Abstract
Capsella bursa-pastoris (L.) Medic (2n = 4x = 32) is a natural double-low (erucic acid < 1%, glucosinolates < 30 μmol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Hybridizations were carried out between two Brassica species viz. B. rapa (2n = 20) and B. napus (2n = 38) as female and C. bursa-pastoris as male parent to introduce these desirable traits into cultivated Brassica species. Majority of F1 plants resembled female parents in morphology and only a few expressed some characters of male parent, including the white petals. Based on cytological observation of somatic cells, the F1 plants were classified into five types: two types from the cross with B. rapa, type I had 2n = 27–29; type II had 2n = 20; three types from the crosses with B. napus, type III was haploids with 2n = 19; type IV had 2n = 29; type V had 2n = 38. One to two chromosomes of C. bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F1 plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in majority of F1 plants, even in Brassica-types and haploids, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum. The cytological and molecular mechanisms behind these results are discussed.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Wide hybridization plays an important role in crop improvement and has been used successfully to transfer desired traits from wild germplasm to large number of crop species, including rape (Peterka et al. 2004; Ma et al. 2006; Snowdon 2007), rice (Amante-Bordeos et al. 1992), wheat (Martin-Sanchez et al. 2003), coffee (Prakash et al. 2004), sunflower (Ronicke et al. 2004). However, investigations on intertribal sexual hybrids are not frequent and reported only for Avena sativa × Zea mays (Riera-Lizarazu et al. 1996) and Brassica napus × Matthiola incana (Luo et al. 2003).
The double-low Brassica varieties have the seed oil with the desired fatty acid composition for human nutrition (i. e., <1% erucic acid and ∼60% oleic acid) and the seed meal suitable for animal feed (<30 μmol glucosinolates/g oil free seed meal). The Brassicaceae family comprises a large number of wild species which are potential sources of nuclear genes for many economically important traits, such as resistance to biotic and abiotic stresses, and novel fatty acid compositions. The crucifer Capsella bursa-pastoris (L.) Medic of tribe Lepidieae is an annual to biennial predominantly autogamous species with worldwide distribution. It has been used traditionally as vegetable and medicinal plant in China and some other countries for many centuries (Zhou 1987). Capsella, Brassica and Arabidopsis show close genetic relationships. The divergence times for the species pairs Arabidopsis-Brassica, Capsella-Brassica and Capsella-Arabidopsis are 12.2–24 (Yang et al. 1999; Koch et al. 2000; Acarkan et al. 2002), 12.4–19.5 (Acarkan et al. 2002) and 6.2–14 (Acarkan et al. 2002; Koch and Kiefer 2005) million years ago, respectively. The seed oil of C. bursa-pastoris has considerably lower erucic acid content compared to other cruciferous plants (Park 1967). A population of this species collected in the campus of Huazhong Agricultural University is found to be a natural double-low germplasm (0.68% erucic acid and 15.68 μmol glucosinolates/g oil free seed meal). C. bursa-pastoris has been reported to be highly resistant to Alternaria brassicae (Conn et al. 1988; Sigareva and Earle 1999). In the present study we have observed that it possesses high resistance to Sclerotinia sclerotiorum, one of the most devastating diseases of rapeseed in China. It has the ability to tolerate cold, salt and drought (Liu et al. 2004a). These facts indicate the utility of C. bursa-pastoris as a potential source of agronomic important traits for introgression into Brassica crops. Intertribal somatic hybrids between C. bursa-pastoris and B. oleracea have been produced, but no progeny plants could be obtained because of sterility, thus their use in further breeding program is limited (Sigareva and Earle 1999). The present investigation reports production of sexual hybrids between Brassica species (B. rapa, B. napus) and C. bursa-pastoris and their morphological and chromosomal/genomic characterizations for the first time.
Materials and methods
Plant materials and crosses
Cultivars used in the present study are B. rapa var. chinensis cv. Aijuehuang and B. napus cvs. Oro (the first B. napus cultivar with low content of erucic acid in the world), Huashuang no. 3 (double-low cultivar), Zhongyou 821 (high content of erucic acid and glucosinolates, but high yield and resistance to S. sclerotiorum).The double-low C. bursa-pastoris was collected in Huazhong Agricultural University. B. rapa and B. napus had yellow petals and black seeds, while C. bursa-pastoris had small deep-green leaves, basal clustering branches, short plant stature (30–50 cm), white petals and very small brown seeds in the heart-shaped pods. The crosses between Brassica species and C. bursa-pastoris with the latter as pollen parent were performed in the fields by hand emasculation and pollination at Wuhan in 2002 and 2003, at Xining, Qinghai Province in 2004. About 2–3 weeks after pollination, some immature embryos were cultured on MS agar medium (Murashige and Skoog 1962) and others left on plants to obtain mature seeds.
Cytology
To determine the chromosome numbers of hybrids, young ovaries were treated with 2 mM 8-hydroxyquinoline for 3–4 h at 22°C, and fixed in a mixture of ethanol: acetic acid (3:1, v:v) for 24 h, stored at −20°C. For meiotic analysis, flower buds were fixed in a mixture of ethanol: acetic acid (3:1, v:v) for 24 h, transferred to fresh mixture and stored at −20°C. Mitotic and meiotic observations were made according to the methods of Li et al. (1995). Pollen fertility was determined as the percentage of pollen grains stained with 1% acetocarmine.
Probe labeling and GISH analyses
DNA was extracted and purified from young leaves according to the method of Dellaporta et al. (1983). The DNA of Brassica species was sheared to 300–500 bp fragments by boiling for 15 min and used as block. The DNA of C. bursa-pastoris was labeled with bio-11-dUTP (SABC in China) by nick translation method and used as probe. The length of the probe DNA fragments averaged approximately 500 bp.
The young ovaries and anthers with pollen mother cells (PMCs) at suitable stages were digested in an enzyme mixture containing 0.6% cellulase Onozuka RS (YAKULT HONSHA Co., LTD, Japan), 0.4% pectinase (MERCK, Germany) and 0.5% snailase (Beijing Baitai Biochem Co., China) at 37°C for about 63 and 68 min, respectively. The chromosome preparations for GISH mainly followed the procedures of Zhong et al. (1996). In situ hybridization was carried out according to the protocol by Leitch et al. (1994). Hybridization signals of the C. bursa-pastoris probe were detected using Cy3-labeled streptavidin (Sigma, USA), and chromosomes were counterstained with 0.2% 4’-6-Diamidino-2-phenylindole (DAPI) solution (Roche, Basel, Switzerland), mounted in antifade solution (Citifluor) and examined under a Leica DMLB fluorescent microscope (Wetzlar, Germany) equipped with CCD (LEICA DC 300F).
AFLP analysis
AFLP fingerprints were generated based on the protocol of Vos et al. (1995), and DNA bands were visualized by silver staining (Bassam et al. 1991). The bands with 80–800 bp were scored.
Fatty acids and glucosinolates analysis
Fatty acids of the seed oil were analyzed on gas chromatography machine (HP 6890, Germany). A bulk seed sample (0.2 g) per plant was crushed and transferred into glass tube with 1 ml mixture of diethyl ether: petroleum ether (1:1, v:v) added, for extraction of seed oil at room temperature over 8 h. After 1 ml methanol (with 5% KOH) was added to the tube for esterification for 40 min and 2 ml H2O added, 0.5 μl of the upper phase containing fatty acid methyl esters as sample was injected into the gas chromatography machine equipped with a fused-silica capillary column (30 m × 0.25 mm). The injector and flame ionization detector were held at 250 and 180°C, respectively. The carrier gas flow was 30 ml/min (H2), 300 ml/min (air) and 25 ml/min (N2). To directly determine the content of glucosinolates, about 3 g seeds were scanned by near-infrared reflectance spectroscopy (NIRS) (Vector 22/N, Bruker, Germany, OPUS/QUANT4.0 software).
Culture of Sclerotinia sclerotiorum and infection
Sclerotinia sclerotiorum isolate was collected from infected B. napus plants in the fields of Huazhong Agricultural University. Fungal mycelia were cultured on solid Potato/Dextrose/Agar (PDA, 20% potato, 2% dextrose and 1.5% agar) medium. Mycelial agar disks of 5-mm diameter punched from the growing periphery of the 2-day old culture of S. sclerotiorum on PDA were used as inoculums to infect the plants.
Leaves excised from plants at the 9–12 leaf stage were inoculated with inoculums, covered with plastic bags to provide adequate humidity for infection at 20°C. The lesion diameter was measured at 48 h after inoculation to evaluate the level of resistance. Plants in the field were inoculated 3 weeks before harvest. The inoculums were affixed to the stems surface with parafilm and remained in contact with the stems surface until lesion developed (Li et al. 2004). The lesion length along the stems was measured 8 days after inoculation.
Results
Crossability and morphology of F1 plants
B. rapa × C. bursa-pastoris
From 7,513 pollinations, 185 F1 plants were obtained (0.025 seeds/silique). Fourteen plants were distinguished by their phenotypes and cytology and grouped according to their chromosome numbers into two types, viz. type I (no. 1) and type II (nos. 2–14) (Table 1). The plant no. 1 was morphologically intermediate between the two parents (Fig. 1a1, a5), but conspicuous in expressing some traits of male parent, such as small deep-green leaves (Fig. 1a2), nanism, basal clustering branches and white petals (Fig. 1c2). The original seed-plant was multiplied in vitro by culturing its buds on MS medium and the cloned plants showed the same phenotype and chromosome number (see below). Some plants exhibited purple petiole (Fig. 1a3) and cleft leaves (Fig. 1a4) of C. bursa-pastoris. Two plants (nos. 1, 4) had yellow seeds. Except plant no. 1, all F1 plants with various pollen fertility (47.6–98.4%) had good seed-set after selfing. The seed-, or cloned- plant no. 1 had very poor pollen (16.7%) and seed fertility. No seed was obtained after selfing, and only few seeds could be obtained following pollination with the female parent.
B. napus × C. bursa-pastoris
From 9,248 pollinations, 169 F1 plants were obtained (0.018 seeds/silique). Of these, 22 were selected and grouped according to their chromosome numbers into three types, type III (nos. 15, 16), type IV (nos. 17–19) and type V (nos. 20–36) (Table 1). These plants exhibited some traits of C. bursa-pastoris (Fig. 1a5, c1, d3) having small sized dark-green, deeply divided leaves (Fig. 1b), nanism, basal clustering branches and white petals (Fig. 1d2), and the flowers of one plant had curly petals (Fig. 1c3). Plants of types III and IV had poor pollen fertility and produced no seeds after selfing and only one to three seeds in a silique after pollination by the female parent. Except plant no. 30, all F1 plants of type V with varying pollen fertility (66.8–98.5%) had good seed-set after selfing. The plant no. 30 which resembled female parent (Fig. 1d1) in morphology was male sterile with rudimentary stamens (Fig. 1c4), however, the pistil was normal and had good seed-set after pollination by the female parent. Most hybrids had black seeds as female (Fig. 1e1), however, four plants had red brown or yellow brown seeds (Fig. 1e2, e3, Table 1), were similar to male parent (Fig. 1e5), and some progenies of plant no. 30 produced yellow seeds (Fig. 1e4). All hybrid plants from the crosses with B. napus and B. rapa produced the pods of Brassica-type, not the heart-shaped pods of C. bursa-pastoris.
Cytogenetic and GISH analyses of F1 plants
B. rapa × C. bursa-pastoris
Capsella bursa-pastoris had 2n = 32 (Fig. 2a1) and the PMCs at diakinesis had 16 bivalents (Fig. 2a2). The single plant of type I (no. 1) had 2n = 27–29 with a preponderance of 29 in ovary cells (85.3%). Majority of its PMCs at diakinesis showed 13 II + 3 or 4 I, 14 II + 3 or 4 I. However, the sum of chromosomes in two polar groups of PMCs at anaphase I (AI) were 2n = 28–36, though 2n = 29 was still the most frequent (34.4%), 2n > 29 appeared in 63.6% cells. One to five laggards were observed in 71.2% AI PMCs (Fig. 2b) and rarely in second divisions. Plants of type II (nos. 2–14) had 2n = 20, same as B. rapa (Table 1).
As C. bursa-pastoris probe was applied to the preparations of B. rapa cv. Aijuehuang (the DNA of itself as block), signals of large size and strong intensity were mainly located at two terminals of one v-shaped bivalent and centromeric part of another one at diakinesis (Fig. 2d1, d2), at terminal or centromeric parts of two chromosomes in each polar group of AI PMCs (Fig. 2e1, e2). The same hybridization pattern on ‘Aijuehuang’ was also observed using Orychophragmus violaceus probe (Liu and Li 2007). Judged from its morphology, the bivalent with its two terminals being strongly labeled was most likely the satellited chromosome pair of B. rapa (Cheng et al. 1994; Liu and Li 2007). This made it more reliable to detect C. bursa-pastoris chromosomes/chromosomal segments in these hybrids. Extensive GISH investigations showed that one chromosomal arm in ovary cells of plant no. 1 was fully covered by signals of the C. bursa-pastoris probe (Fig. 2f1, f2). One chromosome was fully labeled in 36.8% AI PMCs with various chromosome numbers (Fig. 2g1, g2), and two chromosomes in 8.7% PMCs (Fig. 2h1, h2). In plants of type II, signals of small size or weak intensity were located mainly at terminal and centromeric parts of the chromosomes in mitotic and meiotic cells, which showed that no intact chromosomes or large segments of C. bursa-pastoris origin were contained.
B. napus × C. bursa-pastoris
Plants of type III (nos. 15, 16) were haploids with 2n = 19, however, plant no. 15 had 2n = 13–18 (Fig. 2c) in 9.4% somatic cells. Plants of type IV (nos. 17–19) had 2n = 29; type V (nos. 20–36) had 2n = 38, same as B. napus (Table 1). In plants of type IV, 60.5% PMCs at diakinesis had 1 III + 9 II + 8 I, the remaining had 10 II + 9 I and showed segregations 14:15, 13:16, 12:17, 11:18 and 10:19. However, in plant no. 17 of type IV, 4.8% PMCs had 2n = 30 and 31, and 26.7% AI PMCs had 1–2 lagged chromosomes. The chromosome pairing (19 II) and segregation (19:19) were normal in PMCs of type V. Only one chromosomal arm was fully covered by the signal from the probe of C. bursa-pastoris in ovary cells of plant no. 15 (Fig. 2i1, i2), while weak or minor signals appeared at terminal or centromeric parts of some chromosomes in ovary cells and PMCs of these plants.
AFLP analyses of F1 plants
Polymorphic AFLP bands were amplified in the hybrids from the randomly selected fifteen pairs of primers. Three kinds of bands, i.e., specific for C. bursa-pastoris, novel for two parents and absent in Brassica parents were detected in F1 plants except for three plants (nos. 13, 24, 32) which had no specific bands, and the respective numbers in individual plants were 0–28, 39–168 and 32–80 for the cross with B. rapa, and 0–11, 25–65 and 23–60 for the cross with B. napus. The numbers of the specific (28) and novel (168) bands of plant no. 1 were the highest among F1 plants with 55.9% polymorphic bands. The percentages of polymorphic bands in plants from the cross with B. rapa were all over 30% expect for plant no. 2, being higher than in plants from cross with B. napus (about 20%) (Table 1). In haploid plants of type III, three kinds of bands were also detected with comparable percentages to other plants. Some polymorphic loci were the same in F1 plants (Fig. 3, arrowed), indicating that the introgressions were not entirely random.
Erucic acid and glucosinolate contents of F1 plants and progenies
B. rapa × C. bursa-pastoris
Reduced erucic acid content of varying degrees from 51.7% of B. rapa to 8.58–37.78% was observed in F1 plants of type II (Table 2). Similarly, the glucosinolates content in seeds also decreased from 116.57 μmol/g oil free meal of B. rapa to 41.53–85.39 μmol/g, but none reached the level of C. bursa-pastoris (15.68 μmol/g). Most profiles of the selfed seeds of F2 plants derived from one F1 plant were similar to each other and their F1 plants (data not shown).
B. napus × C. bursa-pastoris
The changes in erucic acid content were observed in some F1 plants of type V (Table 2). For the cross with B. napus cv. Oro, the content of glucosinolates was reduced in most F1 plants and progenies, some being <30 μmol/g; however, the content of erucic acid of two plants (nos. 23, 25) increased. For the cross with Huashuang no. 3, F1 plants still had the double-low quality as the female, and the content of glucosinolates was reduced in some plants. Most F1 plants from the cross with Zhongyou 821 possessed reduced contents of erucic acid and glucosinolates, and one plant (no. 35) reached double-low standard. The content of glucosinolates of F2 plants was deviated from those of some F1 plants, but the content of erucic acid remained same (data not shown).
Resistance to Sclerotinia sclerotiorum in progenies
Some lines (F2 or F3) derived from some F1 plants showed significantly higher resistance to S. sclerotiorum compared to female parents (Table 3). Plants nos. 1 and 13, derivatives of the cross with B. rapa cv. Aijuehuang, showed the smallest leaf and stem lesions, 1.8 and 4.3 cm, respectively. Four lines derived from plant nos. 1, 2, 4, 13 had significantly higher resistance expect for leaf infection of plant no. 13, one line derived from plant no. 1 showed significantly (P < 0.01) lower damage on leaves and stems than female parent. For cross with B. napus cv. Oro, the smallest leaf and stem lesions were 1.1 and 3.3 cm on plant nos. 29 and 17, respectively. Six lines derived from plant nos. 17, 20, 21, 22, 29, 30 showed significant resistance except for stem infection of plant no. 29, two lines derived from plant nos. 17 and 30 showed significantly (P < 0.01) lower damage on leaves and stems than female parent. For crosses with Huashuang no. 3 and Zhongyou 821, only few lines showed significantly lower damage than female parents, probably due to the high resistance of female parents.
Discussion
Progenies from the intertribal sexual hybridizations between Brassica species and C. bursa-pastoris were investigated on morphology, cytology and molecular characteristics, which enabled us to determine the hybridity status of each plant and to quantify the level of hybridization occurrence in these crosses. However, the progenies produced were not the hybrids with the expected chromosome complements. Only few F1 plants were morphologically intermediate between the parents (Table 1, Fig. 1) and the rest resembled the female parents. GISH (Fig. 2) and AFLP (Table 1, Fig. 3) analyses revealed that the hybridization events occurred at various levels. Some B. napus-like progenies were also obtained after crossings with C. bursa-pastoris and new lines with higher yield and resistance to S. sclerotiorum were selected (Zhao et al. 1995), however, no genetic study was carried out.
Plant no. 1 of type I from cross with B. rapa expressing some morphological characters of the male C. bursa-pastoris (Table 1, Fig. 1) was a mixoploid with 2n = 27–29 in somatic cells, while 63.6% PMCs had more chromosomes (2n = 30–36). GISH analysis demonstrated that only one or two C. bursa-pastoris chromosomes were included and chromosomal fragments translocated in PMCs and somatic cells (Fig. 2). These results suggested that the original hybrid cells (2n = 26) underwent chromosome duplication once or twice during mitotic divisions of the zygotes or plants, eliminating most male chromosomes, and extra duplication of partial chromosomes during meiotic DNA synthesis phase. The similar results were observed in B. rapa × O. violaceus intergeneric cross, where the chromosome doubling in hybrid cells and successive elimination of O. violaceus chromosomes accompanied by the introgression and recombination were responsible for producing B. rapa-type plants with modified genetic constitutions and phenotypes (Liu and Li 2007). This mechanism would be valid for explaining the present results. Partial hybrids with a haploid complement from female parent (oat) and some chromosomes (1–4) of male parent (maize) were reported in oat × maize cross (Riera-Lizarazu et al. 1996).
AFLP analysis performed on 474 loci for plant no. 1 indicated that it contained 5.9% DNA fragments putatively derived from C. bursa-pastoris, however, 55.9% genomic loci were changed (Table 1), suggesting that other reasons were also involved in these genomic variations. Extensive alteration in DNA methylation patterns (Natali et al. 1998; Liu et al. 2004b; Wang et al. 2005), some mobile genetic elements (transposons and retrotransposons) (Liu and Wendel 2000; Shan et al. 2005; Wang et al. 2005), rapid sequence elimination in synthetic hybrids and allopolyploids (Song et al. 1995; Shaked et al. 2001), and genomic rearrangements in the hybrids were the causes for the genomic variations. The high frequency of the novel bands (35.4%) for two parents and deleted bands (14.6%) in B. rapa might be due to some of these reasons.
Matroclinous plants of types II (AA, 2n = 20) and V (AACC, 2n = 38) could arise due to the complete elimination of the C. bursa-pastoris chromosomes accompanied by alien introgression, and doubling the haploid genome during embryo development. The morphological traits of male parent and specific bands for C. bursa-pastoris indicated the occurrence of alien introgression. This kind of partial hybrids with the same chromosome numbers of female parents but altered genomic compositions had been reported in coffee (Lashermes et al. 2000), rapeseed (Cheng et al. 2002; Hua et al. 2006), and sunflower (Faure et al. 2002). Though the loci of C. bursa-pastoris were only 0–3.6% in these plants, 11.8–39.6% genomic loci were changed. Similarly, extensive genomic variations detected by AFLP analysis were up to 30% loci in rice recombinant inbred lines with <0.1% alien introgressed DNA (Wang et al. 2005). Plants of type III were B. napus haploids and were outcome of complete elimination of the C. bursa-pastoris chromosomes, together with fragment translocations (one chromosomal arm was labeled in ovary cells of plant no. 15) (Fig. 2) and introgressions (0.8–1.3% specific bands for C. bursa-pastoris). In plant no. 15, 9.4% somatic cells had 2n < 19 (Fig. 2), suggesting that several chromosomes of B. napus in some cells were also eliminated.
Plants of type IV had 2n = 29 and their PMCs at diakinesis had 10 II + 9 I and 1 III + 9 II + 8 I, suggesting that the genome of these plants consisted of ten bivalents and nine univalents. Cheng et al. (2002) obtained this kind of plants from B. napus × O. violaceus with similar results of cytology and molecular markers. Because these plants were obtained following crosses in 3 years and they expressed some traits of C. bursa-pastoris, such as basal clustering branches, small deep-green leaves and nanism and on the other hand had 0.6–2.1% specific bands for C. bursa-pastoris, suggested that they originated from true hybridizations with C. bursa-pastoris, not from the pollen contamination of B. rapa. According to their chromosome pairing configurations, their genomic constitution was proposed as 20A + 9C where one C genome was lost from the complement of B. napus. The plants (2n = 29) with all chromosomes of B. napus origin were also obtained in the crosses of B. napus with O. violaceus (2n = 24) (Hua and Li 2006) and Lesquerella fendleri (2n = 12) (Du et al., unpublished). One possible reason for this could be attributed to the dominance of rRNA genes from the two ancestors of B. napus, for the hierarchy of rRNA gene transcriptional dominance is B. nigra >B. rapa >B. oleracea and B. rapa rRNA transcripts are readily detected in natural B. napus, but B. oleracea transcripts are not detectable (Chen and Pikaard 1997). Similarly, more chromosomes from B. oleracea than from B. nigra were lost in cells with partial B. carinata complements (2n < 34) in hybrids between B. carinata and O. violaceus (Hua et al. 2006). The expression of B. rapa rRNA genes might help to stabilize the chromosomes of A genome in B. napus (Li and Ge 2007). Same as the plants of other types, genomic alterations of type IV were obvious and 19.1–26.4% genomic loci were changed.
The first B. napus cultivar ‘Oro’ with low erucic acid was selected from one local variety ‘Liho’ in Germany, while the only donor conferring the low glucosinolates in almost all the B. napus varieties was ‘Bronowski’ from Poland. Thus, the search for new gene source for double-low quality of rapeseed through suitable approaches including wide hybridization is pivotal for further genetic improvement. New B. napus inbred lines with increased levels of oleic and linoleic acids derived from one B. napus cv. Oro × O. violaceus hybrid, and reduced content of glucosinolates (<30 μmol/g oil free meal) was obtained (Ma et al. 2006). The genomic compositions of these new lines were substantially altered from that of B. napus cv. Oro, as revealed by AFLP analysis. These changes should be extensive and affected many genes including plant phenotypes, the synthesis of fatty acids and glucosinolates. The reduction of erucic acid and glucosinolates content in our hybrids and their progenies (Table 2) might be caused by the introgression of related genes from C. bursa-pastoris or the genomic alteration consecutive to hybridization. Progenies with yellow seeds could be used in rapeseed breeding for higher oil content (Daun and DeClercq 1988).
The combination of cytological and molecular techniques was successful to determine the chromosomal/genomic constitutions of partial/introgressive Brassica hybrids (Cheng et al. 2002; Hua et al. 2006; Ma et al. 2006; Liu and Li 2007). In the present study, application of GISH and AFLP techniques better characterized the intertribal hybrids with very limited amount of alien genetic elements. In conclusion, the introgressive hybrids (types II and V) provided an opportunity to rapidly and successfully introduce useful traits of C. bursa-pastoris into Brassica species and to produce lines with improved oil quality and higher resistance to S. sclerotiorum.
References
Acarkan A, Rossberg M, Koch M, Schmidt R (2002) Comparative genome analysis reveals extensive conservation of genome organisation for Arabidopsis thaliana and Capsella rubella. Plant J 23:55–62
Amante-Bordeos A, Sitch LA, Nelson R, Dalmacio RD, Oliva NP, Aswidinnoor H, Leung H (1992) Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice, Oryza sativa. Theor Appl Genet 84:345–354
Bassam BJ, Caetano-Anolles G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196:80–83
Chen ZJ, Pikaard CS (1997) Transcriptional analysis of nucleolar dominance in polyploid plants: biased expression/silencing of progenitor rRNA genes is developmentally regulated in Brassica. Proc Natl Acad Sci USA 94:3442–3447
Cheng BF, Heneen WK, Chen BY (1994) Meiotic studies on a Brassica campestris-alboglabra monosomic addition line and derived B. campestris primary trisomics. Genome 37:584–589
Cheng BF, Seguin-Swartz G, Somers DJ (2002) Cytogenetic and molecular characterization of intergeneric hybrids between Brassica napus and Orychophragmus violaceus. Genome 45:110–115
Conn KL, Tewari JP, Dahiya JS (1988) Resistance to Alternaria brassicae and phytoalexin-elicitation in rapeseed and other crucifers. Plant Sci 56:21–25
Daun JK, DeClercq DR (1988) Quality of yellow and dark seeds in Brassica campestris canola varieties Candle and Tobin. J Am Oil Chem Soc 65:122–126
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA mini preparation: version II. Plant Mol Biol Rep 1:19–21
Faure N, Serieys H, Berville A, Cazaux E, Kaan F (2002) Occurrence of partial hybrids in wide crosses between sunflower(Helianthus annuus) and perennial species H. mollis and H. orgyalis. Theor Appl Genet 104:652–660
Hua YW, Li ZY (2006) Genomic in situ hybridization analysis of Brassica napus × Orychophragmus violaceus hybrids and production of B. napus aneuploids. Plant Breed 125:144–149
Hua YW, Liu M, Li ZY (2006) Parental genome separation and elimination of cells and chromosomes revealed by GISH and AFLP analyses in a Brassica carinata × Orychophragmus violaceus cross. Ann Bot 97:993–998
Koch MA, Kiefer M (2005) Genome evolution among cruciferous plants: a lecture from the comparison of the genetic maps of three diploid species-Capsella rubella, Arabidopsis lyrata subsp. petraea, and A. thaliana. Am J Bot 92:761–767
Koch MA, Haubold B, Mitchell-Olds T (2000) Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis, and related genera (Brassicaceae). Mol Biol Evol 17:1483–1498
Lashermes P, Andrzejewski S, Bertrand B, Combes MC, Dussert S, Graziosi G, Trouslot P, Anthony F (2000) Molecular analysis of introgressive breeding in coffee ( Coffea arabica L.). Theor Appl Genet 100:139–146
Leitch AR, Schwarzacher T, Jackson D, Leitch IJ (1994) Microscopy handbook. In situ hybridization: a practical guide. Bios Scientific, Oxford, pp 27
Li ZY, Ge XH (2007) Unique chromosome behavior and genetic control in Brassica × Orychophragmus wide hybrids: a review. Plant Cell Rep. doi:10.1007/s00299-006-0290–7
Li Z, Liu HL, Luo P (1995) Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus. Theor Appl Genet 91:131–136
Li R, Rimmer R, Buchwaldt L, Sharpe AG, Seguin-Swartz G, Coutu C, Hegedus DD (2004) Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: expressed sequence tag analysis identifies genes associated with fungal pathogenesis. Fungal Genet Biol 41:735–753
Liu M, Li ZY (2007) Genome doubling and chromosome elimination with fragment recombination leading to the formation of Brassica rapa-type plants with genomic alterations in crosses with Orychophragmus violaceus. Genome (in press)
Liu B, Wendel JF (2000) Retrotransposon activation followed by rapid repression in introgressed rice plants. Genome 43:874–880
Liu S, Wang X, Fan Z, Pang Y, Sun X, Wang X, Tanga K (2004a) Molecular cloning and characterization of a novel cold-regulated gene from Capsella bursa-pastoris. DNA Seq 15:262–268
Liu ZL, Wang YM, Shen Y, Guo WL, Hao S, Liu B (2004b) Extensive alterations in DNA methylation and transcription in rice caused by introgression from Zizania latifolia. Plant Mol Biol 54:571–582
Luo P, Fu HL, Lan ZQ, Zhou SD, Zhou HF, Luo Q (2003) Phytogenetics studies on intergeneric hybridization between Brassica napus and Matthiola incana. Acta Bot Sin 45:432–436
Ma N, Li ZY, Cartagena JA, Fukui K (2006) GISH and AFLP analyses of novel Brassica napus lines derived from one hybrid between B. napus and Orychophragmus violaceus. Plant Cell Rep 25:1089–1093
Martin-Sanchez JA, Gomez-Colmenarejo M, Del Moral J, Sin E, Montes MJ, Gonzalez-Belinchon C, Lopez-Brana I, Delibes A (2003) A new Hessian fly resistance gene (H30) transferred from the wild grass Aegilops triuncialis to hexaploid wheat. Theor Appl Genet 106:1248–1255
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–479
Natali L, Giordani T, Polizzi E, Pugliesi C, Fambrini M, Cavallini A (1998) Genomic alterations in the interspecific hybrid Helianthus annuus × Helianthus tuberosus. Theor Appl Genet 97:1240–1247
Park RJ (1967) The occurrence of mustard oil glucosides in Lepidium hyssopifolium Desv., L. bonariense (L.) and Capsella bursa pastoris (L.). Medic Aust J Chem 20:2799–2801
Peterka H, Budahn H, Schrader O, Ahne R, Schütze W (2004) Transfer of resistance against the beet cyst nematode from radish (Raphanus sativus) to rape (Brassica napus) by monosomic chromosome addition. Theor Appl Genet 109:30–41
Prakash NS, Marques DV, Varzea VM, Silva MC, Combes MC, Lashermes P (2004) Introgression molecular analysis of a leaf rust resistance gene from Coffea liberica into C. arabica L. Theor Appl Genet 109:1311–1317
Riera-Lizarazu O, Rines HW, Phillips RL (1996) Cytological and molecular characterization of oat × maize partial hybrids. Theor Appl Genet 93:123–135
Ronicke S, Hahn V, Horn R, Grone I, Brahm L, Schnabl H, Friedt W (2004) Interspecific hybrids of sunflower as a source of Sclerotinia resistance. Plant Breed 123:152–157
Shaked H, Kashkush K, Ozkan H, Feldman M, Levy AA (2001) Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy in wheat. Plant Cell 13:1749–1759
Shan XH, Liu ZL, Dong ZY, Wang YM, Chen Y et al (2005) Mobilization of the active mite transposons mPing and Pong in rice by introgression from wild rice (Zizania latifolia Griseb.). Mol Biol Evol 22:976–990
Sigareva MA, Earle ED (1999) Regeneration of plants from protoplasts of Capsella bursa-pastoris and somatic hybridization with rapid cycling Brassica oleracea. Plant Cell Rep 18:412–417
Snowdon RJ (2007) Cytogenetics and genome analysis in Brassica crops. Chromosome Res 15:85–95
Song K, Lu P, Tang K, Osborn TC (1995) Rapid genome change in synthetic polyploids of Brassica and its implications for polyploidy evolution. Proc Natl Acad Sci USA 92:7719–7723
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M et al (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–4414
Wang YM, Dong ZY, Zhang ZJ, Lin XY, Shen Y, Zhou D, Liu B (2005) Extensive de Novo genomic variation in rice induced by introgression from wild rice (Zizania latifolia Griseb.). Genetics 170:1945–1956
Yang YW, Lai KN, Tai PY, Li WH (1999) Rates of nucleotide substitution in angiosperm mitochondrial DNA sequences and dates of divergence between Brassica and other angiosperm lineages. J Mol Evol 48:597–604
Zhao HJ, Huang YJ, Wang YY (1995) Comparison experiments of new lines from the intergeneric crosses between Brassica napus and Capsella bursa-pastoris, Isatis indigotica. Hubei J Agri Sci 1:8–11
Zhong XB, Hans de Jong J, Zabel P (1996) Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescence in situ hybridization (FISH). Chromosome Res 4:24–28
Zhou TY (1987) Flora of China, vol 33. Science Press, Beijing, pp 84–85
Acknowledgments
The study was supported by Hubei Provinc Natural Science Foundation (2002AC015) and by a grant from Education Ministry of PR China and by PCSIRT (IRT0442). We thank Honghai Guo for S. sclerotiorum infection and Dr. Xianhong Ge for discussions. The critical reading of the manuscript by Prof. Shyam Prakash from Indian Agricultural Research Institute, New Delhi is greatly appreciated.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by K. Toriyama.
Rights and permissions
About this article
Cite this article
Chen, HF., Wang, H. & Li, ZY. Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris . Plant Cell Rep 26, 1791–1800 (2007). https://doi.org/10.1007/s00299-007-0392-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-007-0392-x