Ayako Okuzaki

Relationship between hybridization frequency of Brassica juncea × B. napus and distance from pollen source (B. napus) to recipient (B. juncea) under field conditions in Japan

Several imported transgenic canola (Brassica napus) seeds have been spilled and have grown along roadsides around import ports. B. juncea, a relative of B. napus with which it has high interspecific crossability, is widely distributed throughout Japan. There is public concern about the harmful impacts of feral B. napus plants on biodiversity, but spontaneous hybridization between spilled B. napus and weedy B. juncea populations is hardly revealed. We evaluated the relationship between the hybridization frequency of B. juncea × B. napus and their planting distance in field experiments using the mutagenic herbicide-tolerant B. napus cv. Bn0861 as a pollen source for hybrid screening. The recipient B. juncea cv. Kikarashina was planted in an experimental field with Bn0861 planted in the center. No hybrids were detected under natural flowering conditions in 2009. However, the flowering period was artificially kept overlapping in 2010, leading to a hybridization frequency of 1.62% in the mixed planting area. The hybridization frequency decreased drastically with distance from the pollen source, and was lower under field conditions than estimated from the high crossability, implying that spontaneous hybridization between spilled B. napus and weedy B. juncea is unlikely in the natural environment.

Occurrence of metaxenia and false hybrids in Brassica juncea L. cv. Kikarashina × B. napus

Imported genetically modified (GM) canola (Brassica napus) is approved by Japanese law. Some GM canola varieties have been found around importation sites, and there is public concern that these may have any harmful effects on related species such as reduction of wild relatives. Because B. juncea is distributed throughout Japan and is known to be high crossability with B. napus, it is assumed to be a recipient of B. napus. However, there are few reports for introgression of cross-combination in B. juncea × B. napus. To assess crossability, we artificially pollinated B. juncea with B. napus. After harvesting a large number of progeny seeds, we observed false hybrids and metaxenia of seed coats. Seed coat color was classified into four categories and false hybrids were confirmed by morphological characteristics and random amplified polymorphic DNA (RAPD) markers. Furthermore, the occurrence of false hybrids was affected by varietal differences in B. napus, whereas that of metaxenia was related to hybridity. Therefore, we suggest that metaxenia can be used as a marker for hybrid identification in B. juncea L. cv. Kikarashina × B. napus. Our results suggest that hybrid productivity in B. juncea × B. napus should not be evaluated by only seed productivity, crossability ought to be assessed the detection of true hybrids.

The Non-Mendelian Green Cotyledon Gene in Soybean Encodes a Small Subunit of Photosystem II

Molecular cloning of a cytoplasmic stay-green mutant gene revealed that a small subunit of PSII is involved in chlorophyll b degradation. Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.

CRISPR/Cas9-mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated genome editing system has been widely applied as a powerful tool for modifying preferable endogenous genes. This system is highly expected to be further applied for the breeding of various agronomically important plant species. Here we report the modification of a fatty acid desaturase 2 gene (FAD2), which encodes an enzyme that catalyzes the desaturation of oleic acid, in Brassica napus cv. Westar using the CRISPR/Cas9 system. Two guide RNAs were designed for BnaA.FAD2.a (FAD2_Aa). Of 22 regenerated shoots with FAD2_Aa editing vectors, three contained mutant alleles. Further analysis revealed that two of three mature plants (Aa1#13 and Aa2#2) contained the mutant alleles. The mutant fad2_Aa allele had a 4-bp deletion, which was inherited by backcross progenies (BC1) in the Aa1#13 line. Furthermore, plants with the fad2_Aa allele without transgenes were selected from the BC1 progenies and plants homozygous for fad2_Aa were then produced by self-crossing these BC1 progenies (BC1S1). Fatty acid composition analysis of their seeds revealed a statistically significant increase in the content of oleic acid compared with that in wild-type seeds. These results showed that the application of the CRISPR/Cas9 system is useful to produce desirable mutant plants with an agronomically suitable phenotype by modifying the metabolic pathway in B. napus.

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer’s fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F1 hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC1 obtained from B. juncea × F1 hybrid, and this BC1 exhibited a plant type like that of B. juncea. Most markers were deleted in BC2 and BC3 plants, with only two markers persisting in the BC3. These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.

Development and characterization of transgenic dominant male sterile rice toward an outcross-based breeding system

Genomic selection is attracting attention in the field of crop breeding. To apply genomic selection effectively for autogamous (self-pollinating) crops, an efficient outcross system is desired. Since dominant male sterility is a powerful tool for easy and successive outcross of autogamous crops, we developed transgenic dominant male sterile rice (Oryza sativa L.) using the barnase gene that is expressed by the tapetum-specific promoter BoA9. Barnase-induced male sterile rice No. 10 (BMS10) was selected for its stable male sterility and normal growth characteristics. The BMS10 flowering habits, including heading date, flowering date, and daily flowering time of BMS10 tended to be delayed compared to wild type. When BMS10 and wild type were placed side-by-side and crossed under an open-pollinating condition, the seed-setting rate was <1.5%. When the clipping method was used to avoid the influence of late flowering habits, the seed-setting rate of BMS10 increased to a maximum of 86.4%. Although flowering synchronicity should be improved to increase the seed-setting rate, our results showed that this system can produce stable transgenic male sterility with normal female fertility in rice. The transgenic male sterile rice would promote a genomic selection-based breeding system in rice.