Production and characterization of intergeneric hybrids between Brassica oleracea and a wild relative Moricandia arvensis

Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F₁ hybrid plants were generated in the cross‐combination of B. oleracea × M. arvensis from 64 pods by the placenta‐embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n = 23 chromosomes in mitosis and a meiotic chromosome association of (0–3)II + (17–23)I at metaphase I (M I). In the backcross with B. oleracea, some of these hybrids developed sesquidiploid BC₁ plants with 2n = 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC₂ plants to B. oleracea generated 48 BC₃ progeny with somatic chromosomes from 2n = 19 to 2n = 41. The 2n = 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9¹/₂ + 9¹/₂) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.     

Production of high yield short duration Brassica napus by interspecific hybridization between B. oleracea and B. rapa

Brassica napus is a leading oilseed crop throughout many parts of the world. It is well adapted to long day photoperiods, however, it does not adapt well to short day subtropical regions. Short duration B. napus plants were resynthesized through ovary culture from interspecific crosses in which B. rapa cultivars were reciprocally crossed with B. oleracea. From five different combinations, 17 hybrid plants were obtained in both directions. By self-pollinating the F₁ hybrids or introgressing them with cultivated B. napus, resynthesized (RS) F₃ and semi-resynthesized (SRS) F₂ generations were produced, respectively. In field trial in Bangladesh, the RS B. napus plants demonstrated variation in days to first flowering ranging from 29 to 73 days; some of which were similar to cultivated short duration B. napus, but not cultivated short duration B. rapa. The RS and SRS B. napus lines produced 2–4.6 and 1.6–3.7 times higher yields, respectively, as compared to cultivated short duration B. napus. Our developed RS lines may be useful for rapeseed breeding not only for subtropical regions, but also for areas such as Canada and Europe where spring rapeseed production can suffer from late spring frosts. Yield and earliness in RS lines are discussed.     

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.     

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.)

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. ‘Akiriso’, a clubroot-resistant F₁ cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in ‘Akiriso’. CR in ‘Akiriso’ segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar ‘CR Shinki’. We developed additional markers around CRb and constructed partial genetic maps of this region in ‘Akiriso’ and ‘CR Shinki’. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F₂ populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in ‘Akiriso’ is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.     

Modes of inheritance of two apomixis components,diplospory and parthenogenesis,in Chinese chive (Allium ramosum) revealed by analysis of the segregating population generated by back-crossing between amphimictic and apomictic diploids

To investigate the mode of inheritance of apomixis in Chinese chive, the degrees of diplospory and parthenogenesis were evaluated in F₁ and BC₁ progenies derived from crosses between amphimictic and apomictic diploids (2n = 16, 2x). The F₁ population was generated by crossing three amphimictic diploids 94Mo13, 94Mo49 and 94Mo50 with an apomictic diploid KaD2 and comprised 110 diploids and 773 triploids. All the diploid F₁ plants examined were completely or highly eusporous and completely syngamic. All the triploid F₁ plants examined were highly diplosporous and highly parthenogenetic. KaD2 could not transmit its high level of apomixis via monoploid pollen grains. The BC₁ population, generated by crossing 94Mo49 with apomictic triploids found in the F₁ offspring, exhibited heteroploidy; it comprised haploid, diploid, triploid, tetraploid and various aneuploid individuals. In this generation, clear segregation was observed between diplospory and parthenogenesis. Analysis of the BC₁ population suggests that diplospory and parthenogenesis are each controlled by single dominant genes, D and P, respectively. However, all the BC₁ plants characterized as parthenogenetic were diplosporous. The absence of phenotypically eusporous parthenogenetic plants can be explained by assuming that the presence of diplospory gene is a prerequisite for the parthenogenesis gene expression in Chinese chive.     

Major QTLs associated with green stem disorder insensitivity of soybean (Glycine max (L.) Merr.)

Green stem disorder (GSD) is one of the most serious syndromes affecting soybean (Glycine max) cultivation in Japan. In GSD, stems remain green even when pods mature. When soybean plants develop GSD, seed surfaces are soiled by tissue fluid and seed quality is deteriorated during machine harvesting. We performed quantitative trait locus (QTL) analyses for GSD insensitivity using recombinant inbred lines (RILs; n = 154) derived from a cross between an insensitive line (‘Touhoku 129’) and a sensitive leading cultivar (‘Tachinagaha’) during a 6-year evaluation. Three effective QTLs were detected. The influences of these QTLs were in the following order: qGSD1 (LG_H) > qGSD2 (LG_F) > qGSD3 (LG_L). At these three QTLs, ‘Touhoku 129’ genotypes exhibited more GSD insensitivity than ‘Tachinagaha’ genotypes. The lower incidence of GSD for ‘Touhoku129’ was attributable primarily to these three QTLs because RILs harboring a ‘Touhoku 129’ genotype at the three QTLs exhibited a GSD incidence similar to that of ‘Touhoku 129.’ Although a limitation of this study is that only one mapping population was evaluated, this QTL information and the flanking markers of these QTLs would be effective tools for resolving GSD in soybean breeding programs.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Development and validation of DNA markers linked to Sdvy-1, a common bean gene conferring resistance to the yellowing strain of Soybean dwarf virus

The yellowing strain of Soybean dwarf virus (SbDV-YS) causes yellowing and yield loss in common bean (Phaseolus vulgaris). The most effective control is achieved through breeding for resistance. An indeterminate climbing cultivar with a white seed coat, ‘Oofuku’, is resistant to SbDV-YS in inoculation tests. We crossed ‘Oofuku’ with an elite cultivar, ‘Taisho-Kintoki’, which is SbDV-YS-susceptible, determinate dwarf with a red-purple seed coat, and performed amplified-fragment-length polymorphism analysis of F₃ lines. From nucleotide sequences of the resistant-specific fragments and their flanking regions, we developed five DNA markers, of which DV86, DV386, and DV398 were closely linked to Sdvy-1, a resistance gene. Using the markers, we developed ‘Toiku-B79’ and ‘Toiku-B80’, the near-isogenic lines (NILs) incorporating Sdvy-1 in the background of ‘Taisho-Kintoki’. The NILs had similar growth habit, maturity date and seed shape to those of ‘Taisho-Kintoki’. The quality of boiled beans was also similar, except that the NILs had more seed coat cracking than ‘Taisho-Kintoki’. The NILs showed no SbDV-YS infection in inoculation tests. We suggest that Sdvy-1 is a useful source of SbDV-YS resistance in common bean.     

Analyses of phenotype and ARGOS and ASY1 expression in a ploidy Chinese cabbage series derived from one haploid

The aim of this research was to improve our understanding of how ploidy level influences phenotype and gene expression in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Haploid plants (2n = 10) was induced by 0.2% colchicine to produce diploid (2n = 20) and tetraploid plants (2n = 40). The aneuploid (2n = 24) was also obtained by hybridization between diploid plants as the female and tetraploid plants. The ploidy levels of all plants were identified through chromosome counts and flow cytometry. Leaves and petals became larger as the ploidy level increased from haploid to diploid, and from aneuploid to tetraploid. Similarly, expression of ARGOS was regulated by genome size, increasing in parallel with the level of ploidy. Among the four ploidy types, expression was stronger in the floral buds than in the leaves. Expression by ASY1 also differed according to ploidy level, being highest in diploid plants, followed in order by tetraploids. Expression was similar between haploids and aneuploids at two stages—prior to and after meiosis—but was higher in the haploids during meiosis. When buds were compared within the same ploidy type at different stages, ASY1 expression was obviously higher during meiosis than either before or after. Our study demonstrated the generation and phenotype of a ploidy Chinese cabbage series derived from one haploid. Expression of genes ARGOS and ASY1 were modulated by genome size in this ploidy series, and the regulated patterns of the two genes was different.     

Differential resistances to anthracnose in Capsicum baccatum as responding to two Colletotrichum pathotypes and inoculation methods

Chili anthracnose, caused by Colletotrichum spp., is one of the major diseases to chili production in the tropics and subtropics worldwide. Breeding for durable anthracnose resistance requires a good understanding of the resistance mechanisms to different pathotypes and inoculation methods. This study aimed to investigate the inheritances of differential resistances as responding to two different Colletotrichum pathotypes, PCa2 and PCa3 and as by two different inoculation methods, microinjection (MI) and high pressure spray (HP). Detached ripe fruit of Capsicum baccatum ‘PBC80’ derived F₂ and BC₁s populations was assessed for anthracnose resistance. Two dominant genes were identified responsible for the differential resistance to anthracnose. One was responsible for the resistance to PCa2 and PCa3 by MI and the other was responsible for the resistance to PCa3 by HP. The two genes were linked with 16.7 cM distance.     

Identification of qRL7, a major quantitative trait locus associated with rice root length in hydroponic conditions

Root system development is an important target for improving yield in rice. Active roots that can take up nutrients more efficiently are essential for improving grain yield. In this study, we performed quantitative trait locus (QTL) analyses using 215 recombinant inbred lines derived from a cross between Xieqingzao B (XB), a maintainer line with short roots and R9308, a restorer line with long roots. Only a QTLs associated with root length were mapped on chromosomes 7. The QTL, named qRL7, was located between markers RM3859 and RM214 on chromosome 7 and explained 18.14–18.36% of the total phenotypic variance evaluated across two years. Fine mapping of qRL7 using eight BC₃F₃ recombinant lines mapped the QTL to between markers InDel11 and InDel17, which delimit a 657.35 kb interval in the reference cultivar Nipponbare. To determine the genotype classes for the target QTL in these BC₃F₃ recombinants, the root lengths of their BC₃F₄ progeny were investigated, and the result showed that qRL7 plays a crucial role in root length. The results of this study will increase our understanding of the genetic factors controlling root architecture, which will help rice breeders to breed varieties with deep, strong and vigorous root systems.     

Transfer of powdery mildew resistance from Brassica carinata to Brassica oleracea through embryo rescue

Interspecific hybrid plants and backcross 1 (BC₁) progeny were produced through sexual crosses and embryo rescue between Brassica carinata accession PI 360883 and B. oleracea cvs Titleist’and‘Cecile’to transfer resistance to powdery mildew to B. oleracea. Four interspecific hybrids were obtained through application of embryo rescue from crosses with B. carinata as the maternal parent, and their interspecific nature confirmed through plant morphology and random amplified polymorphic DNA (RAPD) analysis. Twenty‐one BC₁ plants were obtained through sexual crosses and embryo rescue although embryo rescue was not necessary to produce first backcross generation plants between interspecific hybrids and B. oleracea. All interspecific hybrids and eight of the BC₁ plants were resistant to powdery mildew.     

Evaluation of the effects of five QTL regions on Fusarium head blight resistance and agronomic traits in spring wheat (Triticum aestivum L.)

Fusarium head blight (FHB) is an important disease of wheat (Triticum aestivum L.). The aim of this study was to determine the effects of quantitative trait locus (QTL) regions for resistance to FHB and estimate their effects on reducing FHB damage to wheat in Hokkaido, northern Japan. We examined 233 F₁-derived doubled-haploid (DH) lines from a cross between ‘Kukeiharu 14’ and ‘Sumai 3’ to determine their reaction to FHB during two seasons under field conditions. The DH lines were genotyped at five known FHB-resistance QTL regions (on chromosomes 3BS, 5AS, 6BS, 2DL and 4BS) by using SSR markers. ‘Sumai 3’ alleles at the QTLs at 3BS and 5AS effectively reduced FHB damage in the environment of Hokkaido, indicating that these QTLs will be useful for breeding spring wheat cultivars suitable for Hokkaido. Some of the QTL regions influenced agronomic traits: ‘Sumai 3’ alleles at the 4BS and 5AS QTLs significantly increased stem length and spike length, that at the 2DL QTL significantly decreased grain weight, and that at the 6BS QTL significantly delayed heading, indicating pleiotropic or linkage effects between these agronomic traits and FHB resistance.     

Genetic dissection of black grain rice by the development of a near isogenic line

Rice (Oryza sativa L.) can produce black grains as well as white. In black rice, the pericarp of the grain accumulates anthocyanin, which has antioxidant activity and is beneficial to human health. We developed a black rice introgression line in the genetic background of Oryza sativa L. ‘Koshihikari’, which is a leading variety in Japan. We used Oryza sativa L. ‘Hong Xie Nuo’ as the donor parent and backcrossed with ‘Koshihikari’ four times, resulting in a near isogenic line (NIL) for black grains. A whole genome survey of the introgression line using DNA markers suggested that three regions, on chromosomes 1, 3 and 4 are associated with black pigmentation. The locus on chromosome 3 has not been identified previously. A mapping analysis with 546 F₂ plants derived from a cross between the black rice NIL and ‘Koshihikari’ was evaluated. The results indicated that all three loci are essential for black pigmentation. We named these loci Kala1, Kala3 and Kala4. The black rice NIL was evaluated for eating quality and general agronomic traits. The eating quality was greatly superior to that of ‘Okunomurasaki’, an existing black rice variety. The isogenicity of the black rice NIL to ‘Koshihikari’ was very high.     

Confirming a major QTL and finding additional loci responsible for field resistance to brown spot (Bipolaris oryzae) in rice

Brown spot is a devastating rice disease. Quantitative resistance has been observed in local varieties (e.g., ‘Tadukan’), but no economically useful resistant variety has been bred. Using quantitative trait locus (QTL) analysis of recombinant inbred lines (RILs) from ‘Tadukan’ (resistant) × ‘Hinohikari’ (susceptible), we previously found three QTLs (qBS2, qBS9, and qBS11) that conferred resistance in seedlings in a greenhouse. To confirm their effect, the parents and later generations of RILs were transplanted into paddy fields where brown spot severely occurred. Three new resistance QTLs (qBSfR1, qBSfR4, and qBSfR11) were detected on chromosomes 1, 4, and 11, respectively. The ‘Tadukan’ alleles at qBSfR1 and qBSfR11 and the ‘Hinohikari’ allele at qBSfR4 increased resistance. The major QTL qBSfR11 coincided with qBS11 from the previous study, whereas qBSfR1 and qBSfR4 were new but neither qBS2 nor qBS9 were detected. To verify the qBSfR1 and qBSfR11 ‘Tadukan’ resistance alleles, near-isogenic lines (NILs) with one or both QTLs in a susceptible background (‘Koshihikari’) were evaluated under field conditions. NILs with qBSfR11 acquired significant field resistance; those with qBSfR1 did not. This confirms the effectiveness of qBSfR11. Genetic markers flanking qBSfR11 will be powerful tools for marker-assisted selection to improve brown spot resistance.     

Study on lily introgression breeding using allotriploids as maternal parents in interploid hybridizations

Based on a recent hypothesis, “Five same genomes of endosperm are essential for its development in Lilium”, it is expected that allotriploid lily (OTO) can be hybridized with diploid Oriental lily (OO) for introgression breeding in Lilium L.. To test the hypothesis, OTO lilies, ‘Belladonna’, ‘Candy Club’ and ‘Travatore’, were used as the maternal parents and crossed with two diploid OO cultivars, ‘Siberia’ and ‘Sorbonne’, and the species L. regale Wilson (TT). Results showed that capsules of all OTO × OO hybridizations developed well and 0.8~3.3 viable seedlings per ovary were obtained through normal pollination and embryo rescue; however, all OTO × TT crosses failed. Genomic in situ hybridization showed that the progenies of the OTO × OO hybridizations were aneuploid and a variable number of T-genome chromosomes were introduced into the progenies through the allotriploid lilies. The present results not only demonstrate that allotriploid OTO lilies, although male sterile, can be used as maternal parents to produce aneuploid progenies, but also strongly support the new hypothesis in lily breeding.     

Male sterility in alloplasmic Brassica rapa L. carrying Eruca sativa cytoplasm

For the development of a new cytoplasmic male sterility (CMS) system in Brassica rapa (2n= 20, AA), intergeneric hybridization was performed in Eruca sativa (2n= 22, EE)×B. rapa. The original amphihaploid F₁ plant (2n= 21, EA) generated via embryo rescue produced a sesquidiploid F₁ plant (2n= 31, EAA), from which the alloplasmic F₃ plants were generated. In F₃, some progenies with malformed anthers were maintained as male-sterile lines up to the F5 generation. In the F₆ and F₇ generations, the alloplasmic male-sterile plants were bred by backcrossing to several B. rapa genotypes and were then classified into the three distinctive types: petaloid, antherless and brown anther, in addition to three intermediate types between them. By southern blot analysis, each plant of the petaloid and antherless types was shown to carry the cytoplasm genome of E. sativa. These male-sterile plants produced as many viable seeds as the corresponding male-fertile plants, although their nectary gland development was minimal. Consequently, CMS lines of the petaloid and antherless types with enhanced seed fertility and nectary gland development could provide promising plant materials for F₁ seed production in B. rapa.     

N2O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies

Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N₂O) gas to obtain 2n male gametes. N₂O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N₂O treatment restores fertility in sterile hybrids. To establish optimal N₂O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N₂O gas at different anther developmental stages from fertile and sterile hybrid lilies. N₂O treatment using the optimal 1–4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N₂O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC₁ plants. Thus N₂O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.     

Intergeneric crosses for the transfer of resistance to the beet cyst nematode from Raphanus sativus to Brassica napus

Summary The possibilities to transfer important traits and in particular resistance to the beet cyst nematode (Heterodera schachtii, abbrev. BCN) from Raphanus sativus to Brassica napus were investigated. For these studies B. napus, R. sativus, the bridging hybrid ×Brassicoraphanus (Raparadish) as well as offspring of the cross ×Brassicoraphanus (Raparadish) ×B. napus were used. Reciprocal crosses between B. napus and R. sativus were unsuccessful, also with the use of embryo rescue. Crosses between ×Brassicoraphanus as female parent and B. napus resulted in a large number of F₁ hybrids, whereas the reciprocal cross yielded mainly matromorphic plants. BC₁, BC₂ and BC₃ plants were obtained from backcrosses with B. napus, which was used as the male parent. F₁ hybrids and BC plants showed a large variation for morphology and male and female fertility. Cuttings of some F₁ and BC₁ plants, obtained from crosses involving resistant plants of ×Brassicoraphanus, were found to possess a level of resistance similar to that of the resistant parent. These results and indications for meiotic pairing between chromosomes of genome R with those of the genomes A and/or C suggest that introgression of the BCN-resistance of Raphanus into B. napus may be achieved.     

Interspecific cross of Brassica oleracea var. alboglabra and B. napus : effects of growth condition and silique age on the efficiency of hybrid production, and inheritance of erucic acid in the self-pollinated backcross generation

Interspecific hybrids were produced from reciprocal crosses between Brassica napus (2n = 38, AACC) and B. oleracea var. alboglabra (2n = 18, CC) to introgress the zero-erucic acid alleles from B. napus into B. oleracea. The ovule culture embryo rescue technique was applied for production of F₁ plants. The effects of silique age, as measured by days after pollination (DAP), and growth condition (temperature) on the efficiency of this technique was investigated. The greatest numbers of hybrids per pollination were produced under 20°/15°C (day/night) at 16 DAP for B. oleracea (♀) × B. napus crosses, while under 15°/10°C at 14 DAP for B. napus (♀) × B. oleracea crosses. Application of the ovule culture technique also increased the efficiency of BC₁ (F₁ × B. oleracea) hybrid production by 10-fold over in vivo seed set. The segregation of erucic acid alleles in the self-pollinated backcross generation, i.e. in BC₁S₁ seeds, revealed that the gametes of the F₁ and BC₁ plants carrying a greater number of A-genome chromosomes were more viable. This resulted in a significantly greater number of intermediate and a smaller number of high-erucic acid BC₁S₁ seeds.