* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Parental Genome Separation and Elimination of Cells and
Genetic engineering wikipedia , lookup
Skewed X-inactivation wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Genomic imprinting wikipedia , lookup
Human genome wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Molecular Inversion Probe wikipedia , lookup
Genome (book) wikipedia , lookup
Genome evolution wikipedia , lookup
Genomic library wikipedia , lookup
Y chromosome wikipedia , lookup
Genome editing wikipedia , lookup
Microevolution wikipedia , lookup
X-inactivation wikipedia , lookup
History of genetic engineering wikipedia , lookup
Neocentromere wikipedia , lookup
Annals of Botany 97: 993–998, 2006 doi:10.1093/aob/mcl073, available online at www.aob.oxfordjournals.org Parental Genome Separation and Elimination of Cells and Chromosomes Revealed by AFLP and GISH analyses in a Brassica carinata · Orychophragmus violaceus Cross Y U - W E I H U A , M I N L I U and Z A I - Y U N L I * National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding Technology, National Center of Oil Crop Improvement (Wuhan), Huazhong Agricultural University, Wuhan 430070, China Received: 5 January 2005 Returned for revision: 8 June 2005 Accepted: 10 February 2006 Published electronically: 19 April 2006 Background and Aims The phenomenon of parental genome separation during the mitotic divisions of hybrid cells was proposed to occur under genetic control in intergeneric hybrids between cultivated Brassica species and Orychophragmus violaceus (2n = 24). To elucidate further the cytological and molecular mechanisms behind parental genome separation, Brassica carinata (2n = 34) · O. violaceus hybrids were resynthesized and their chromosome/genomic complements analysed. Methods F1 hybrids of the cross were obtained following embryo rescue, and were investigated for their cytological behaviour and subjected to genomic in situ hybridization (GISH) and amplified fragment length polymorphism (AFLP) to determine the contribution of parental genomes. Key Results All the F1 plants with high fertility closely resembled B. carinata in morphological attributes. These were mixoploids with 2n chromosome numbers ranging from 17 to 35; however, 34, the same number as in B. carinata, was the most frequent number of chromosomes in ovary and pollen mother cells (PMCs). GISH clearly identified 16 chromosomes of B. nigra in ovary cells and PMCs with 2n = 34 and 35. However, no O. violaceus chromosome was detected, indicating the presence of the intact B. carinata genome and elimination of the entire O. violaceus genome. However, some AFLP bands specific for O. violaceus and novel for the two parents were detected in the leaves. Cells with fewer than 34 chromosomes had lost some B. oleracea chromosomes. F2 plants were predominantly like B. carinata, but some contained O. violaceus characters. Conclusions The cytological mechanism for the results involves complete and partial genome separation at mitosis in embryos of F1 plants followed by chromosome doubling, elimination of cells with O. violaceus chromosomes and some introgression of O. violaceus genetic information. Key words: Brassica carinata, Orychophragmus violaceus, intergeneric hybrids, genomic in situ hybridization, amplified fragments length polymorphism, genome separation, chromosome doubling, chromosome elimination, introgression. INTROD UCTION Hybridization between two different species with differentiated genomes is a major mechanism for the origin of plant species leading to the formation of allopolyploids (Soltis and Soltis, 1995; Otto and Whitton, 2000). Sometimes it also leads to the formation of a new species with features derived from both parents but without including the whole genomes of both parents through the process of introgressive hybridization (Rieseberg et al., 2000; Guttman, 2001), where one or more chromosomes or chromosomal segments of one parent species are incorporated into the genome of the other (Guttman, 2001). Wild and cultivated allopolyploids are well adapted; however, man-made ones are typically unstable and are rarely used as crops (Comai, 2000). In artificial crosses, chromosome instability may result in the production of partial hybrids with unexpected chromosome numbers (Riera-Lizarazu et al., 1996), mixoploids consisting of cells with different chromosome numbers (Schulz-Schaeffer, 1980) and haploids after elimination of one of the parental genomes (Kasha and Kao, 1970). Orychophragmus violaceus (2n = 24, genomes OO), a member of Cruciferae, is cultivated as an ornamental plant in China. This species has been reported to be valuable * For correspondence. E-mail [email protected] for the genetic improvement of Brassica crops, because of its high seed yield potential and good quality seed oil (Luo et al., 1994). Intergeneric hybrids between the six cultivated Brassica species in the U-triangle (U, 1935) and O. violaceus were obtained previously and the cytogenetics were documented (Li et al., 1995, 1998; Li and Heneen, 1999; Li and Liu, 2001). As a continuation of these studies, B. carinata · O. violaceus hybrids were resynthesized. F1 plants and their progeny were investigated cytologically and were subjected to genomic in situ hybridization (GISH) and amplified fragment length polymorphism (AFLP) analyses, and the results are reported here. MATERIALS AND METHODS Plant material and crosses The cross Brassica carinata (accession no. GO-7) · Orychophragmus violaceus (L.) O. E. Schulz [syn. Moricandia sonchifolia (Bunge) Hook Fil.] was made by hand emasculation and pollination as reported earlier (Li et al., 1998); the reciprocal cross was not attempted as previous experiments showed it to be unsuccessful. About 2–3 weeks after pollination, the immature embryos were cultured on MS agar medium (Murashige and Skoog, 1962). The leaves of young plants were collected for AFLP The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross 994 T A B L E 1. Phenotypes, cell components and AFLP patterns of F1 plants from a B. carinata · O. violaceus cross Type I II F1 plant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 III B. carinata O. violaceus a Stem coloura Seed coat coloura Percentage of ovary cells (2n = 34) Types of ovary cells Percentage of PMCs (2n = 34) Types of PMCs g p p g g g g g g g g g g g g g g g g g g g g g g g g/p y y b y y y y y y y y y y y y y y y y y y y y y y y b 34.8 44.0 88.9 37.5 56.0 50.0 53.3 45.5 51.5 57.1 30.0 48.4 51.2 50.0 46.9 31.9 45.2 38.7 30.0 23.3 40.5 37.5 43.4 39.4 27.3 8 10 2 12 9 11 11 8 7 6 8 7 9 7 12 16 8 13 11 12 10 9 12 9 9 62.4 89.3 100 80.9 93.8 85.4 86.5 92.0 91.9 85.5 66.7 96 91.4 100 100 72.7 100 90.2 83.3 91.7 72.2 94.4 88.2 75.7 86.4 4 2 1 3 2 3 3 3 3 3 3 2 2 1 1 3 1 4 2 3 3 2 3 3 2 Pollen stainabiltity No. of O. violaceus bands No. of deleted bands in B. carinata No. of novel bands 89.9 90.1 87.9 94.2 82.4 92.9 98.4 95.6 91.5 89.3 95.9 75 96.3 95.2 71.9 93.1 90.1 89.1 83.5 85.4 91 83.5 91.5 79.7 94.6 1 16 16 1 0 2 3 4 2 9 3 1 5 1 4 14 7 1 5 3 0 3 2 4 3 0 4 3 0 0 0 0 8 0 7 1 0 0 0 1 13 0 0 0 0 2 2 0 2 0 0 4 4 0 0 0 1 2 0 1 0 1 0 0 4 7 1 0 0 0 0 0 0 1 0 g = green; p = purple; y = yellow, b = brown. analyses. A population of ten F2 plants was raised from each F1 plant. DNA extraction and probe labelling DNA was extracted and purified from young leaves according to the method of Dellaporta et al. (1983). The DNA from B. oleracea var. alboglabra and B. carinata (GO-7) was sheared by boiling for 15 min and used as a block. The DNA from O. violaveus was labelled with digoxigenin-11-dUTP (Roche) or biotin-11-dUTP (Sabc in China) by the nick translation method, and the DNA from B. nigra ‘Giebra’ was labelled with biotin-11-dUTP using the same method, after purification, and dissolved in TE (10 M Tris.HCl, 0.5 M EDTA) at a concentration of 100 ng mL–1. The length of probe DNA fragments averaged approx. 500 bp. GISH and AFLP analyses The determination of chromosome numbers and meiotic observations were carried out according to Li et al. (1995). The slide preparation of chromosomes for GISH mainly followed the procedures of Zhong et al. (1996). In situ hybridization was carried out according to the protocols of Leitch et al. (1994). 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). RESULTS Morphology of F1 plants and progeny At least three F1 plants from each of the 25 surviving embryos from the B. carinata · O. violaceus cross were established in the field. The plants derived from the same embryo were morphologically indistinguishable. Based on the morphology and chromosome number in ovary cells, F1 plants were classified into three types: type I (2n = 24–35), type II (2n = 20–34) and type III (2n = 17–34) (Table 1). All F1 plants and their progeny had good seed-set on selfing or open pollination, although certain differences in pollen stainability were observed (Table 1). Plants of type I (no. 1) and III (nos 4–25) had matroclinous morphology. F2 plants were morphologically similar to the respective F1 plants. However, two plants of type II (nos 2 and 3) had morphological traits of B. carinata in addition to some characters from O. violaceus, such as the light purple stems in both plants and brown seed coat in plant no. 3. These characters were also observed in F2 plants, as among ten F2 plants in type II, half still maintained the light purple stems and two from plant no. 3 produced brown seeds. Cytogenetics and GISH analyses of F1 plants and their progeny All the F1 plants were mixoploids (2n=17–34), with 2n = 34 being the most frequent chromosome number as Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross A1 A2 B1 995 B2 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3 F I G . 1. Genomic in situ hybridization analyses on the ovary cells and PMCs in the hybrids between B. carinata and O. violaceus. Blue signals were due to 40 ,6-diamidino-2-phenylindole (DAPI) staining, red signals were due to Cy3 and green signals were due to fluorescein isothiocyanate (FITC). (A1and A2) The metaphase chromosomes in one root tip cell (2n = 24) of O. violaceus (A1) were fully labelled by its own genomic DNA probe (A2). (B1 and B2) In one AI PMC of plant no. 1 with 17 : 17 segregation and two lagging fragments (B1), the terminal parts of two chromosomes in each polar group were labelled by the O. violaceus probe (B2). In the four cells below, the B. nigra (red) and O. violaceus probes (green) were used and the green signals overlapped with the red signals. (C1–C3) In one root tip cell (2n = 35) from selfed seeds of plant no. 1 (C1), 16 chromosomes were labelled red (C2), but the minor chromosome (C1, red arrow) was unlabelled by both probes (C2 and C3). Large signals of both types appeared on satellites (C1, white arrows). (D1–D3 and E1–E3) One metaphase (2n = 34/30) and one interphase nucleus (D1 and E1) in one ovary of plant nos 2 and 7, respectively, with 16 and 15 chromosomes, respectively, labelled red (D2 and E2), and four and three chromosomes, respectively, labelled green at their terminal parts (D3 and E3) and both signals in the nucleus (D2, D3, E2 and E3). (F1–F3) One AI PMC of plant no. 9 with 17 : 17 segregation (F1) had eight large red signals (F2) and two small ones at terminals in each polar group (F3). Scale bar = 5 mm. 996 Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross in B. carinata. The majority of their pollen mother cells (PMCs) exhibited regular formation of 17 bivalents and 17 : 17 segregation as in B. carinata. This factor contributed to the high fertility of these F1 plants and production of B. carinata-type progeny. However, plant no. 1 (2n = 24–35) was an exception where some cells had 2n = 35, i.e. the full complement of B. carinata and an additional minor chromosome. This minor chromosome remained as univalent and was lagged or divided at anaphase I (AI) (Fig. 1B1 and B2). The root tip cells (Fig. 1C1–C3) and ovary cells of F2 plants also retained this minor chromosome. When the O. violaceus genomic probe was applied to O. violaceus preparations, signals distributed along the whole length of chromosomes (Fig. 1A1 and A2). In contrast, GISH in Brassica was normally characterized by strong signals at centromeric heterochromatin and only very weak hybridization on chromosome arms (Snowdon et al., 1997). This made it easier to distinguish the parental chromosomes in their hybrids. In all, 219 ovary cells with different chromosome numbers were observed. Signals from the O. violaceus probe showed the variation in size, number and intensity, but the majority of signals were located at the terminal parts or on one arm of the chromosome. This indicated that no intact O. violaceus chromosomes were included in ovary cells. When B. nigra and O. violaceus probes were used simultaneously, the signals from the B. nigra probe were at centromeric parts and were generally larger and stronger than those from the O. violaceus probe, which showed the existence of chromosomes of B. nigra origin. In all cells with 2n=34 and 35, 16 B. nigra chromosomes were easily identified (Fig. 1D1–D3). In some cells (2n < 34), the 16 chromosomes of B. nigra origin were clearly identified, suggesting the loss of some B. oleracea chromosomes, while in other cells (2n < 34) more chromosomes of B. nigra origin than B. oleracea origin were preferentially maintained (Fig. 1E1–E3), showing that most of the chromosomes lost from the complements of B. carinata were from the C genome of B. oleracea. In PMCs at diakinesis, metaphase I (MI) and AI, signals from the O. violaceus probe were again located at terminal or small and centrimeric parts of bivalents or chromosomes, and exhibited much larger variation in size, number and intensity than those of the B. nigra probe. The signals of the O. violaceus probe again confirmed no inclusion of the bivalents or chromosomes from O. violaceus in all PMCs with various pairings and segregations. In PMCs with 17 bivalents and 17 bivalents plus one univalent, eight bivalents from B. nigra were easily detected from the large and strong centromeric signals, and the signals from the O. violaceus probe were located at the end of the bivalents and overlapped with those of the B. nigra probe. Some signals of the O. vioalceus probe covered half of the bivalent. Eight bivalents of B. nigra origin were maintained in some PMCs with fewer than 17 bivalents. In PMCs at AI with 17 chromosomes in each polar group and occasionally one or two extra laggards, eight chromosomes from B. nigra were identified and two signals of different sizes from the O. violaceus probe appeared, the stronger one overlapping one from the M P1 P2 F1 Plants M F1 Plants M 500bp 400bp 300bp 200bp * A M P1 P2 300bp 200bp B F I G . 2. AFLP profiles generated from the primer combinations 50 -GACTGCGTACCAATTCATT-30 and 50 -GATGAGTCCTGAGTAACCC-30 (A) and 50 -GACTGCGTACCAATTCATG-30 and 50 -GATGAGTCCTGAGTAACAC-30 (B) in F1 plants nos 1–25. M = 100 bp DNA ladder, P1 = B. carinata, P2 = O. violaceus. O. violaceus-specific and deleted for B. carinata bands are indicated by black and white arrowheads, respectively (A), and the novel bands for two parents by black arrowheads (B). The asterisk marks the faint B. carinata band (A). B. nigra probe and the weaker one located on the nonB. nigra chromosome (Fig. 1F1–F3). The minor chromosome in 2n=35 of ovary cells and PMCs was not labelled by any probe. Among F2 plants, no chromosomes were wholly labelled by the O. violaceus probe, and the minor chromosome also remained unlabelled with any probe. Judging from the morphology of the fragment and its transmission between generations, it might be that this minor chromosome belongs to the C genome of B. oleracea. Moreover, in ovary cells and PMCs at diakinesis and AI, the larger signals from the O. violaceus probe overlapped with those from the B. nigra probe in most cases, and the weak signals were located on the remaining chromosomes of B. oleracea origin, which might result from the blocking of DNA from B. oleracea var. alboglabra. More signals from the O. violaceus probe overlapped with the chromosomes of B. nigra origin than those of B. oleracea origin, suggesting the higher homology between the dispersed repeats of B. nigra and O. violaceus. The strong signals or unique signals came from the satellites and nucleolar organizer regions (NORs) in all cells observed using the O. violaceus probe, if the satellites appeared. In cells (2n < 34) with recognizable satellites, the two satellite chromosomes were always included. AFLP analyses Using 35 pairs of primers, bands between 80 and 1000 bp were scored in the two parents and F1 plants. The numbers of bands amplified in B. carinata and O. violaceus were 1780 and 1858, respectively; of these, 338 bands (approx. 20 %) were common for the two parents. The 1780 bands Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross I II III BB × OO BB × OO BB × OO BO BO BO B O B+1O O–1 BB OO BB+2O OO–2 B O BB B O BO OO B+1O BB+2O B–1B +1O Hybrid cells O–1O+1B GS BB–2B+2O OO –2O+2B B–1B +1O BB–2B+2O 997 CD Meiosis Selfing F I G . 3. Possible situations of genome separation (GS) followed by chromosome duplication (CD) in the hybrids between Brassica (BB) species and O. violaceus (OO). When the parental genomes were completely separated during the mitotic and meiotic divisions of the hybrid cells (I), theoretically three types of plants, i.e. Brassica parents, hybrids and those with the O. violaceus genome and the Brassica cytoplasm, should be produced by the hybrids; however, the third type was not found, probably due to the low frequency and viability of the cells and gametes with the O. violaceus genome. The addition (II) and exchange (III) of some chromosomes between parental genomes during partial genome separation resulted in the production of Brassica species–O. violaceus additions and substitutions (exemplified by one chromosome). were averagely amplified in the leaves of each hybrid plant, and three types of special bands appeared in the F1 plants: O. violaceus-specific bands; deleted bands in B. carinata; and novel bands for two parents (Table 1; Fig. 2). Plant no. 1 had one O. violaceus-specific band but no other bands. Plants nos 2 and 3 with some O. violaceus characters exhibited 16 O. violaceus-specific bands out of 1797 and 1796 bands, respectively, the highest number among all F1 plants. In type III, plants nos 8, 10, 15, 16 and 24 had three types of bands, others had one or two typess of bands and plant no. 5 exhibited the same bands as B. carinata. In particular, plant no. 16 with the maximal number (16) of types of ovary cell showed 14 O. violaceus-specific, 13 deleted and seven novel bands, the latter two numbers being the highest among all F1 plants and much higher than those of others in this group. DISCUSSION The hybrids derived from the same cross of B. carinata (GO-7) · O. violaceus in the present and previous studies (Li et al., 1998) were cytologically similar and were mixoploids, consisting of cells with various chromosome numbers. The number of chromosomes with the highest frequency in somatic cells of ovaries and PMCs was 2n=34, the same number as in the female parent B. carinata. Cytogenetic results showed that the chromosome behaviour in these hybrids was repeatable and not an occasional event. The remarkable cytological characteristic of these F1 mixoploids was that all ovary cells (2n = 17–34) and PMCs contained B. carinata chromosomes without intact O. violaceus chromosomes (Fig. 1B–F). The majority of PMCs showed the same chromosome pairing and segregation as in normal B. carinata plants even though limited O. violaceus DNA was detected by AFLP (Table 1; Fig. 2). It is proposed that complete and partial separation of the parental genomes during mitosis reported earlier for the cross (Li et al., 1998) could well explain the present results, as illustrated in Fig. 3. The GISH identification of cells (2n = 34) with the whole B. carinata complement substantiated the proposal that cells with the O. violaceus complement or its chromosomes are eliminated during the process of embryo development and plant growth. The identification of cells with a complete and partial complement of B. carinata in one plant indicated the multiple origins of cells from different events. In future, efforts should be directed to determine the genomic complements of hybrids at early stages using GISH and molecular markers. Although no O. violaceus chromosomes were detected in ovary cells and PMCs, some O. violaceus-specific bands, deleted bands in B. carinata and new AFLP bands in the leaves of F1 plants indicated the existence of partial O. violaceus DNA, loss of some B. carinata genetic material and occasional recombination. Moreover, some hybrids and progeny (2n=34) showed some characters specific for O. violaceus, such as the brown seed coat and the purple colour on the stems and leaves, suggesting the introgression of O. violaceus genetic material or genes but beyond GISH resolution. The GISH results showed that more chromosomes from B. oleracea than from B. nigra (B. carinata evolving from the hybridization between B. oleracea and B. nigra) were lost in cells with partial B. carinata complements (2n < 34), which was the opposite to what was expected from the cytology in the hybrids of O. violaceus with B. oleracea and B. nigra (Li and Heneen, 1999). It is also interesting to note that the intact B. nigra genome was maintained in hybrids (B. carinata ·B. rapa, AABBCC) · O. violaceus 998 Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross (2n = 40–44) (X.-H. Ge and Z.-Y.Li, unpubl. res.). One possible reason for this could be attributed to the dominance of rRNA genes from the two ancestors of B. carinata because the hierarchy of rRNA gene transcriptional dominance was B. nigra > B. rapa > B. oleracea, and B. nigra rRNA transcripts were readily detected in both natural and synthetic lines of B. carinata, but B. oleracea transcripts were not detectable (Chen and Pikaard, 1997). The two satellite chromosomes which were always observed in the cells (2n < 34) with partial complements of B. carinata were of B genome origin and their retention helped to stabilize other chromosomes of this genome. The complete or partial elimination of the chromosomes from one parent during the mitotic divisions of the zygotes in hybrids from widely separated species was reported to result in haploid embryos and plants of one parent (Kasha and Kao, 1970) or partial hybrids with a haploid complement from one parent and some chromosomes of the other parent (Riera-Lizarazu et al., 1996). The cytological mechanisms in the present investigation are different from pseudogamy (Jörgensen, 1928) and semigamy (Coe, 1953; Turcotte and Feaster, 1967), or simply chromosome elimination during embryo development, and therefore seem to be intermediate between the classical hybrids of allopolyploids and chromosome elimination. ACKNOWLEDGEMENTS The study was supported by the grants from Hubei Province (2002AC015) and National Natural Science Foundation (30070413) and Education Ministry of the P.R. China and by PCSIRT (IRT0442). The constructive suggestions of two anonymous reviewers to revise the manuscript and the critical reading and modifying by Professor Shyam Prakash from the Indian Agricultural Research Institute, New Delhi were greatly appreciated. LITERATURE CITED Bassam B, Caetano-Anolles G, Gresshoff PM. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry 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. Proceedings of the National Academy of Sciences of the USA 94: 3442–3447. Coe GE. 1953. Cytology of reproduction in Cooperia pedonculata. American Journal of Botany 40: 335–343. Comai L. 2000. Genetic and epigenetic interactions in allopolyploid plants. Plant Molecular Biology 43: 387–399. Dellaporta SL, Wood J, Hicks JB. 1983. A plant DNA mini preparation: version II. Plant Molecular Biology Reports 1: 19–21. Guttman B. 2001. Evolution. In: Brenner S, Miller JH, eds. Encyclopedia of genetics. San Diego: Academic Press, Vol. 2, 663–666. Jörgensen CA. 1928. The experimental formation of heteroploid plants in the genus Solanum. Genetics 19: 133–211. Kasha KJ, Kao KN. 1970. High frequency haploid production in barley (Hordeum vulgare L.). Nature 225: 874–876. Leitch AR, Schwarzacher T, Jackson D, Leitch IJ. 1994. In situ hybridization: a practical guide. Microscopy handbook No. 27. Oxford: BIOS Scientific Publishers. Li Z, Heneen WK. 1999. Production and cytogenetics of intergeneric hybrids between the three cultivated Brassica diploids and Orychophragmus violaceus. Theoretical and Applied Genetics 99: 694–704. Li ZY, Liu Y. 2001. Cytogenetics of intergeneric hybrids between Brassica species and Orychophragmus violaceus. Progress in Natural Science 11: 721–727. Li Z, Liu HL, Luo P. 1995. Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus. Theoretical and Applied Genetics 91: 131–136. Li Z, Wu JG, Liu Y, Liu HL, Heneen WK. 1998. Production and cytogenetics of intergeneric hybrids Brassica juncea · Orychophragmus violaceus and B. carinata · O. violaceus. Theoretical and Applied Genetics 96: 251–265. Luo P, Lan ZQ, Li ZY. 1994. Orychophragmus violaceus, a potential edible-oil crop. Plant Breeding 113: 83–85. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum 15: 473–479. Otto SP, Whitton J. 2000. Polyploid incidence and evolution. Annual Review of Genetics 34: 401–437. Riera-Lizarazu O, Rines HW, Phillips RL. 1996. Cytological and molecular characterization of oat · maize partial hybrids. Theoretical and Applied Genetics 93: 123–135. Rieseberg LH, Bair SJE, Dardner KA. 2000. Hybridization, introgression, and linkage evolution. Plant Molecular Biology 42: 205–224. Schulz-Schaeffer J. 1980. Cytogenetics—Plants, Animals, Humans. New York: Springer-Verlag, 267–271. Snowdon RJ, Köhler W, Friedt W, Köhler A. 1997. Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids. Theoretical and Applied Genetics 95: 1320–1324. Soltis PS, Soltis DE. 2000. The role of genetics and genomic attributes in the success of polyploids. Proceedings of the National Academy of Sciences of the USA 97: 7051–707. Turcotte EL, Feaster CV. 1967. Semigamy in cotton. Journal of Heredity 58: 54–57. U N. 1935. Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Japanese Journal of Botany 7: 389–452. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A. 1995. Pot AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23: 4407–4414. 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 Research 4: 24–28.