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Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNAAFLP Techniques
Agricultural Sciences in China
October 2008
2008, 7(10): 1181-1188
Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNAAFLP Techniques FAN Shu-ying, LE Jian-gang, CHENG Guang-jie and WU Cai-jun College of Agronomy, Jiangxi Agricultural University, Nanchang 330045, P.R.China
Abstract Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) transcriptome map with cDNA-AFLP techniques was constructed. The inbred line Aijiaohuang 97-3-2, the inbred line Baimanjing 001-24 of turnip [B. campestris ssp. rapifera (Matzg.) Sinsk] and 183 F6 (recombinant inbred population) plants were used as experimental materials. cDNAs were synthesized from total RNA extracted from young leaves at rosette stage. 256 pairs of cDNA-AFLP primers were used to detect the polymorphisms between parents Aijiaohuang 97-3-2 and Baimanjing 001-24. 56 pairs of cDNA-AFLP primers with high polymorphisms were screened from 256 pairs of primer by DNA-AFLP techniques. The genetic diversity of parents and 183 F6 progenies was detected by 56 pairs of cDNA-AFLP primers. The segregation and distribution of cDNA-AFLPs molecular marker were analyzed to construct transcriptome map amongst parents and F6 plants. A total of 164 cDNA-AFLPs marker loci were mapped into 13 linkage groups which covered 1 401.2 cM with an average distance of 9.7 cM. It was the first transcriptome map of Chinese cabbage using cDNA-AFLP technique. Key words: Brassica campestris ssp. chinensis, cDNA-AFLP, transcriptome map
INTRODUCTION Chinese cabbage (Brasscia campestris ssp. chinensis), which originates in China, is one of the most important vegetable crops; it is cultivated extensively and distributed widely in China and is very important in agriculture. So, it is necessary to study genetic improvement with molecular map in this crop. In the past, the research of classical genetics in Chinese cabbage lagged because of lack of morphological marker and no classical genetic map was constructed. Now the development of molecular marker makes it possible to construct Chinese cabbage molecular map. Song et al. (1991) constructed the first RFLP map with F2 population of an intercross between michili and spring broccoli. Pakchoi rape, turnip type rape and Jingshuicai
were used to research corresponding molecular genetic map respectively (Chyi et al. 1992; Kole et al. 1997; Novakova et al. 1996; Nozaki et al. 1997; Tanhuanpaa et al. 1996; Teutonico and Osbom 1994). Ajisaka et al. (1995) constructed Chinese cabbage RAPD molecular map which covered 860 cM with 115 RAPD and 2 isozyme markers using combinations between varieties. Matsumoto et al. (1998) constructed a Chinese cabbage genetic map which covered 735 cM with 63 RFLP markers. Zhang et al. (2000) constructed the first Chinese RAPD map with F2 population which derived from an intercross between turnip and Chinese cabbage. Lu et al. (2002) constructed a molecular map covering 1 810.9 cM with 131 genetic markers by RAPD and AFLP methods, using F2 segregating population of the cross between Chinese cabbage and turnip. With AFLP and RAPD genetic analysis, Yu et al. (2003) constructed
This paper is translated from its Chinese version in Scientia Agricultura Sinica. FAN Shu-ying, Professor, E-mail: [email protected]; Correspondence WU Cai-jun, E-mail: [email protected]
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
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a molecular map of 2 665.7 cM including 352 genetic markers. Zhang et al. (2005) constructed a 826.3 cM molecular linkage map using DH lines obtained by microspore culture from the F1 between parents white heart Chinese cabbage of high-resistant TuMV 91-112 and orange red heart of high-sensitive TuMV T12-19, the map included 10 linkage groups and 406 marker loci with an average genetic interval of 2.0 cM; the number of linkage groups and chromosomes was same. Amongst the finished Chinese cabbage molecular linkage maps, most of them were constructed with F 2 population. Since F2 population is impermanency, it is difficult to make a comparative study between these molecular linkage maps and to locate the quantitative characters of Chinese cabbage exactly. In international, DH population and recombinant inbred lines are widely used to construct molecular map. Plant genetic map construction with kinds of molecular markers is now popular, but the report about constructing transcriptome map is less presently. Kurata (1994) constructed the first plant gene expression map with 833 expressed sequence tags (ESTs) which came from the root tissue and callus of rice. Suarez et al. (2000) analyzed a pair of parents in cassava groups by cDNA-AFLP, and got 500 transcript-derived fragments (TDFs) which could express in their parents, respectively; they cloned and analyzed 50 in 500 TDFs, and located some markers in their genetic map. Brugmans et al. (2002) constructed transcriptome map of potato with cDNA-AFLP. Li et al. (2003) constructed transcriptome map by SRAP (related sequence amplified polymorphism), using broccoli and cauliflower, and believed constructing transcriptome map was a valid method to compare sequence for the genomes with no prior sequence information. Transcriptome map is a kind of molecular genetic map constructed using ESTs. ESTs not only provide many molecular markers for constructing genetic map of genome, but also the ESTs which come from different species, different periods and different tissues and organs, to offer valid information for comparing gene functions and discovering and identifying new genes (Zhang et al. 2003). In this study, recombinant inbred lines were prepared with cross combinations between aijiaohuang inbred line 97-3-2 and Baimanjing turnip inbred line 001-24, transcriptome map was constructed using cDNA-AFLP.
FAN Shu-ying et al.
MATERIALS AND METHODS Materials 250 seeds were randomly selected from F2 progeny of an intercross between Aijiaohuang (B. campestris L. ssp. chinensis Makino cv. Aijiaohuang) inbred line 973-2 and Baimanjing turnip [B. campestris ssp. rapifera (Matzg.) Sinsk cv. Baimanjing] inbred line 001-24, and produced 183 recombinant inbred lines of F6 by single seed descent. The groups of parents, F1 and F6 were sowed on seedbed, respectively, then transplanted in the experimental plot, which was repeated three times according to randomized block design. Field managements were conventional. Tender leaves were samples and numbered plants at rosette phase. TRIzol®, Taq DNA polymerase, and SMARTTM PCR cDNA Synthesis Kit were bought from Life Technologies, Promega (Shanghai) and Clontech, respectively. EDTA, DEPC, Acrylamide, Bis-adrylamide, carbamide, ammonium persulfate, TEMED, and so on were all bought from Shanghai Sangon, joints and primers of cDNA-AFLP were synthesized by Shanghai Sangon, the sequences were: Taq I joints: 5´-GACGATGAGTCCTGAC-3´; 5´CGGTCAGGACTCAT-3´; Taq I pre-amplification primers: 5´-GACGATG AGTCCTGACCGA-3´; Taq I selective amplification primers: 5´-GATGAGT CCAGACCGANN-3´ (N presents any type of ATCG, a total of 16 pairs of primers between T4-T9); Ase I joints: 5´-GCGTAGACTGCGTACC-3´; 5´-TA GGTACGCAGTC-3´; Ase I pre-amplification primers: 5´-CTCGTA GACTGCGTACCTAAT-3´; Ase I selective amplification primers: 5´-GACTG CGTACCTAATNN-3´ (a total of 16 pairs of primers between A4- A19).
METHODS Completed RNA extraction and cDNA synthesis The completed RNA was extracted by TRIzo1® from respective tender leaves of parents, hybrid and recom-
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNA-AFLP Techniques
binant inbred line in accordance with the TRIzol® User Manual. The first and second chains of cDNA were synthesized following the SMARTTM PCR cDNA Synthesis Kit User Manual.
Methods of cDNA-AFLP cDNA-AFLP was carried out according to cDNA-AFLP Procotol on http://www.dpw.wau.nl/pv/index.htm. Elect r o p h o r e s i s w a s p e r f o r m e d w i t h 6 % PA G E (polyacrylamide gel electrophoresis), 1 × TBE electrophoretic buffer, and 1 700 V, and ceased when the distance was 10 cm between xylene cyanol and the fringe of below, then silver-staining was detected.
cDNA-AFLP markers separation and detection of F6 population Polymorphism was detected between parents 97-3-2 and 001-24 with 256 pairs of cDNA-AFLP primers, high polymorphism primers were selected to analyze F6 population. Each amplified reaction which was selected included a group of parents, F 1 and blank, respectively, as control to confirm the amplified polymorphism markers resources and eliminate the bands that appeared when compared.
Data collection and coding AFLP are dominant markers, the bands “1” represent progenies derived from female parent Aijiaohuang inbred line 97-3-2, “2” represent progenies derived from male parent Baimanjing turnip 001-24, and “0” represent the unclear or no data bands. The names of cDNA-AFLP markers consist of primer combinations names and approximate length of amplified fragments.
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= 4, largest interval was 35 cM), then sorted with command “order” and “ripple”, inserted by “try” and “build”. Third, the recombination rate was transformed into genetic map interval unite (cM) with function of Kosambi. Finally, the linkage map was constructed by Mapchart2.0.
RESULTS Selection of polymorphism primers 256 pairs of primer combinations were used for cDNAAFLP markers of parents which were at rosette phase, 11 571 bands were amplified in total, with an average of 45.2 bands for per primer combination, and 1 135 polymorphism bands were detected. The number of polymorphism bands was quite different for different primers, the least was 1, the most was up to 13, and the average was 3.7 per primer. 56 pairs of primers were selected according to the number, intensive and repeatability of amplified polymorphism bands (Table 1). Polymorphism bands of the 56 pairs of primers were verified further with some individuals of F 1 and F 6 populations, the results indicated high polymorphism (Fig.1).
cDNA-AFLP markers of F6 population separation 56 pairs of primers with high polymorphism were used to analyze F6 population, 264 high definition polymorphism bands in which 139 bands were from female parent Chinese cabbage and 125 bands were from male parent turnip were obtained. The F2 tests indicated that 226 bands segregated at 1:1 ratio and 38 bands (14.4%) showed segregation distortion.
cDNA-AFLP molecular map construction Map construction on computer with Mapmaker/ Exp version 3.0 The separated data was tested for goodness of fit, the markers which segregated at 1:1 ratio were selected to construct linkage map with software Mapmaker/Exp (version 3.0) on PC. First, the markers were divided into different linkage groups by command “group” (LOD
226 cDNA-AFLP markers which segregated at 1:1 ratio in recombinant inbred line population were used for linkage analysis; a genetic map with 13 linkage groups which had 34, 24, 19, 15, 14, 13, 9, 8, 7, 6, 6, 5, and 4 cDNA-AFLP markers, respectively, was constructed (Fig.2). This map covered 1 401.2 cM and included 164 cDNA-AFLP markers, the max interval was 32.6
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Table 1 Selected primer combinations for cDNA-AFLP analysis of F6 population derived from the cross of Brassica campestris L. ssp. chinensis and Brassica campestris L. ssp. rapifera No.
Primer combination
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 26 27 28
A4T14 A4T16 A5T5 A5T9 A5T16 A6T7 A6T11 A6T15 A7T8 A7T12 A7T15 A7T16 A7T18 A8T6 A8T10 A8T16 A8T17 A8T18 A9T10 A9T14 A9T16 A10T6 A10T7 A11T7 A11T8 A11T10 A12T7 A12T9
Number of polymorphism bands 7 7 9 6 9 10 6 6 7 6 6 7 6 8 6 7 9 7 6 9 6 10 8 10 12 8 9 6
No. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
Primer combination A12T11 A12T13 A13T5 A13T10 A14T7 A14T12 A14T16 A14T17 A14T18 A15T9 A15T10 A15T12 A15T16 A16T13 A17T4 A17T6 A17T10 A17T14 A17T17 A17T18 A17T19 A18T4 A18T6 A18T19 A19T4 A19T7 A19T9 A19T19
Number of polymorphism bands 7 13 7 9 6 8 7 7 8 6 6 8 9 6 8 8 7 7 8 6 7 8 9 7 6 10 6 7
markers, about 37.9% markers were not involved in linkage groups. The cDNA-AFLP markers of this map were unequal distributed in linkage groups; the interval was more than 20 cM in group 3, group 4, group 5 and group 10. Group 3, group 5, group 10, group 11 and group 13 were unsaturated, in which the average interval was more than 10 cM. Group 1 had the most markers of 34; group 13 had the least markers of 4. Of the 164 cDNA-AFLP markers in this map, 91 markers were from female parent and 73 markers were from male parent.
DISCUSSION
Fig. 1 Identification of the selected cDNA-AFLP primer combination using F6 plants of the cross between B. campestris L. ssp. chinensis and B. campestris L. ssp. rapifera with cDNA-AFLP markers. 1-3, female parent 97-3-2, male parent 001-24, and hybrid; 4-12, F6 plant. The primer is A10T6.
cM, the mini interval was 3.8 cM and the average was 9.7 cM (Table 2). Amongst the 264 polymorphism
Feasibility of using polymorphism of cDNA-AFLP as molecular marker Transcriptome map (also expression map or exon map) is a molecular genetic map constructed by ESTs. It could be used to analyze functions of genes because EST is a product of genetic template for protein synthesis and ultimate decision of morphological traits and
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Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNA-AFLP Techniques
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Table 2 The molecular map of Brassica campeastris L. ssp. chinensis and linkage groups with F6 population derived from the cross of B. campestris L. ssp. chinensis and Brassica campeastris L. ssp. rapifera with cDNA-AFLP markers Linkage group Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Group 11 Group 12 Group 13 Average Total
Number of marker
Length of group (cM)
Mean interval (cM)
Max interval (cM)
Min interval (cM)
34 24 19 15 14 13 9 8 7 6 6 5 4
262.7 211.2 188.2 131.2 135.0 99.9 72.3 67.0 45.3 63.1 52.6 38.2 34.5
8.0 9.2 10.5 9.4 10.4 8.3 9.0 9.6 7.6 12.6 10.5 9.6 11.5 9.7
14.6 18.1 32.6 21.5 22.8 11.7 12.5 15.6 11.6 21.3 16.3 13.2 16.2
3.8 4.5 5.3 4.3 5.2 4.8 6.2 6.2 4.5 6.3 6.2 8.4 6.5
164
1 401.2
characteristics of tissues and organs (Zhang et al. 2003). Since invented by Bachem et al. (1996), the cDNAAFLP technique, during which cDNA reversely transcripted from mRNA is used as template and polymorphism differentiation of genomic expression sequences are mainly displayed while advantages of AFLP such as abundant polymorphism, high stability and dispensation with sequence information are preserved, has been developing as a common method to display gene differential expression, construct genetic linkage map of expression genes and clone genes (Bachem et al. 1996, 1998, 2001; Dellagi et al. 2000). Brugmans et al. (2002) analyzed the differences of the transcriptions between individuals of double haploid population of asexual potato and individuals of Arabidopsis F2 population derived from the cross between inbred lines, and constructed the transcriptome map using individual transcripts as genetic markers. It was found that the transcript markers were distributed all around the genome but not just in the centromere area; moreover, most of them showed single nucleotide polymorphism. The length of these cDNA-AFLP polymorphic fragments was around 50-500 bp. As compared to genome linkage map, though the transcript markers did not appear as a cluster in some specific location of chromosome, the cDNA-AFLP markers were found frequently in active transcription area. The same results they got from the research of Arabidopsis using cDNA-AFLP. In their study, a lane output 1-7 clear cDNA-AFLP polymorphism bands, with these bands as a transcriptome map was constructed easily, and the transcript mark-
ers were uniformly distributed on all chromosomes. Based on the results, they believed, as a molecular technique, cDNA-AFLP had the same reliability as the other molecular techniques of genome.
Characteristics of transcription map with polymorphism of cDNA-AFLP markers In this article, 183 plants of F6 recombinant inbred line population derived from Chinese cabbage × turnip were used to get 264 clear polymorphism bands by cDNAAFLP; most of the bands were 100-500 bp. 164 cDNAAFLP markers included in 13 linkage groups (Fig.2) were used to construct transcriptome map. Compared to Brugmans’ map (Brugmans et al. 2002), common features were obtained: some linkage groups had just several markers, and some had tens of markers; furthermore, the interval between markers in some groups was more than 30 cM. And the comparison to another map constructed with broccoli and cauliflower by Li et al. (2003) got similar results. In this study, the longest group had 34 markers and the max interval was 32.6 cM; in the map constructed by Li et al. (2003), the longest linkage group included 49 markers, the max interval was 43.8 cM, and most of cDNA-AFLP polymorphic fragments were 100-500 bp. Li et al. (2003) believed constructing transcriptome map could locate genes accurately, but also could detect directly the functions of genes. Besides, the target-genes could be easily found since the transcriptome map takes cDNA as molecular markers and eliminates
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Fig. 2 Transcriptome map of cDNA-AFLP markers with F6 population derived from the cross of B. campestris L. ssp. chinensis and B. campestris L. ssp. rapifera.
the introns and repeated genes; moreover, it shorts the genome and closes the markers and functional genes by splicing. Brugmans et al. (2002) held that transcriptome map could locate transcript factors and analyze the quantitative characters’ change from the transcription level. The transcription map constructed
using cDNA molecular markers enables to select parents on mRNA molecular level; it enhances the efficiency and security of parents selection and accelerates the process of breeding. Although the transcriptome map has many advantages, it also has shortages, compared to genomic
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNA-AFLP Techniques
linkage map: (1) The interval between markers was too large in some groups. For Arabidopsis, the interval was 39 cM, and 43.8 cM for cauliflower, and for this study, it was 32.6 cM; (2) many markers could not be involved in map. Brugmans et al. (2002) reported 331 markers, but only 218 markers were used to construct the map. Li et al. (2003) constructed map using 247 out of 281 markers. In this article, 226 markers showed a 1:1 segregation ratio, and constructed map had 164 markers; (3) in this study, some markers were found in two or more linkage groups. Such as the marker A11T10_326 found in group 1 and group 2, the marker A8T16_275 were found in group 2 and group 6. The main reasons could be: (1) currently, all of the transcriptome maps do not cover genomes of plants. This could be the main reason that some markers are not involved in map and the interval was too large; (2) lack of professional software for constructing transcriptome map; (3) multi-copy or transcription regulators might lead to some markers appearing in 2 or more linkage groups. Besides, Li et al. (2003) found that a few transcript markers were RNA (non-coding protein) which might regulate transcription; these markers could exist in different linkage groups. Because accurate QTL location and map-based cloning need more transcript markers, three measures could be taken in practice: First, target markers should be selected according to research goal, such as targeted genes location and clone; second, the markers should be selected in the max interval region or the parents with great difference in max interval region to construct map population; third, to find new transcript markers. The transcriptome map of this article included 13 linkage groups; Chinese cabbage and turnip have 10 pairs of chromosomes, so at least 3 pairs of chromosomes could have the regions of frequent exchange or vacant marker. The corresponding relationship between linkage groups and chromosomes is still unclear, but the development of in situ hybridization provides a feasible way to solve it.
CONCLUSION The map in this article covered 1 401.2 cM with a max interval of 32.6 cM, a minimum of 3.8 cM and an average of 9.7 cM, a total of 164 cDNA-AFLP markers
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were mapped into 13 linkage groups in which the longest group had 34 markers and the shortest had 4 markers. It was the first transcriptome map of Chinese cabbage (B. campestris L. ssp. Chinensis) using cDNA-AFLP technique.
Acknowledgements The study was funded by the National Natural Science Foundation of China (30560087).
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October 2008
2008, 7(10): 1181-1188
Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNAAFLP Techniques FAN Shu-ying, LE Jian-gang, CHENG Guang-jie and WU Cai-jun College of Agronomy, Jiangxi Agricultural University, Nanchang 330045, P.R.China
Abstract Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) transcriptome map with cDNA-AFLP techniques was constructed. The inbred line Aijiaohuang 97-3-2, the inbred line Baimanjing 001-24 of turnip [B. campestris ssp. rapifera (Matzg.) Sinsk] and 183 F6 (recombinant inbred population) plants were used as experimental materials. cDNAs were synthesized from total RNA extracted from young leaves at rosette stage. 256 pairs of cDNA-AFLP primers were used to detect the polymorphisms between parents Aijiaohuang 97-3-2 and Baimanjing 001-24. 56 pairs of cDNA-AFLP primers with high polymorphisms were screened from 256 pairs of primer by DNA-AFLP techniques. The genetic diversity of parents and 183 F6 progenies was detected by 56 pairs of cDNA-AFLP primers. The segregation and distribution of cDNA-AFLPs molecular marker were analyzed to construct transcriptome map amongst parents and F6 plants. A total of 164 cDNA-AFLPs marker loci were mapped into 13 linkage groups which covered 1 401.2 cM with an average distance of 9.7 cM. It was the first transcriptome map of Chinese cabbage using cDNA-AFLP technique. Key words: Brassica campestris ssp. chinensis, cDNA-AFLP, transcriptome map
INTRODUCTION Chinese cabbage (Brasscia campestris ssp. chinensis), which originates in China, is one of the most important vegetable crops; it is cultivated extensively and distributed widely in China and is very important in agriculture. So, it is necessary to study genetic improvement with molecular map in this crop. In the past, the research of classical genetics in Chinese cabbage lagged because of lack of morphological marker and no classical genetic map was constructed. Now the development of molecular marker makes it possible to construct Chinese cabbage molecular map. Song et al. (1991) constructed the first RFLP map with F2 population of an intercross between michili and spring broccoli. Pakchoi rape, turnip type rape and Jingshuicai
were used to research corresponding molecular genetic map respectively (Chyi et al. 1992; Kole et al. 1997; Novakova et al. 1996; Nozaki et al. 1997; Tanhuanpaa et al. 1996; Teutonico and Osbom 1994). Ajisaka et al. (1995) constructed Chinese cabbage RAPD molecular map which covered 860 cM with 115 RAPD and 2 isozyme markers using combinations between varieties. Matsumoto et al. (1998) constructed a Chinese cabbage genetic map which covered 735 cM with 63 RFLP markers. Zhang et al. (2000) constructed the first Chinese RAPD map with F2 population which derived from an intercross between turnip and Chinese cabbage. Lu et al. (2002) constructed a molecular map covering 1 810.9 cM with 131 genetic markers by RAPD and AFLP methods, using F2 segregating population of the cross between Chinese cabbage and turnip. With AFLP and RAPD genetic analysis, Yu et al. (2003) constructed
This paper is translated from its Chinese version in Scientia Agricultura Sinica. FAN Shu-ying, Professor, E-mail: [email protected]; Correspondence WU Cai-jun, E-mail: [email protected]
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
1182
a molecular map of 2 665.7 cM including 352 genetic markers. Zhang et al. (2005) constructed a 826.3 cM molecular linkage map using DH lines obtained by microspore culture from the F1 between parents white heart Chinese cabbage of high-resistant TuMV 91-112 and orange red heart of high-sensitive TuMV T12-19, the map included 10 linkage groups and 406 marker loci with an average genetic interval of 2.0 cM; the number of linkage groups and chromosomes was same. Amongst the finished Chinese cabbage molecular linkage maps, most of them were constructed with F 2 population. Since F2 population is impermanency, it is difficult to make a comparative study between these molecular linkage maps and to locate the quantitative characters of Chinese cabbage exactly. In international, DH population and recombinant inbred lines are widely used to construct molecular map. Plant genetic map construction with kinds of molecular markers is now popular, but the report about constructing transcriptome map is less presently. Kurata (1994) constructed the first plant gene expression map with 833 expressed sequence tags (ESTs) which came from the root tissue and callus of rice. Suarez et al. (2000) analyzed a pair of parents in cassava groups by cDNA-AFLP, and got 500 transcript-derived fragments (TDFs) which could express in their parents, respectively; they cloned and analyzed 50 in 500 TDFs, and located some markers in their genetic map. Brugmans et al. (2002) constructed transcriptome map of potato with cDNA-AFLP. Li et al. (2003) constructed transcriptome map by SRAP (related sequence amplified polymorphism), using broccoli and cauliflower, and believed constructing transcriptome map was a valid method to compare sequence for the genomes with no prior sequence information. Transcriptome map is a kind of molecular genetic map constructed using ESTs. ESTs not only provide many molecular markers for constructing genetic map of genome, but also the ESTs which come from different species, different periods and different tissues and organs, to offer valid information for comparing gene functions and discovering and identifying new genes (Zhang et al. 2003). In this study, recombinant inbred lines were prepared with cross combinations between aijiaohuang inbred line 97-3-2 and Baimanjing turnip inbred line 001-24, transcriptome map was constructed using cDNA-AFLP.
FAN Shu-ying et al.
MATERIALS AND METHODS Materials 250 seeds were randomly selected from F2 progeny of an intercross between Aijiaohuang (B. campestris L. ssp. chinensis Makino cv. Aijiaohuang) inbred line 973-2 and Baimanjing turnip [B. campestris ssp. rapifera (Matzg.) Sinsk cv. Baimanjing] inbred line 001-24, and produced 183 recombinant inbred lines of F6 by single seed descent. The groups of parents, F1 and F6 were sowed on seedbed, respectively, then transplanted in the experimental plot, which was repeated three times according to randomized block design. Field managements were conventional. Tender leaves were samples and numbered plants at rosette phase. TRIzol®, Taq DNA polymerase, and SMARTTM PCR cDNA Synthesis Kit were bought from Life Technologies, Promega (Shanghai) and Clontech, respectively. EDTA, DEPC, Acrylamide, Bis-adrylamide, carbamide, ammonium persulfate, TEMED, and so on were all bought from Shanghai Sangon, joints and primers of cDNA-AFLP were synthesized by Shanghai Sangon, the sequences were: Taq I joints: 5´-GACGATGAGTCCTGAC-3´; 5´CGGTCAGGACTCAT-3´; Taq I pre-amplification primers: 5´-GACGATG AGTCCTGACCGA-3´; Taq I selective amplification primers: 5´-GATGAGT CCAGACCGANN-3´ (N presents any type of ATCG, a total of 16 pairs of primers between T4-T9); Ase I joints: 5´-GCGTAGACTGCGTACC-3´; 5´-TA GGTACGCAGTC-3´; Ase I pre-amplification primers: 5´-CTCGTA GACTGCGTACCTAAT-3´; Ase I selective amplification primers: 5´-GACTG CGTACCTAATNN-3´ (a total of 16 pairs of primers between A4- A19).
METHODS Completed RNA extraction and cDNA synthesis The completed RNA was extracted by TRIzo1® from respective tender leaves of parents, hybrid and recom-
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Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNA-AFLP Techniques
binant inbred line in accordance with the TRIzol® User Manual. The first and second chains of cDNA were synthesized following the SMARTTM PCR cDNA Synthesis Kit User Manual.
Methods of cDNA-AFLP cDNA-AFLP was carried out according to cDNA-AFLP Procotol on http://www.dpw.wau.nl/pv/index.htm. Elect r o p h o r e s i s w a s p e r f o r m e d w i t h 6 % PA G E (polyacrylamide gel electrophoresis), 1 × TBE electrophoretic buffer, and 1 700 V, and ceased when the distance was 10 cm between xylene cyanol and the fringe of below, then silver-staining was detected.
cDNA-AFLP markers separation and detection of F6 population Polymorphism was detected between parents 97-3-2 and 001-24 with 256 pairs of cDNA-AFLP primers, high polymorphism primers were selected to analyze F6 population. Each amplified reaction which was selected included a group of parents, F 1 and blank, respectively, as control to confirm the amplified polymorphism markers resources and eliminate the bands that appeared when compared.
Data collection and coding AFLP are dominant markers, the bands “1” represent progenies derived from female parent Aijiaohuang inbred line 97-3-2, “2” represent progenies derived from male parent Baimanjing turnip 001-24, and “0” represent the unclear or no data bands. The names of cDNA-AFLP markers consist of primer combinations names and approximate length of amplified fragments.
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= 4, largest interval was 35 cM), then sorted with command “order” and “ripple”, inserted by “try” and “build”. Third, the recombination rate was transformed into genetic map interval unite (cM) with function of Kosambi. Finally, the linkage map was constructed by Mapchart2.0.
RESULTS Selection of polymorphism primers 256 pairs of primer combinations were used for cDNAAFLP markers of parents which were at rosette phase, 11 571 bands were amplified in total, with an average of 45.2 bands for per primer combination, and 1 135 polymorphism bands were detected. The number of polymorphism bands was quite different for different primers, the least was 1, the most was up to 13, and the average was 3.7 per primer. 56 pairs of primers were selected according to the number, intensive and repeatability of amplified polymorphism bands (Table 1). Polymorphism bands of the 56 pairs of primers were verified further with some individuals of F 1 and F 6 populations, the results indicated high polymorphism (Fig.1).
cDNA-AFLP markers of F6 population separation 56 pairs of primers with high polymorphism were used to analyze F6 population, 264 high definition polymorphism bands in which 139 bands were from female parent Chinese cabbage and 125 bands were from male parent turnip were obtained. The F2 tests indicated that 226 bands segregated at 1:1 ratio and 38 bands (14.4%) showed segregation distortion.
cDNA-AFLP molecular map construction Map construction on computer with Mapmaker/ Exp version 3.0 The separated data was tested for goodness of fit, the markers which segregated at 1:1 ratio were selected to construct linkage map with software Mapmaker/Exp (version 3.0) on PC. First, the markers were divided into different linkage groups by command “group” (LOD
226 cDNA-AFLP markers which segregated at 1:1 ratio in recombinant inbred line population were used for linkage analysis; a genetic map with 13 linkage groups which had 34, 24, 19, 15, 14, 13, 9, 8, 7, 6, 6, 5, and 4 cDNA-AFLP markers, respectively, was constructed (Fig.2). This map covered 1 401.2 cM and included 164 cDNA-AFLP markers, the max interval was 32.6
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Table 1 Selected primer combinations for cDNA-AFLP analysis of F6 population derived from the cross of Brassica campestris L. ssp. chinensis and Brassica campestris L. ssp. rapifera No.
Primer combination
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 26 27 28
A4T14 A4T16 A5T5 A5T9 A5T16 A6T7 A6T11 A6T15 A7T8 A7T12 A7T15 A7T16 A7T18 A8T6 A8T10 A8T16 A8T17 A8T18 A9T10 A9T14 A9T16 A10T6 A10T7 A11T7 A11T8 A11T10 A12T7 A12T9
Number of polymorphism bands 7 7 9 6 9 10 6 6 7 6 6 7 6 8 6 7 9 7 6 9 6 10 8 10 12 8 9 6
No. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
Primer combination A12T11 A12T13 A13T5 A13T10 A14T7 A14T12 A14T16 A14T17 A14T18 A15T9 A15T10 A15T12 A15T16 A16T13 A17T4 A17T6 A17T10 A17T14 A17T17 A17T18 A17T19 A18T4 A18T6 A18T19 A19T4 A19T7 A19T9 A19T19
Number of polymorphism bands 7 13 7 9 6 8 7 7 8 6 6 8 9 6 8 8 7 7 8 6 7 8 9 7 6 10 6 7
markers, about 37.9% markers were not involved in linkage groups. The cDNA-AFLP markers of this map were unequal distributed in linkage groups; the interval was more than 20 cM in group 3, group 4, group 5 and group 10. Group 3, group 5, group 10, group 11 and group 13 were unsaturated, in which the average interval was more than 10 cM. Group 1 had the most markers of 34; group 13 had the least markers of 4. Of the 164 cDNA-AFLP markers in this map, 91 markers were from female parent and 73 markers were from male parent.
DISCUSSION
Fig. 1 Identification of the selected cDNA-AFLP primer combination using F6 plants of the cross between B. campestris L. ssp. chinensis and B. campestris L. ssp. rapifera with cDNA-AFLP markers. 1-3, female parent 97-3-2, male parent 001-24, and hybrid; 4-12, F6 plant. The primer is A10T6.
cM, the mini interval was 3.8 cM and the average was 9.7 cM (Table 2). Amongst the 264 polymorphism
Feasibility of using polymorphism of cDNA-AFLP as molecular marker Transcriptome map (also expression map or exon map) is a molecular genetic map constructed by ESTs. It could be used to analyze functions of genes because EST is a product of genetic template for protein synthesis and ultimate decision of morphological traits and
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Table 2 The molecular map of Brassica campeastris L. ssp. chinensis and linkage groups with F6 population derived from the cross of B. campestris L. ssp. chinensis and Brassica campeastris L. ssp. rapifera with cDNA-AFLP markers Linkage group Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Group 11 Group 12 Group 13 Average Total
Number of marker
Length of group (cM)
Mean interval (cM)
Max interval (cM)
Min interval (cM)
34 24 19 15 14 13 9 8 7 6 6 5 4
262.7 211.2 188.2 131.2 135.0 99.9 72.3 67.0 45.3 63.1 52.6 38.2 34.5
8.0 9.2 10.5 9.4 10.4 8.3 9.0 9.6 7.6 12.6 10.5 9.6 11.5 9.7
14.6 18.1 32.6 21.5 22.8 11.7 12.5 15.6 11.6 21.3 16.3 13.2 16.2
3.8 4.5 5.3 4.3 5.2 4.8 6.2 6.2 4.5 6.3 6.2 8.4 6.5
164
1 401.2
characteristics of tissues and organs (Zhang et al. 2003). Since invented by Bachem et al. (1996), the cDNAAFLP technique, during which cDNA reversely transcripted from mRNA is used as template and polymorphism differentiation of genomic expression sequences are mainly displayed while advantages of AFLP such as abundant polymorphism, high stability and dispensation with sequence information are preserved, has been developing as a common method to display gene differential expression, construct genetic linkage map of expression genes and clone genes (Bachem et al. 1996, 1998, 2001; Dellagi et al. 2000). Brugmans et al. (2002) analyzed the differences of the transcriptions between individuals of double haploid population of asexual potato and individuals of Arabidopsis F2 population derived from the cross between inbred lines, and constructed the transcriptome map using individual transcripts as genetic markers. It was found that the transcript markers were distributed all around the genome but not just in the centromere area; moreover, most of them showed single nucleotide polymorphism. The length of these cDNA-AFLP polymorphic fragments was around 50-500 bp. As compared to genome linkage map, though the transcript markers did not appear as a cluster in some specific location of chromosome, the cDNA-AFLP markers were found frequently in active transcription area. The same results they got from the research of Arabidopsis using cDNA-AFLP. In their study, a lane output 1-7 clear cDNA-AFLP polymorphism bands, with these bands as a transcriptome map was constructed easily, and the transcript mark-
ers were uniformly distributed on all chromosomes. Based on the results, they believed, as a molecular technique, cDNA-AFLP had the same reliability as the other molecular techniques of genome.
Characteristics of transcription map with polymorphism of cDNA-AFLP markers In this article, 183 plants of F6 recombinant inbred line population derived from Chinese cabbage × turnip were used to get 264 clear polymorphism bands by cDNAAFLP; most of the bands were 100-500 bp. 164 cDNAAFLP markers included in 13 linkage groups (Fig.2) were used to construct transcriptome map. Compared to Brugmans’ map (Brugmans et al. 2002), common features were obtained: some linkage groups had just several markers, and some had tens of markers; furthermore, the interval between markers in some groups was more than 30 cM. And the comparison to another map constructed with broccoli and cauliflower by Li et al. (2003) got similar results. In this study, the longest group had 34 markers and the max interval was 32.6 cM; in the map constructed by Li et al. (2003), the longest linkage group included 49 markers, the max interval was 43.8 cM, and most of cDNA-AFLP polymorphic fragments were 100-500 bp. Li et al. (2003) believed constructing transcriptome map could locate genes accurately, but also could detect directly the functions of genes. Besides, the target-genes could be easily found since the transcriptome map takes cDNA as molecular markers and eliminates
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Fig. 2 Transcriptome map of cDNA-AFLP markers with F6 population derived from the cross of B. campestris L. ssp. chinensis and B. campestris L. ssp. rapifera.
the introns and repeated genes; moreover, it shorts the genome and closes the markers and functional genes by splicing. Brugmans et al. (2002) held that transcriptome map could locate transcript factors and analyze the quantitative characters’ change from the transcription level. The transcription map constructed
using cDNA molecular markers enables to select parents on mRNA molecular level; it enhances the efficiency and security of parents selection and accelerates the process of breeding. Although the transcriptome map has many advantages, it also has shortages, compared to genomic
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Chinese Cabbage-pak-choi Transcriptome Map Construction with cDNA-AFLP Techniques
linkage map: (1) The interval between markers was too large in some groups. For Arabidopsis, the interval was 39 cM, and 43.8 cM for cauliflower, and for this study, it was 32.6 cM; (2) many markers could not be involved in map. Brugmans et al. (2002) reported 331 markers, but only 218 markers were used to construct the map. Li et al. (2003) constructed map using 247 out of 281 markers. In this article, 226 markers showed a 1:1 segregation ratio, and constructed map had 164 markers; (3) in this study, some markers were found in two or more linkage groups. Such as the marker A11T10_326 found in group 1 and group 2, the marker A8T16_275 were found in group 2 and group 6. The main reasons could be: (1) currently, all of the transcriptome maps do not cover genomes of plants. This could be the main reason that some markers are not involved in map and the interval was too large; (2) lack of professional software for constructing transcriptome map; (3) multi-copy or transcription regulators might lead to some markers appearing in 2 or more linkage groups. Besides, Li et al. (2003) found that a few transcript markers were RNA (non-coding protein) which might regulate transcription; these markers could exist in different linkage groups. Because accurate QTL location and map-based cloning need more transcript markers, three measures could be taken in practice: First, target markers should be selected according to research goal, such as targeted genes location and clone; second, the markers should be selected in the max interval region or the parents with great difference in max interval region to construct map population; third, to find new transcript markers. The transcriptome map of this article included 13 linkage groups; Chinese cabbage and turnip have 10 pairs of chromosomes, so at least 3 pairs of chromosomes could have the regions of frequent exchange or vacant marker. The corresponding relationship between linkage groups and chromosomes is still unclear, but the development of in situ hybridization provides a feasible way to solve it.
CONCLUSION The map in this article covered 1 401.2 cM with a max interval of 32.6 cM, a minimum of 3.8 cM and an average of 9.7 cM, a total of 164 cDNA-AFLP markers
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were mapped into 13 linkage groups in which the longest group had 34 markers and the shortest had 4 markers. It was the first transcriptome map of Chinese cabbage (B. campestris L. ssp. Chinensis) using cDNA-AFLP technique.
Acknowledgements The study was funded by the National Natural Science Foundation of China (30560087).
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