Sexual hybridisation in crosses of cultivated Brassica species with the crucifers Erucastrum gallicum and Raphanus raphanistrum: Potential for gene introgression

Studies were conducted to investigate the crossability of the cultivated Brassica species, Brassica napus (oilseed rape), B. rapa (turnip rape), and B. juncea (brown and oriental mustard), with two related cruciferous weeds that are abundant in certain regions of Canada, Erucastrum gallicum (dog mustard) and Raphanus raphanistrum ssp. raphanistrum (wild radish). Seed was produced without recourse to embryo rescue from all reciprocal crosses except R. raphanistrum × B. juncea. Four hybrid plants were recovered, namely B. napus × E. gallicum, B. napus × R. raphanistrum (two plants), and B. rapa × E. gallicum. The hybrids were characterized by their morphology, RAPD analysis, and cytological examination. The B. rapa × E. gallicum hybrid was extremely vigourous and fertile, and would likely grow in natural habitats. This hybrid produced self-seed and backcrossed readily with the B. rapa parent and, to a lesser extent, with the E. gallicum parent. The B. napus × E. gallicum hybrid was a weak plant, but produced fertile backcross progeny with the E. gallicum parent. The B. napus × R. raphanistrum hybrids were vigourous but mostly sterile. Because of their low vigour and/or sterility, hybrids produced from crosses of B. napus with the cruciferous weeds would not likely be an environmental concern. However, the potential of the B. napus × E. gallicum and B. rapa × E. gallicum hybrids to backcross with E. gallicum may be of concern. This revised version was published online in July 2006 with corrections to the Cover Date.     

Different recombination frequencies in wheat doubled haploid populations obtained through maize pollination and anther culture

This study compared the meiotic recombination frequency between wheat doubled haploid (DH) populations obtained through two different methods, maize pollination (MP♀) and anther culture (AC♂). The comparison was based on a genetic linkage analysis, performed with DNA markers. Thirty-five polymorphic markers (15 SSR, 15 AFLP, 5 RAPD) were screened in MP♀ and AC♂ doubled haploids populations, derived from the same hybrid genotype (F₁ of ‘Eta’ × ‘Darkhan 15’). Nine linkage groups, comprising 35 loci (the MP♀ lines) and 31 loci (the AC♂ lines), were constructed. The linkage groups in both DH populations showed identical orders of markers, except for one group mapping to chromosome 6B. The MP♀ and AC♂ linkage maps differed significantly in recombination frequencies for corresponding intervals. In total, the AC♂ linkage map (495.5 cM) was 40.5% longer than the MP♀ map (352.8 cM), indicating a significantly higher meiotic recombination rate in pollen mother cells. The enhancement in recombination was visible in five of nine linkage groups, and in 7 intervals between individual loci out of 19 compared. Moreover, for 6 other intervals a lack of linkage was observed in the AC♂ population, as compared to the MP♀ map.     

Interspecific hybridization of Brassica napus and B. juncea through ovary,ovule and embryo culture

Summary Employing in vitro culture of ovaries, ovules and embryos, interspecific hybrids have been obtained amongst two important oilseed crops, Brassica napus x B. juncea and their reciprocal. The test-tube hybrid plants have been transferred to the field, and reared to maturity. The F₁ seeds obtained from the hybrid ovaries showed normal germination, and the hybrid plants exhibited a range of variation of characters.     

Molecular characterization of novel resynthesized rapeseed (Brassica napus) lines and analysis of their genetic diversity in comparison with spring rapeseed cultivars*

Resynthesized (RS) rapeseed generated from interspecific hybridization between suitable forms of Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and B. oleracea L. (CC, 2n = 18) represents a potentially important resource to expand genetic diversity in the narrow gene pool of oilseed rape (B. napus L., AACC, 2n = 38). In this study 165 RS rapeseed lines originating from crosses between an Indian Yellow Sarson (B. rapa ssp. trilocularis) and five different cauliflower (B. oleracea convar. botrytis) cultivars were studied using amplified fragment length polymorphism (AFLP) markers and their genetic diversity was compared in relationship to an assortment of 40 diverse spring oilseed and fodder rape varieties. Using three AFLP primer combinations, a total of 467 polymorphic bands were scored. Cluster analysis allowed differentiation among the different RS lines, which, as expected, were genetically highly divergent from the cultivars. The genetic diversity of the material is discussed in relation to its morphological variability with a view to the implementation of RS lines in oilseed rape breeding.     

十字花科芸薹属种间杂种营养优势的利用研究

选用4个油菜雄性不育系和三种类型十字花科芸薹属种, 研究了不同杂交组合的亲和性, 杂种一代的营养优势表现和杂种营养优势的生理基础以及品质性状表现. 结果表明: 利用油菜雄性不育系与其它十字花科芸薹属种间杂交可以显著提高种间杂交的亲和性, 并具有细胞质遗传效应. 通过杂交制种可以获得大量的杂种应用于生产. 十字花科     

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.     

不同基因型油菜锌效率机理Ⅰ ——耐低锌油菜基因型苗期筛选指标研究

采用营养液培养试验比较了低锌营养下芥菜、白菜、甘蓝型3个油菜(Brassica napus L.)品种幼苗的生物量，抗氧化酶活性，根系活力，叶绿素含量及对锌的吸收、利用差异。试验结果表明，3个油菜品种由于基因型不同，在耐低锌营养能力方面存在显著性差异。在低锌时(10-6mol/L Zn2+)，幼苗植株地上部干重以芥菜型>甘蓝型>白菜型，地上部干重分别较无锌对照处理增加17.8%(芥菜型)、9.9%(白菜型)、3.3%(甘蓝型)。芥菜型叶CAT、POD、SOD活性，叶绿素含量及根系活力也明显高于甘蓝型和白菜型油菜。锌积累量及锌利用率也以芥菜型远远大于其它2个品种，锌素利用效率分别为12.3%（芥菜型），8.0%(白菜型)，4.9% (甘蓝型)。苗期油菜叶CAT、POD、SOD等抗氧化酶活性、叶绿素含量及根系活力可以作为筛选耐低锌营养油菜基因型的评价指标之一。     

Production of intergeneric hybrids between Brassica and Sinapis species by means of embryo rescue techniques

Reciprocal crosses were made to produce intergeneric hybrids among seven species of Brassica and three species of Sinapis with the aid of embryo rescue techniques. Ovule culture showed better responses in terms of hybrid plant development. Altogether, hybrids from 8 different combinations were obtained from the crosses between B. campestris var. trilocularis × S. turgida, B. campestris var. pekinensis × S. arvensis, S. arvensis × B. campestris var. pekinensis, B. oleracea var. alboglabra × S. alba, B. oleracea var. alboglabra × S. turgida, B. carinata × S. alba, B. carinata × S. arvensis, and B. carinata × S. turgida. Reciprocal crosses yielded no hybrid except in the combination of S. arvensis × B. campestris var. pekinensis. Among them hybrids from 6 combinations were established in the glass house. The hybridity of the plants was confirmed by morphological characters, pollen stainability, chromosome number and by isozyme analysis. Hybrids of four combinations out of six turned out as true hybrids, one as sesquidiploids and the another one as false hybrid plant. This revised version was published online in August 2006 with corrections to the Cover Date.     

化学杂交剂EXP对油菜的杀雄效果

化学杂交剂EXP的主要成分是一种磺酰脲类化合物,作用靶标是植物的乙酰乳酸合成酶（acetolactate synthase,ALS）,通过抑制酶活性,阻止支链氨基酸缬氨酸、亮氨酸和异亮氨酸的合成。在甘蓝型油菜自交系82089抽薹15～20 cm高,最大花蕾长度1～2 mm时期,单株用药量12～15 mL,用0.05 μg/mL、0.25 μg/mL EXP溶液处理可分别诱发92%和97%不育率。而0.25 μg/mL浓度处理不育株的育性较为稳定,自交结实率为6.6%,可以作为最佳用药浓度的参考值。EXP透导的不育株叶色变浅、株高降低、花蕾密集、花期推迟、花瓣褪色、花药干瘪无花粉。检测结果表明,不育株花蕾叶绿素和类胡萝卜素含量有所减少,处理雄蕊的过氧化物酶和酯酶活性降低,而水溶性蛋白质含量显著减少。同一时期用EXP处理芥菜型油菜和白菜型油菜也可诱导雄性不育,但芥菜型油菜不育持续时段较短,白菜型油菜结实性不好,不宜用于生产杂交种。本研究结果的意义在于,磺酰脲类ALS抑制剂种类非常多,有可能从中筛选到更高效、更持久的化学杂交剂。     

Different Behavior of Brassica juncea and B. carinata as Sources of Phoma lingam Resistance in Experiments of Interspecific Transfer to B. napus

With the aim to transfer Phoma lingam resistance into rape, successful interspecific crosses were made between three oilseed rape varieties (Brassica napus) and the resistant species B. carinata and B. carinata. Although both hybrid types B. napus×B. juncea and B. napus×B. carinata showed the same high level of resistance as the respective resistant parent, the resistance could be only transferred from juncea crosses. After three backcross generations, lines morphologically undistinguishable from rape, fertile, and with a high degree of resistance were obtained. The resistance of B. carinata was practically lost in the first backcross. A possible explanation of this different behavior could be a higher recombination between the genomes B and C (juncea crosses) than between B and A (carinata crosses). The: applied embryo culture increased the yield of hybrids and first backcross plants and reduced considerably the generation time.     

十字花科黑腐病菌hrpW基因导入油菜之研究初报

为了研究十字花科植物黑腐病菌（Xanthomonas campestris pathovar campestris,简称Xcc）hrpW基因的功能及Xcc的致病机理,实验以甘蓝型油菜为试验材料,以5~6d无菌苗的带柄子叶为外植体,建立了高效稳定的带柄子叶再生体系,同时探索了影响根癌农杆菌介导转化甘蓝型油菜子叶柄的各种因素;并用农杆菌介导法将hrpW基因导入甘蓝型油菜中,经PCR检测法分析,证明hrpW基因已整合到油菜基因组中。hrpW基因编码HarpinXcc（十字花科植物黑腐病菌Harpin蛋白）,该转基因植株的获得,为进一步研究十字花科植物黑腐病菌的致病机理提供了材料,也为选育高效抗病油菜种质奠定了基础。     

Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B. campestris)

A simple and reliable method for evaluating the viability of Brassica pollen was established in which the in vitro germination rate of pollen was adopted as the index of the viability of pollen grains. Pollen grains were preincubated in an atmosphere in which the relative humidity (RH) was fixed to 52% or 66% at 20 °C for 5 hours. They were cultured for 16 hours at 25 °C in a liquid Kwack's medium (1964) supplemented with 20% sucrose, and the pH was adjusted to 8.0. They were then observed under a microscope and the number of germinating and unchanged pollen grains were counted. The germination rate of pollen was improved and stabilized by preincubation and the use of a high pH medium. More than 90% of the freshly harvested pollen grains of Brassica rapa (syn. B. campestris) germinated constantly in these conditions Undehisced anthers were collected from flowers at anthesis and dehydrated by incubation at 20 °C for 16–24 hours in an atmosphere where the RH was fixed to 15% or 32%. They were put into a plastic vial and preserved in a freezer at -20 °C. The germination percentage of the preserved pollen was scored at intervals during preservation. The germination rate of the pollen grains preserved at -20°C for 1 year was higher than 50% and the pollen proved to be efficient for seed set. Most of the seeds germinated normally. This revised version was published online in August 2006 with corrections to the Cover Date.     

油菜及其近缘种种子萌发期耐盐碱性差异

为了明确甘蓝型油菜、白菜型油菜、芥菜型油菜、白芥和芸芥种子萌发期的耐盐碱性差异,分别采用0（CK）、0.4%、0.6%、0.8%、1.0%、1.2%和1.5%的NaCl溶液和0（CK）、0.1%、0.2%、0.3%、0.4%、0.5%和0.6%的NaHCO₃溶液进行萌发期胁迫处理,测定了发芽势、发芽率和发芽指数的变化规律。结果表明：不同浓度NaCl溶液处理,油菜及其近缘植物的平均发芽率依次为芥菜型油菜（71.7%）>甘蓝型油菜（64.0%）>白芥（62.4%）>白菜型油菜（48.4%）>芸芥（30.6%）。当NaCl的浓度为1.0%时,芥菜型油菜的发芽率最高,为91%,芸芥最低,仅有2%。不同浓度NaHCO₃溶液胁迫下,5种植物的平均发芽率依次为白芥（85.3%）>甘蓝型油菜（84.3%）>白菜型油菜（59.1%）>芸芥（42.9%）>芥菜型油菜（34.3%）。当NaHCO₃溶液浓度为0.4%时,白芥的发芽率最高,为95%,而芥菜型油菜最低,为0。综上,芥菜型油菜对NaCl胁迫的耐性最强,白菜型油菜和芸芥对NaCl胁迫的耐性差;白芥和甘蓝型油菜对NaHCO₃胁迫的耐性强,而芥菜型油菜对NaHCO₃胁迫的耐性较差。可见,在油菜种植区改良盐渍化土壤,中性盐渍化土壤最好选用芥菜型油菜,偏碱性盐渍化土壤可以选用白芥或甘蓝型油菜。     

Resynthesis of Brassica napus L. for self-incompatibility: self-incompatibility reaction, inheritance and breeding potential

Self-incompatibility (SI) in Brassica has been considered as a pollination control mechanism for commercial hybrid seed production, and so far has been extensively used in vegetable types of Brassicas. Oilseed rape Brassica napus (AACC) is naturally self-compatible in contrast to its parental species that are generally self-incompatible. Introduction of S-alleles from its parental species into oilseed rape is therefore needed to use this pollination control mechanism in commercial hybrid seed production. Self-incompatible lines of B. napus , carrying SI alleles in both A and C genomes, were resynthesized from self-incompatible B. oleracea var. italica (CC) cv.'Green Duke' and self-incompatible B. rapa ssp. oleifera (AA) cv. 'Horizon', 'Colt' and 'AC Parkland'. All resynthesized B. napus lines exhibited strong dominant SI phenotype. Reciprocal cross-compatibility was found between some of these self-incompatible lines. The inheritance of S-alleles in these resynthesized B. napus was digenic confirming that each of the parental genomes contributed one S-locus in the resynthesized B. napus lines. However, the presence of two S-loci in the two genomes was found not to be essential for imparting a strong SI phenotype. Possible use of these dominant self-incompatible resynthesized B. napus lines in hybrid breeding is discussed.     

甘蓝型油菜抗菌核病研究进展

油菜是主要油料作物之一,由核盘菌(Sclerotiniasclerotiorum(Lib.)deBary)引起的菌核病(sclerotiniastemrot)是油菜的主要病害。针对当前与菌核病相关研究的新进展,本研究从4个方面对其进行了概括：（1）核盘菌的侵染方式以及在侵染过程中核盘菌分泌的草酸与寄主中钙离子的动态关系;（2）油菜通过合成植保素、酚类化合物、木质素、几丁质酶和β-1,3-葡聚糖酶等来抵抗核盘菌入侵的抗病机理;（3）现有综合防治技术中抗性种质筛选、无花瓣育种和生物防治;（4）利用与草酸代谢相关基因、抗病相关基因、防御相关转录因子基因和抗菌肽基因开展的油菜基因工程研究成果。并且进一步提出了未来油菜抗菌核病研究的可能方向,这些总结与建议为今后油菜抗菌核病研究提供了有益参考。     

Hybridization of Sinapis alba L. and Brassica napus L. via Embryo Rescue

Embryo rescue techniques were used to obtain hybrids between Sinapis alba L. (white mustard) and Brassica napus L. (oilseed rape) with the goal of improving the disease tolerance of oilseed rape. Hybrid plants with 31 or 43 chromosomes were only recovered, when S. alba, was used as the female parent. One hybrid was obtained from the cross S. alba L. cv. ‘Kirby’×B. napus L. cv. ‘Topas’, while 26 hybrids were obtained, when various S. alba L. cultivars were pollinated with the rapid cycling B. napus line CrGC 5006. All F₁, hybrid plants were male sterile; however, the first generation backcross to B. napus L., also obtained by embryo rescue, produced plants with 50 chromosomes and 61–84 % pollen viability. Second backcross generation seed was produced by normal sexual crossing. Preliminary cytological analyses of pollen mother cells of hybrid plants suggests the possibility of genetic exchange between the two species.     

Leaf yield component combining abilities in mulberry (Morus spp.)

A line × tester analysis was carried out in mulberry (Morus spp.) to determine the genetic interaction in the expression of various quantitative characters including leaf yield. Eight clonal varieties were selected, 3 of them were designated as lines (♀) viz. Berhampore-1, China white and MS-5 and 5 of them were called as testers (♂) viz. Mandalaya, Kosen, Assamjati, MS-1 and Kajli. Combining ability studies were conducted on these parents along with their F₁ hybrids for the variables laminar index, growth rate, weight of 100 dry leaves, number of primary branches per plant, plant height, nodal distance, leaf-twig ratio, aerial biomass, moisture content, moisture retention capacity and leaf yield. Broad genetic variability was observed among the genotypes. The ratio of General Combining Ability (GCA) and Specific Combining Ability (SCA) indicated the predominance of non-additive genes in mulberry. While China white (female) and MS-1 (male) were the best general combiners among the parents, Berhampore-1 × Kajli was the best cross for leaf yield. Results suggest that selective crossing followed by proper screening may be the best approach for breeding of high yielding varieties in mulberry. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glandular hair densities in three perennial Medicago species

Summary Eight triazine resistant (Brassica napus x B. oleracea) x B. oleracea interspecific hybrids with chromosome numbers ranging from 25 to 27 were backcrossed a second time to B. oleracea but no seed was formed. However, in vitro embryo rescue on 77 developing ovules yielded nine BC₂ plants with chromosome numbers between 19 and 25 and in which the herbicide resistance was still strongly expressed. Three of these plants (NOH-8B2B1, 2n=20; NOH-8B2B3 and NOH-8B2B4, 2n=19) were backcrossed again to B. oleracea. Two of the three plants produced seed which germinated to produce triazine resistan BBC₃s with 18, 19 or 20 chromosomes. The triazine resistant B. campestris cytoplasm has now been stabilized in B. oleracea.     

油菜野芥NSa细胞质雄性不育系的特异性分子鉴定

常规细胞质类型鉴定方法花费时间长、工作量大、鉴定效率低、应用性较差。基于基因组重复序列的PCR技术(rep-PCR)在重复序列丰富的线粒体和原核生物的基因组鉴定中效果较好, 本研究对8份油菜资源的细胞质进行分子鉴定,结果表明rep-PCR可以明确区分甘蓝型油菜中常见的6种雄性不育细胞质。同时对甘蓝型油菜和野芥(Sinapis arvensis)体细胞杂交创建的、育性较为稳定的NSa野芥雄性不育胞质进行了分子特异性鉴定,获得了两条NSa不育胞质的特征DNA条带。但波里马和萝卜质不育细胞质的特异性引物在NSa中不能扩增出特异性片段,说明NSa不属于这两种细胞质类型。本研究结果为该不育胞质系统的利用和知识产权保护提供了理论依据和技术支撑。     

Genetic relationships among resynthesized,semi-resynthesized and natural <Emphasis Type="Italic">Brassica napus</Emphasis> L. genotypes

The allopolyploidization event that created cultivated oilseed rape Brassica napus L, followed by intense breeding, reduced its genetic diversity. Resynthesized (RS) B. napus L. obtained by interspecific hybridization between genotypes of B. rapa L. and B. oleracea L. can be a valuable source for broadening genetic diversity in cultivated oilseed rape. In this study, we determined the extent of DNA polymorphism among natural accessions of oilseed rape, resynthesized B. napus, their parental species and double-low quality semi-RS lines carrying the Rfo gene. Using 10 selected primer combinations, 522 polymorphic AFLP markers were scored in the complete set of 100 Brassica sp. To detect relationships between these genotypes, a cluster analysis was performed using the Jaccard’s distance. Resynthesized allopolyploids clustered directly between their diploid parents. Cultivated accessions of oilseed rape created a compact group away from resynthesized allopolyploids as well as semi-RS lines. The natural oilseed rape group, which consists of 49 cultivars and breeding lines of oilseed rape, is characterized by lower genetic diversity than the group of 33 accessions of resynthesized oilseed rape, and the analysis showed that the double-low quality semi-RS lines represent a specific genetic variation of B. napus. The de novo resynthesized B. napus lines and the semi-RS lines of double-low quality generated from them, provide a significant opportunity for enrichment the gene pool of oilseed rape.