior grain quality and high soluble starch in the stem, is highly susceptible to damage by BPH. Here, to enhance its BPH resistance, we developed seven near-isogenic lines (NILs) carrying BPH2, BPH17-ptb, BPH32, BPH3, BPH17, BPH20, and BPH21 through marker-assisted selection and evaluated resistance to two BPH populations. Most lines were more resistant to the Hadano-1966 BPH population than Sagabiyori but were less effective against the highly virulent Koshi-2013 population. Nevertheless, in antixenosis tests, Koshi-2013 settled less on all NILs than on Sagabiyori. In addition, adult mortality and the percentage of fresh weight loss of lines carrying BPH17 and BPH3 indicated that these lines have higher resistance to Koshi-2013 than Sagabiyori. Current study revealed that BPH resistance of Sagabiyori became stronger by transferring BPH3 and BPH17 genes. Thus, BPH3 and BPH17 might be valuable for breeding programs to enhance BPH resistance of high grain quality rice varieties with heat tolerance.

This study aimed to investigate the relationship between low seed set and abnormal embryo sacs lacking normal female organs, such as one egg cell, two assistant cells, and two polar nuclei, in Ipomoea trifida, which is closely related to sweet potato, and sweet potato cultivars and lines, through histological analysis of their ovaries on flowering day. Ovaries of diploid, tetraploid, and hexaploid lines of I. trifida each had four ovules, except for some hexaploid lines with five or six ovules. Almost all sweet potato cultivars and lines had four ovules per ovary, although some sib-cross lines had two or three ovules. The number of ovules per ovary did not have direct effects on low seed set. The frequency of abnormal embryo sac increased with polyploidy in I. trifida. However, it varied among different sweet potato cultivars and lines. Moreover, the variation in abnormal embryo sacs occurred at an earlier stage of gametogenesis (type A) in the tetraploid and hexaploid plants of I. trifida and sweet potato cultivars and lines. These findings suggest that the high frequency of abnormal embryo sacs is a primary cause of low seed set in sweet potato and that it is closely related to the decline in seed propagation that occurs in the evolution process of sweet potato.

Closed fertilization in flowers, or cleistogamy, reduces the risk of fungal infection in Triticeae crops. In barley (Hordeum vulgare), cleistogamy is determined by a single recessive gene, cly1, which results from a single nucleotide polymorphism within the microRNA172 target site of the Apetala2 (AP2) transcription factor gene. The recessive cly1 allele negatively regulates the development of lodicules, keeping florets closed at anthesis. However, cleistogamy is not evident in hexaploid wheat (Triticum aestivum) cultivars. This study aimed at identifying mutations in wheat AP2 orthologs by ethyl methane sulfonate–induced mutagenesis and high-resolution melt analysis. Although flowers of AP2 mutants induced in the A and D genomes opened at anthesis, their lodicule size was significantly smaller, especially in the direction of depth, than that of wild-type plants. One of the mutants that carried a nucleotide replacement in AP2 from the D genome produced a compact spike caused by a substantial decrease in rachis internode length, analogous to the barley dense spike. Cleistogamous hexaploid wheat might be generated by combining effective mutant alleles of AP2-homoeologous genes.

Job’s tears (Coix lacryma-jobi L.) is grown widely in Asian countries and a crop that can fertilize with own pollen and pistils. The grains are used not only for food but also for medicinal purposes. The grain of many cultivars contains glutinous endosperm; only grains with this glutinous endosperm are suitable for use as medicine in Japan. Many wild types have non-glutinous endosperm and can easily cross with cultivar under natural environmental conditions. Because the non-glutinous endosperm trait is dominant to that of glutinous endosperm, F₁ seeds produced by crosses between a cultivar and a wild type have non-glutinous endosperm. To reduce the rate of unwanted crosses, we investigated the pollen dispersal distance by using a red leaf sheath as a morphological marker. When plants were cultivated in rows 70 cm apart, the crossing rate was about 25%–35%. As the distance increased, the crossing rate decreased at a rate that could be fitted to a power approximation in fields without intervening plants and to an exponential equation in fields with intervening plants. Our data could be used as guidelines for preventing unwanted crossing with wild types when growing cultivars.

DNA markers are indispensable tools in genetics and genomics research as well as in crop breeding, particularly for marker-assisted selection. Recent advances in next-generation sequencing technology have made it easier to obtain genome sequences for various crop species, enabling the large-scale identification of DNA polymorphisms among varieties, which in turn has made DNA marker design more accessible. However, existing primer design software is not suitable for designing many types of genome-wide DNA markers from next-generation sequencing data. Here, we describe the development of V-primer, high-throughput software for designing insertion/deletion, cleaved amplified polymorphic sequence, and single-nucleotide polymorphism (SNP) markers. We validated the applicability of these markers in different crops. In addition, we performed multiplex PCR targeted amplicon sequencing using SNP markers designed with V-primer. Our results demonstrate that V-primer facilitates the efficient and accurate design of primers and is thus a useful tool for genetics, genomics, and crop breeding. V-primer is freely available at https://github.com/ncod3/vprimer.