Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids

Genomic in situ hybridization (GISH) methods were used to detect different genome components within Brassica amphidiploid species and to identify donor chromatin in hybrids between Brassica napus and Raphanus sativus. In Brassica juncea and Brassica carinata the respective diploid donor genomes could be reliably distinguished by GISH, as could all R-genome chromosomes in the intergeneric hybrids. The A- and C-genome components in B. napus could not be clearly distinguished from one another using GISH, confirming the considerable homoeology between these genomes. GISH methods will be extremely beneficial for monitoring chromatin transfer and introgression in interspecific Brassica hybrids. Received: 20 May 1997 / Accepted: 28 July 1997     

Development of a molecular marker associated with Verticillium wilt resistance in diploid interspecific potato hybrids

Verticillium wilt (VW) is a widespread and serious potato (Solanum tuberosum) disease caused by the soilborne fungi Verticillium dahliae and V. albo-atrum. Breeding for VW resistance in potato is challenging due to ambiguous symptom expression, a lack of high throughput screening techniques, and variability in colonization by the fungus among and within plants. Genetic studies have identified major genes that confer resistance in diploid Solanum chacoense (V _c ) and interspecific hybrids (V _w and V _t ). However, to date, these genes have not been used to develop molecular markers for the identification of resistant clones. Tomato Ve1 and Ve2 gene sequence information was used to amplify candidate Ve gene orthologs from both resistant and susceptible diploid potato hybrids. A CAPS marker was generated to track VW resistance in a backcross population segregating for resistance. The marker was also tested for its usefulness in other breeding lines. Our results indicate that this marker is effective for selection of the V _w gene in segregating breeding populations.     

Synthesis of Brassica carinata from Brassica nigra x Brassica oleracea hybrids obtained by ovary culture

Summary Brassica carinata was synthesized by hybridization between its diploid progenitor species B. nigra and B. oleracea followed by chromosome doubling. Up to 40 times more hybrids were obtained, using ovary culture than with conventional hand pollination. Two hybrids from the cross B. nigra x B. oleracea var alboglabra were raised to maturity. High chromosome pairing was observed in both the hybrids and the amphidiploid. A range of variability for desirable characters is reported in the synthesized amphidiploids.     

Production and characterization of interspecific hybrids between Brassica maurorum and crop brassicas

 Seven interspecific hybrids were produced between Brassica maurorum (♀), a wild species resistant to Alternaria blight and white rust, and all the monogenomic (B. campestris, B. nigra and B. oleracea) and digenomic (B. juncea, B. napus and B. oleracea) crop brassicas (♂) through embryo rescue. The hybrids were confirmed by means of morphological and cytological studies. All the hybrids were pollen-sterile. Amphidiploids were induced in three of the hybrids: B. maurorum×B. napus, B. maurorum×B. carinata, B. maurorum×B. nigra. The hybrids were also confirmed through DNA analyses for nuclear and organelle genomes using RAPD and RFLP techniques. Received: 31 July 1998 / Accepted: 14 August 1998     

Molecular marker-assisted breeding for improved Ogura cms restorer line (RfoRfo) and mapping of the restorer gene (Rfo) in Brassica juncea

Developing a fully functional hybrid system is a must for hybrid breeding in Brassica juncea. The B. napus Ogura cms hybrid system was transferred into B. juncea by the researchers at INRA, France. The B. juncea restorer (R) line (RfoRfo) exhibited poor vigor, low fertility and was black-seeded due to linkage drag. Our studies indicated that the Rfo gene in B. juncea R line was linked to the 5 C9 markers of B. napus (sN3553F, sS2285, sN3841, sN12905 and At5g58730) and 4 radish markers (At3g27100, At5g25080, At4g13720 and At5g06240) in addition to the 6 radish markers reported before (ScH03, ScA14, OPF3, BolJon, CAB and PGIint). These markers were used to screen for improved restorer plants in the three crosses of B. juncea restorer plant O39-16 (Rforf) × condiment var. Cutlass, O39-16 (Rforf) × canola B. juncea line C668 and O39-16 (Rforf) × resynthesized B. juncea line 15043. One improved homozygous R line VR441 (RfoRfo) with only 1 C9 marker sN12905 and 2 radish markers ScH03 and BolJon was successfully developed via marker-assisted selection in the cross O39-16 × 15043. VR441 had good seed-setting (average: 14.3 seeds/pod), strong growth vigor and was yellow-seeded. Linkage mapping revealed that the Rfo gene was introgressed into chromosome 9 of the A genome in B. juncea. The development of the improved R line VR441 has made the Ogura cms hybrid system fully functional in B. juncea. We are currently using the improved system for developing high yielding hybrid varieties in condiment and canola B. juncea.     

Ogura细胞质雄性不育紫菜苔×萝卜属间杂种F1的获得及细胞遗传学研究

以Ogura细胞质雄性不育紫菜苔(AA,2n＝20)为母本,以不同萝卜品种(RR,2n＝18)为父本进行杂交,获得了大量的属间杂种.杂种F1幼苗在低温下子叶及真叶均不缺绿.以红萝卜为父本获得的杂种F1植株叶柄、叶脉呈紫红色;以白萝卜为父本获得的杂种F1植株叶柄、叶脉不呈紫红色.所有的杂种F1植株都开白花,蜜腺正常,雄配子高度不育,但是雌配子具有部分育性.杂种F1花粉母细胞的染色体数目为预期的2n＝19,染色体平均配对构型为15.53+1.34+0.25+0.01,多数染色体以单价体的形式存在,但也有一些二价体、三价体甚至四价体,最多达到6+3+1,参加配对的染色体数达到13条,表明A染色体组和R染色体组具有一定的同源性.对该杂种的遗传及育种意义进行了讨论.     

Ogura细胞质雄性不育紫菜苔×萝卜属间杂种F_1的获得及细胞遗传学研究

以Ogura细胞质雄性不育紫菜苔(AA, 2n＝20)为母本,以不同萝卜品种(RR,2n＝18)为父本进行杂交,获得了大量的属间杂种.杂种F1幼苗在低温下子叶及真叶均不缺绿.以红萝卜为父本获得的杂种F1植株叶柄、叶脉呈紫红色;以白萝卜为父本获得的杂种F1植株叶柄、叶脉不呈紫红色.所有的杂种F1植株都开白花,蜜腺正常,雄配子高度不育,但是雌配子具有部分育性.杂种F1花粉母细胞的染色体数目为预期的2n＝19,染色体平均配对构型为15.53+1.34+0.25+0.01,多数染色体以单价体的形式存在,但也有一些二价体、三价体甚至四价体,最多达到6+3+1,参加配对的染色体数达到13条,表明A染色体组和R染色体组具有一定的同源性.对该杂种的遗传及育种意义进行了讨论.     

Camalexin induction in intertribal somatic hybrids between Camelina sativa and rapid-cycling Brassica oleracea

  Camelina sativa, a wild relative of Brassica crops, is virtually immune to blackspot disease caused by Alternaria brassicicola. Intertribal somatic hybrids were produced between C. sativa and rapid-cycling Brassica oleracea as a step toward the transfer of resistance to this disease into Brassica vegetable crops. The plants recovered were confirmed as somatic hybrids by flow cytometry and RAPD analysis. All hybrids showed a morphology intermediate between the two parents. Rooted plants grew in soil up to 4–5 weeks, and some produced sterile flowers. Two of three hybrids tested showed a high level of resistance to  A. brassicicola. Resistance was correlated with the induction of high levels of the phytoalexin camalexin 48 h after inoculation, as in the resistant Camelina fusion partner. In contrast, susceptible somatic hybrids produced much lower levels of camalexin. Received: 2 April 1998 / Accepted: 14 July 1998     

Genome discrimination in progeny of interspecific hybrids between Brassica napus and Raphanus raphanistrum.

Genomic in situ hybridization (GISH) applied to the F1 interspecific hybrid between oilseed rape (Brassica napus, AACC, 2n = 38) and wild radish (Raphanus raphanistrum, RrRr, 2n = 18) showed the predicted 19 chromosomes from B. napus and 9 chromosomes from R. raphanistrum. The very low female fertility of these interspecific hybrids when backcrossed to R. raphanistrum led to only two descendants. Their chromosome number varied between 45 and 48. Both of these progenies showed only 9 chromosomes from R. raphanistrum and 36-39 chromosomes from B. napus. These results indicate the efficiency and limits of GISH as a suitable tool to assess and interpret the behavior of chromosomes after such interspecific crosses. The unexpected chromosome combination is discussed.     

A Lactuca universal hybridizer, and its use in creation of fertile interspecific somatic hybrids

A Lactuca sativa cv. Ardente line heterozygous for a gene encoding resistance to kanamycin, a positive and dominant trait, was crossed with cv. Girelle, which is heterozygous for a recessive albinism marker. The resulting seeds yielded 25% albino seedlings, of which 50% were also resistant to kanamycin. Such plantlets (KR, a) grown in vitro were used for preparation of universal hybridizer protoplasts, since green buds that can develop on kanamycin containing-medium should result from fusion with any wild-type protoplast. To test the practicability of this selection scheme, we fused L. sativa KR, a protoplasts with protoplasts derived from various wild Lactuca as well as various other related species. Protoplast-derived cell colonies were selected for resistance to kanamycin at the regeneration stage. Green buds were regenerated after fusion with protoplasts of L. tatarica and of L. perennis. So far, 9 interspecific hybrid plants have been characterized morphologically. In addition, random amplified polymorphic DNA (RAPD) analysis with selected primers confirmed that these plants are indeed interspecific hybrids. Some plants are female-fertile and production of backcross progenies with L. sativa is in progress. Since many desirable traits such as resistances to viruses, bacteria and fungi (Bremia lactucae) have been characterized in wild Lactuca species, the use of somatic hybridization in breeding programmes now appears a practical possibility.     

Direct transfer of a cold-tolerant Ogura male-sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion

Cold tolerant cytoplasmic male-sterile (CMS) cabbage (Brassica oleracea var. capitata) was produced by the fusion of leaf protoplasts from fertile cabbage and cold-tolerant Ogura CMS broccoli lines. The cabbage lines tested showed great variation in plant regeneration from unfused protoplasts; three with high regenerability were selected as the fusion partners. Several procedures for eliminating the nuclear DNA of the broccoli fusion partner were tested. Diploid cabbage plants were identified by flow cytometry and morphological characters. Gamma-irradiation (30 krad) was the most successful procedure; isolation of cytoplasts from broccoli leaf protoplasts, followed by gamma-irradiation of the cytoplast fraction, also produced diploids. UV-irradiation of the broccoli protoplasts was less effective. PCR using primers for an Ogura CMS-specific mitochondrial DNA sequence permitted the identification of cybrids likely to be CMS. Over 200 diploid plants with the CMS-specific sequence were obtained from 66 independent fusion products and three cabbage lines. Plants were ready for transfer into soil within 8 months after fusion. The plants identified as CMS by PCR produced male-sterile flowers. Our procedures permit the transfer of a desirable male-sterile cytoplasm into cabbage much more rapidly than conventional backcrossing procedures. Received: 4 June 1996 / Accepted: 2 August 1996     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

In vitro pollen germination of species and two interspecific hybrids from the genus Brassica

In vitro pollen germination of five species and two interspecific hybrids from the genus Brassica was tested in four media. Genetically fixed differences in the demands for optimal pollen germination among species were found. The experiments were designed to define optimal content of mineral salts, sugar, and PEG for every investigated species or hybrids. The differences found among species are discussed in relation to the evolutionary trend.     

ZmGrp3: identification of a novel marker for root initiation in maize and development of a robust assay to quantify allele-specific contribution to gene expression in hybrids

This study comprises a comprehensive gene expression analysis of the root tip specific maize gene ZmGrp3. In the first part of this paper expression of ZmGrp3 was studied in maize inbred lines. First, RNA in situ hybridization experiments confined the expression of ZmGrp3 to the columella and the epidermis of all embryonic and postembryonic root types. Second, Northern-blot analyses of the maize root initiation mutants rtcs and lrt1 revealed that the ZmGrp3 gene is not expressed prior to root initiation, thus providing a novel marker for this developmental process. Finally, a comprehensive expression profiling in 42 tissues via the Lynx MPSS system revealed almost exclusive expression of ZmGrp3 in maize roots. In the second part of this survey, ZmGrp3 expression was assayed in maize hybrids. In this context, a novel approach to quantify allele-specific contribution to gene expression in maize hybrids was developed. This assay combines RT–PCR amplification of polymorphisms between two alleles and subsequent quantification of allele-specific gene expression via a combination of didesoxyterminator assays and capillary electrophoresis. Allelic expression of the ZmGrp3 gene in six reciprocal hybrids generated from three ZmGrp3 alleles was analyzed via a new statistical mixed model approach.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Production and characterization of interspecific somatic hybrids between Brassica oleracea var. botrytis and B. nigra and their progenies for the selection of advanced pre-breeding materials

Somatic hybridization is a potential method for gene transfer from wild relatives to cultivated crops that can overcome sexual incompatibilities of two distantly related species. In this study, interspecific asymmetric somatic hybrids of Brassica oleracea var. botrytis (cauliflower) and Brassica nigra (black mustard) were obtained by protoplast fusion and their backcrossed (BC₃) and selfed (S₃) offspring were analyzed. Cytological analysis showed that the B. nigra chromosomes were successively eliminated in the backcrosses with cauliflower. The fertility of the hybrid progenies was quite different due to the asynchronous and abnormal chromosome behavior of pollen mother cells (PMC) during meiosis. Analysis of sequence-related amplified polymorphism (SRAP) showed that all of these hybrids mainly had the DNA banding pattern from the two parents with some alterations. Genetically, the selfed generations were closer to B. nigra, while the backcrossed generations were closer to the cauliflower parent. Analysis of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) showed that all somatic hybrids in this study contained chloroplast (cp) DNA of the donor parent black mustard, while mitochondrial (mt) DNA showed evidence of recombination and variations in the regions analyzed. Furthermore, three BC₃ plants (originated from somatic hybrids 3, 4, 10) with 2–8 B. nigra-derived chromosomes shown by genomic in situ hybridization (GISH) displayed a more cauliflower-like morphology and high resistance to black-rot. These plants were obtained as bridge materials for further analysis and breeding.     

Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis

Interspecific chimeras between tuber mustard and red cabbage were obtained by in vitro graft-culture method. Before grafting, 6-day-old seedlings of tuber mustard and red cabbage were vertically half-cut and treated with different concentrations of 6-BA and NAA for 1 min, then, they were symmetrically fit together. As a result, sectorial chimeras were initially produced from the united shoot tips. The maximum frequency of chimeral bud formation reached 6.33% when the vertical sections of tuber mustard and cabbage were treated with 2 mg/l 6-BA and 1 mg/l NAA. When sectorial chimeras were propagated on MS medium containing 1 mg/l 6-BA, periclinal and mericlinal chimeras gradually developed. Chimeral shoots were rooted on half-strength MS medium containing 0.1 mg/l NAA. The rooted chimeras were acclimatized and transferred to the field for cytological and morphological analysis. The results showed that stomata density in the chimeras was significantly higher than that of their parents, while chloroplast size, starch grain size and number were intermediate between the two parents. The chimeras were further analyzed by flow cytometry, and the results indicated that they contained both sets of parental chromosomes. Moreover, chimeral plants possessed valuable characters from the two parents.     

Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea

Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe an efficient protocol for Agrobacterium-mediated transformation using seedling explants that is applicable to various Brassica varieties; this protocol has been used to genetically engineer commercial cultivars of canola and cauliflower in our laboratory. Young seedling explants are inoculated with Agrobacterium on the day of explant preparation. Explants are grown for 1 week in the absence of a selective agent before being transferred to a selective medium to recover transgenic shoots. Transgenic shoots are subjected to an additional round of selection on medium containing higher levels of the selective agent and a low-carbohydrate source; this helps to eliminate false-positive plants. Use of seedling explants offers flexible experiment planning and a convenient explant source. Using this protocol, transgenic plants can be obtained in 2.5 to 3.5 months.     

Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea

Background

Cabbage Fusarium wilt is a major disease worldwide that can cause severe yield loss in cabbage (Brassica olerecea). Although markers linked to the resistance gene FOC1 have been identified, no candidate gene for it has been determined so far. In this study, we report the fine mapping and analysis of a candidate gene for FOC1 using a double haploid (DH) population with 160 lines and a F₂ population of 4000 individuals derived from the same parental lines.

Results

We confirmed that the resistance to Fusarium wilt was controlled by a single dominant gene based on the resistance segregation ratio of the two populations. Using InDel primers designed from whole-genome re-sequencing data for the two parental lines (the resistant inbred-line 99–77 and the highly susceptible line 99–91) and the DH population, we mapped the resistance gene to a 382-kb genomic region on chromosome C06. Using the F₂ population, we narrowed the region to an 84-kb interval that harbored ten genes, including four probable resistance genes (R genes): Bol037156, Bol037157, Bol037158 and Bol037161 according to the gene annotations from BRAD, the genomic database for B. oleracea. After correcting the model of the these genes, we re-predicted two R genes in the target region: re-Bol037156 and re-Bol0371578. The latter was excluded after we compared the two genes’ sequences between ten resistant materials and ten susceptible materials. For re-Bol037156, we found high identity among the sequences of the resistant lines, while among the susceptible lines, there were two types of InDels (a 1-bp insertion and a 10-bp deletion), each of which caused a frameshift and terminating mutation in the cDNA sequences. Further sequence analysis of the two InDel loci from 80 lines (40 resistant and 40 susceptible) also showed that all 40 R lines had no InDel mutation while 39 out of 40 S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage.

Conclusions

This work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.