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A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var. capitata L.)
Journal of Integrative Agriculture
April 2013
2013, 12(4): 596-602
RESEARCH ARTICLE
A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var. capitata L.) CHEN Chen1, 2, ZHUANG Mu1, FANG Zhi-yuan1, WANG Qing-biao1, ZHANG Yang-yong1, LIU Yu-mei1, YANG Li-mei1 and CHENG Fei2 1 2
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China Horticulture College of Qingdao Agricultural University, Qingdao 266109, P.R.China
Abstract The dominant genic male sterility (DGMS) gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage (Brassica oleracea var. capitata L.), has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants (CDMs399-3/CDMs399-3) is a laborious and time-consuming process. For marker-assisted selection (MAS) of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats (EST-SSR) and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis (BSA). By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygous male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding. Key words: cabbage (Brassica oleracea var. capitata L.), dominant genic male sterility (DGMS), expressed sequence tag-simple sequence repeats (EST-SSR), bulked segregant analysis (BSA), marker-assisted selection (MAS)
INTRODUCTION Cabbage (Brassica oleracea var. capitata L.) is cultivated widely around China and other countries such as USA, UK, Japan, Korea, India and etc. Because of exhibiting obvious heterosis, most cabbage cultivars released for market are F1 hybrids produced via self-incompatible lines or male-sterile lines. The production of hybrid seed using male-sterile lines is a reliable and economical choice for the efficient utilization of hetReceived 5 December, 2011
erosis in cabbage. The dominant genic male sterility (DGMS) gene CDMs399-3, also called as Ms-cd1 (Wang et al. 2005), was identified as a spontaneous mutant in an early spring cabbage line 79-399-3 in 1979 (Fang et al. 1997) and has been utilized well in cabbage commercial hybrid seed production in China (Fang et al. 2004). To date, the following aspects of the DGMS controlled by CDMs399-3 have been elucidated: (i) The development of male sterility is associated with abnormal degeneration of the cellulose cell wall and failure of microspore separation from tetrads during anther devel-
Accepted 28 February, 2012
Correspondence ZHUANG Mu, Tel: +86-10-82108756, Fax: +86-10-62174123, E-mail: [email protected]
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60277-4
A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage
opment (Fang et al. 1997; Lou et al. 2007); (ii) the male sterility in 79-399-3 is sensitive to temperature below 15°C, and can develop a few viable pollen grains in certain genetic background. Thus, homozygous malesterile line can be developed by selfing heterozygous male-sterile plants (Fang et al. 1997); (iii) the identification of the homozygous male-sterile plants from an F2 population, derived from selfing a heterozygous malesterile plant with a few viable pollen grains, is a timeconsuming and laborious process. The normal process is as follows: maintain all the male-sterile plants including homozygous male-sterile plants (1/4 of the F2 population) and heterozygous male-sterile plants (1/2 of the F2 population) by tissue culture while all the malesterile plants are test-crossing with a male-fertile tester (CDms399-3/CDms399-3), then cultivate each testcross plant with a population not less than 12 progeny plants to flower in next spring after vernalization, investigate the male-sterility segregating ratio of testcross progeny, finally obtain the homozygous genotype based on that testcross progeny showed 100% male-sterile (Fang et al. 2004). A reduction of the number of plants that need to be test-crossed, vernalized, and maintained by tissue culture will greatly reduce the cost of the development of superior male-sterile lines and facilitate the hybrid seed production. Molecular markers closely linked to the gene CDMs399-3 can make marker-assisted selection (MAS) feasible at the young-seedling stage. Based on bulked segregant analysis (BAS) method, A RAPD marker OPT11900 linked to the CDMs399-3 gene with the distance of 7.48 cM was identified (Wang et al. 1998), and converted into a sequence characterized amplified region (SCAR) marker (Wang et al. 2000b) and extended random primer amplified region (ERPAR) marker (Wang et al. 2000a). Using two backcross populations with the recurrent male parents of two flat-headed autumn cabbage inbred lines, a RFLP marker pBN11 linked to the CDMs399-3 gene was identified (Liu et al. 2003). Due to the limitation of RFLP technology, pBN11 has not been applied to MAS in cabbage yet. Three amplified fragment length polymorphism (AFLP) markers and three SCAR markers that linked to CDMs399-3 were mapped onto linkage group 9, corresponding to chromosome 3 of B. oleracea (Wang et al. 2005). 14 sequence-related amplified polymorphism (SRAP) markers and one simple sequence repeat (SSR) marker linked to CDMs399-3 were iden-
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tified using a segregating BC4 population with an inbred line of Chinese kale (B. oleracea var. alboglabra) as the recurrent male parent (Zhang et al. 2011). Most of the previous markers linked to CDMs399-3 were dominant markers, and no marker had been identified in a fertility-segregated population of spring cabbage line. SSRs are easy, high-efficient, co-dominant and reliable PCR-based markers. Because they are located in transcribed region of the genome, expressed sequence tag-simple sequence repeats (EST-SSR) can be developed rapidly and inexpensively from the EST database by data mining, and they could increase the efficiency of MAS (Gupta et al. 2004). Based on the development of EST-SSR in our lab (Chen et al. 2010), here we reported the identification of an EST-SSR marker linked to CDMs399-3 and use of this marker to rapidly obtain the homozygous male sterile plants of an earlymatured spring cabbage line 397 with a round head.
RESULTS Fertility survey of the two populations For the F2 segregating population P921, 56 plants were male-sterile and 17 were male-fertile. The segregation ratio of male sterility and male fertility was consistent with the expected ratio of 3:1 in a χ2 test (χ2=0.114 and P>0.05). Out of the 56 male-sterile plants of population P921, 7 plants were homozygous and the other 49 plants were heterozygous, indicating a distorted segregation ratio of CDMs399-3 may be resulted from competition among gametes for preferential fertilization (Lyttle 1991). The F2 segregating population P1170 consisted of 7 homozygous male-sterile plants, 9 heterozygous male-sterile plants and 4 fertile plants, which were consistent with 1:2:1 segregation (χ 2=1.1 and P>0.05). The results confirmed that the deduction that the male sterility in B. oleracea var. capitata L. is controlled by one single dominant gene (Fang et al. 1997).
Identification of SSR markers linked to the gene CDMs399-3 978 EST-SSR and 395 SSR primer pairs were screened to identify polymorphisms between the homozygous
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male sterile bulk and the male fertile bulk. The primer pairs that produced polymorphic bands were tested for a second time. Only one EST-SSR marker BoE332 (Appendix) was identified as a candidate marker possibly linked to the DGMS gene CDMs399-3. Scoring of BoE332 in bulks showed that only one approximate 270 bp band could be amplified in homozygous male-sterile plants, and one approximate 290 bp band in male-fertile plants. 73 plants of population P921 were screened with BoE332. BoE332 in population P921 showed that 8 of 73 plants were homozygous male-sterile, 48 of 73 plants were heterozygous male-sterile and 17 of 73 plants were male-fertile (Fig. 1). Compared with the results of the field investigation of male fertility, 2 of 56 male-sterile plants and 2 of 17 male-fertile plants tested by marker BoE332 were not consistent with the fertility results of field investigation. The linkage distance was calculated by scoring BoE332 for all 73 plants of the P921 population that had been identified phenotype for male fertility. The co-dominant marker BoE332 closely flanked the gene CDMs399-3 at 3.6 cM.
The MAS of CDMs399-3 using marker BoE332 Due to the marker BoE332 was not 100% linked to the
CHEN Chen et al.
gene CDMs399-3, the homozygous male-sterile plants identified using BoE332 might include heterozygous ones. Therefore, to further identify the real homozygous male-sterile plants, 56 male-sterile plants of population P921 were test-crossed, no less than 12 plants of each test-crossing population were cultivated, marker BoE332 was applied to check the genotypes of the 56 test-crossing populations before field evaluation, and the fertility investigation at flowering were carried out in greenhouse. The male-sterile plant was scored as homozygous male-sterile if all the test crossing plants showed both approximate 270 and 290 bp amplification bands (Fig. 2-A), while the male-sterile plant was scored as heterozygous if at least one of the test crossing plants showed only one approximate 290 bp band (Fig. 2-B). Based on the testing of marker BoE332, 7 homozygous male-sterile plants in population P921 were obtained finally. The result was 100% coincided with that of the fertility investigation. The MAS of homozygous male-sterile plants using marker BoE332 was established: firstly screen the plants of F2 population segregating in fertility; secondly screen no less than 12 plants for each test-crossing population derived from the male-sterile plants of F2 population; finally determine the results by combining results of the two steps.
Fig. 1 PCR products of the partial plants of F2 population P921 with EST-SSR marker BoE332. M, 100 bp DNA ladder; S, homozygous male-sterile bulk; F, male-fertile bulk; a, homozygous male-sterile plant in population P921; b, male-fertile plant in population P921; h, heterozygous male-sterile plant in population P921. *, the recombinant plant.
Fig. 2 PCR products of test crossing plants derived from homozygous male-sterile plant (A) and heterozygous male-sterile plant (B) with EST-SSR marker BoE332. M, 100 bp DNA ladder; S, homozygous male-sterile plant; F, male-fertile plant; b, male-fertile plant of test crossing population; h, heterozygous male-sterile plant of test crossing population.
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.
A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage
To further test the utility of BoE332, the same procedure was applied to F2 population P1170. The PCR screening results using BoE332 marker indicated that 5 plants were homozygous male-sterile, 11 plants were heterozygous male-sterile and 4 were male-fertile. The 16 male-sterile plants of population P1170 were then followed by test-crossing and male-sterile genotypic status was determined using BoE332 screening. 7 homozygous male-sterile plants were identified, which were coincided 100% with the field observation. The results indicated that marker BoE332 was valid for cabbage inbred line 397.
DISCUSSION Cabbage inbred line 397, a key parent material growing in spring with excellent desired characters such as round head, early maturity and good quality, has been successfully converted into several DGMS lines. These lines has been applied in hybrid seed production of several cultivars in china, such as Zhonggan17, Zhonggan18 and Zhonggan21, which have been released in almost all the areas of China (Fang et al. 2004, 2007; Yang et al. 2004). For cultivar improvement in cabbage, especially for key parent inbred lines as well as key line 397, more novel homozygous male-sterile lines were needed. Because the conventional selection is relatively long procedure, the availability of inexpensive, rapid, and reliable markers linked to CDMs399-3 would greatly improve the efficiency of cabbage breeding programs via MAS. The previously obtained markers linked to CDMs399-3 such as RAPD marker OPT11900 and ERPAR marker EPT11900 were dominant markers and identified from segregating populations of flat cabbage or other B. oleracea crops. They could not assist selection of the homozygous male-sterile plants in spring cabbage lines (Wang et al. 1998, 2000b). Because CDMs399-3 was mutated from the spring cabbage line 79-399-3, the high similarity of its genetic basis made development of practical marker linked to it in spring cabbage lines much difficult. The only previously reported SSR marker was 8C0909, which was linked to the MS-cd1 gene (the other name of CDMs399-3) with a distance of 2.06 cM in a kale population (Zhang et al. 2011). However, this marker showed no polymorphism in the populations we tested. After screening 978 EST-SSRs
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and 395 SSRs, we only obtained one co-dominant marker BoE332 that linked to the CDMs399-3 with a distance of 3.6 cM, indicating that the polymorphism surrounding the male sterility gene is very low in the genetic background of 397. To our knowledge, this is the first SSR maker identified in early spring round cabbage. Even though molecular markers linked to genic male sterility (GMS) have been reported in Brassica family (Lu et al. 2004; Lei et al. 2007; He et al. 2008; Feng et al. 2009; Zhang et al. 2011), there is little information of practical utilization in MAS. BoE332 has potential to be practically used to select homozygous male-sterile lines in fertility segregating population of cabbage key line 397. For example, using marker BoE332, 19 of 67 plants from a F2 population P1171 segregating in male fertility derived from cabbage line 397 were selected as homozygous male-sterile at first, 16 of 67 plants were identified finally as homozygous male-sterile combining the result of test-crossing populations of each male sterile plant (data not shown), and the MAS result was coincided with the field investigation during the flowering period. Therefore, although the linkage distance of BoE332 to CDMs399-3 is 3.6 cM, BoE332 was effective for assisted selection of homozygous male-sterile plants for spring cabbage line 397. SSR marker BoE332 might not be applied directly in MAS of the DGMS transferring of the other cabbage inbred lines (data not shown). The utilization of marker BoE332 in MAS was probably limited to its distance linked to the gene CDMs399-3. In order to obtain more closely linked markers, it might be necessary to employ PCR walking and screen much molecular markers such as SNP and InDel based on the latest advances of genome sequencing and resequencing in B. oleracea var. capitata in which our lab is participating. The molecular markers linked to the gene CDMs399-3 have been mapped onto linkage group O9, corresponding to chromosome 3 of B. oleracea and the region corresponds to chromosome 5 in Arabidopsis thaliana (Wang et al. 2005). In this study, the sequences of the PCR amplification bands of BoE332 correspond also to chromosome 5 in Arabidopsis thaliana (data not shown). Up to now, the DGMS system has been an effective hybrid seed production system in B. oleracea. However, very little is known regarding the molecular basis of the DGMS trait, which limits its wide appli-
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.
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cation to hybrid breeding. Based on the information of genome sequencing and resequencing in cabbage, cloning and characterization of CDMs399-3 gene in the future will greatly improve the development of homozygous male-sterile lines and further its application in hybrid breeding.
CONCLUSION In this study, we obtained a co-dominant EST-SSR marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the spring cabbage line 397 (Brassica oleracea var. capitata L.). BoE332 has potential to be practically used to MAS of homozygous male-sterile lines in fertility segregating population of cabbage key line 397.
CHEN Chen et al.
DNA isolation and SSR analysis Genomic DNA was isolated from fresh leaves of all the plants using a cetyltrimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1990) with minor modifications. The quality and concentration of extracted DNA were tested by using 1% agarose gel electrophoresis. BSA method was employed to identify SSR markers linked to the gene CDMs399-3. 978 EST-SSRs, designed from the published EST sequence of B. oleracea in NCBI (Appendix), and 395 SSRs (http://www.brassica.info) were used for SSR analysis. PCR amplification was carried out in a volume of 20 µL, containing 1 U Taq polymerase, 4 pmol of each EST-SSR or SSR primer, 200 µmol L-1 dNTPs, 1×PCR buffer (contains 1.5 mmol L-1 MgCl2) and 50 ng genomic DNA as the template.
MATERIALS AND METHODS Plant materials and populations 4 homozygous male-sterile plants and 5 male-fertile plants from an F2 segregating population P725 were used to form bulks (Michelmore et al. 1991) for screening EST-SSR and SSR markers. The F2 population P725 was generated by selfing a temperature sensitive heterozygous male-sterile plant from eight generations of backcrossing the early spring cabbage line 397 into the original male-sterile line 79-399-3. 73 plants of the F2 population P921 derived from selfing a temperature sensitive heterozygous male-sterile plant of the population P725, were used to obtain the ESTSSR marker linked to the DGMS gene CDMs399-3. 20 plants from another F2 population P1170 derived from selfing a different temperature sensitive male-sterile plant of the population P725 was used to further test the utilization of the obtained marker linked to the gene CDMs399-3 (Fig. 3). All the male-sterile plants of the population P921 and P1170 were maintained by tissue culture, and were test-crossed with the fertile plant, to determine whether male-sterile plants were homozygous or not. All plants were grown in the open field, and transplanted in the greenhouse of Institute of Vegetables and FLowers, Chinese Academy of Agricultural Sciences, Beijing, China, and cultivation management was conducted normally. Male fertility was investigated in both F2 populations and test cross populations in the spring. During the flowering period, fertility was characterized based on the observation of pollen grains in anthers of open flowers at least three times (Fang et al. 1997; Wang et al. 2000a).
Fig. 3 Segregation population of male fertility in cabbage. MsMs, homozygous male-sterile genotype; Msms, heterozygous malesterile genotype; msms, male-fertile genotype; Ts1 and Ts2 and Ts3, different temperature sensitive male-sterile plants; Bulk-1, homozygous male-sterile bulk; Bulk-2, male-fertile bulk.
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.
A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage
All the reagents were bought from Beijing Biomed Co., Ltd. (Bejing, China). The PCR profile was as follows: 94°C for 4 min, followed by 35 cycles at 94°C for 0.5 min, appropriate annealing temperature for 0.5 min, 72°C for 1 min, ending with a final extension step of 7 min at 72°C. The PCR products were separated on a 8% polyacrylamide gel (acrylamide:bis=29:1) and visualized with silver staining as described by Panaud et al. (1996).
Linkage analysis Polymorphic SSR marker bands were scored as homozygous sterile (a), fertile (b) and heterozygous sterile (h) for all samples. Linkage distance analysis was performed with Joinmap 4.0, with a minimum LOD score of 3 (Lander et al. 1987).
Acknowledgements We thank Prof. Elizabeth Earle, Department of Plant Breeding and Genetics, Cornell University, USA, for constructive advice on writing the manuscript. This research was supported by the National Science and Technology Ministry of China (2008BADB1B02 and 2009BADB8B03), the Core Research Budget of the Non-profit Governmental Research Institution (ICS, CAAS) (1610032011011), the China Agriculture Research System (CARS-25), and the National High Technology Research and Development Program of China (863 Program, 2012AA100101). This work was conducted at the Key Laboratory of Horticultural Crop Biology and Germplasm Innovation of the Ministry of Agriculture, China. Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm
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40, 320-324. (in Chinese) Fang Z Y, Sun P T, Liu Y M, Yang L M, Wang X W, Hou A F, Bian C S. 1997. A male-sterile line with dominant gene (Ms) in cabbage and its utilization for hybrid seed production. Euphytica, 97, 265-268. Feng H, Wei P, Piao Z Y, Liu Z Y, Li C Y, Wang Y G, Ji R Q, Ji S J, Zou T, Choi S R, et al. 2009. SSR and SCAR mapping of a multiple-allele male-sterile gene in Chinese cabbage (Brassica rapa L.). Theoretical and Applied Genetics, 119, 333-339. Gupta P K, Rustgi S. 2004. Molecular markers from the transcribed expressed region of the genome in higher plants. Functional and Integrative Genomics, 4, 139162. He J P, Ke L P, Hong D F, Xie Y Z, Wang G C, Liu P W, Yang G S. 2008. Fine mapping of a recessive genic male sterility gene (Bnms3) in rapeseed (Brassica napus) with AFLPand Arabidopsis-derived PCR markers. Theoretical and Applied Genetics, 117, 11-18. Lander E S, Green P, Abrahamson J, Barlow A, Daly M J, Lincon S E, Newbury L, et al. 1987. MAPMAKER: an interactive computing package for constructing primary genetic linkages of experimental and natural populations. Genomics, 1, 174-181. Lei S L, Yao X Q, Yi B, Chen W, Ma C Z, Tu J X, Fu T D. 2007. Towards map-based: fine mapping of a recessive genic male-sterile gene (BnMs2) in Brassica napus L. and syntenic region identification based on the Arabidopsis thaliana genome sequences. Theoretical and Applied Genetics, 115, 643-651. Liu Y M, Fang Z Y, McMullen M, Zhuang M, Yang L M, Wang X W, Zhang Y Y, Sun P T. 2003. Identification of a RFLP marker linked to a dominant male sterile gene in cabbage. Acta Horticulturae Sinica, 30, 549-553. (in Chinese) Lou P, Kang J G, Zhang G Y, Bonnema G, Fang Z Y, Wang X W. 2007. Transcript profiling of a dominant male sterile mutant (Ms-cd1) in cabbage during flower bud development. Plant Science, 172, 111-119. Lu G Y, Yang G S, Fu T D. 2004. Linkage map construction and mapping of a dominant genic male sterility gene (Ms) in Brassica napus. Acta Genetica Sinica, 31, 13091315. (in Chinese) Lyttle T. 1991. Segregation distorters. Annual Review of Genetics, 25, 511-557. Michelmore R W, Paran I, Kesseli R V. 1991. Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions using segregating populations. Proceedings of the National Academy of Sciences of the United States of America, 88, 98289832. Panaud O, Chen X, McCouch S. 1996. Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.). Molecular and General Genetics, 252, 597-
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607. Wang X W, Fang Z Y, Huang S W, Sun P T, Liu Y M, Yang L M, Zhuang M, Qu D Y. 2000a. An extended random primer amplified region (ERPAR) marker linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata). Euphytica, 112, 267-273. Wang X W, Fang Z Y, Sun P T, Liu Y M, Yang L M, Zhuang M. 2000b. A SCAR marker applicable in marker assisted selection of a dominant male sterility gene in cabbage. Acta Horticulturae Sinica, 27, 143-144. (in Chinese) Wang X W, Fang Z Y, Sun P T, Liu Y M, Yang L M. 1998. Identification of a RAPD marker linked to a dominant male sterile gene in cabbabe. Acta Horticulturae Sinica, 25, 197-198. (in Chinese)
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Wang X W, Lou P, Bonnema G, Yang B J, He H J, Zhang Y G, Fang Z Y. 2005. Linkage mapping of a dominant male sterility gene ms-cd1 in Brassica oleracea. Genome, 48, 848-854. Yang L M, Fang Z Y, Liu Y M, Wang X W, Zhuang M, Zhang Y Y, Sun P T. 2004. ‘Zhonggan 18’ - A new cabbage hybrid variety with the hybridization of dominant male sterile line and inbred line. Acta Horticulturae Sinica, 31, 837. (in Chinese) Zhang X M, Wu J, Zhang H, Ma Y, Guo A G, Wang X W. 2011. Fine mapping of a male sterility gene MS-cd1 in Brassica oleracea. Theoretical and Applied Genetics, 123, 231-238. (Managing editor SUN Lu-juan)
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.
April 2013
2013, 12(4): 596-602
RESEARCH ARTICLE
A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var. capitata L.) CHEN Chen1, 2, ZHUANG Mu1, FANG Zhi-yuan1, WANG Qing-biao1, ZHANG Yang-yong1, LIU Yu-mei1, YANG Li-mei1 and CHENG Fei2 1 2
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China Horticulture College of Qingdao Agricultural University, Qingdao 266109, P.R.China
Abstract The dominant genic male sterility (DGMS) gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage (Brassica oleracea var. capitata L.), has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants (CDMs399-3/CDMs399-3) is a laborious and time-consuming process. For marker-assisted selection (MAS) of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats (EST-SSR) and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis (BSA). By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygous male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding. Key words: cabbage (Brassica oleracea var. capitata L.), dominant genic male sterility (DGMS), expressed sequence tag-simple sequence repeats (EST-SSR), bulked segregant analysis (BSA), marker-assisted selection (MAS)
INTRODUCTION Cabbage (Brassica oleracea var. capitata L.) is cultivated widely around China and other countries such as USA, UK, Japan, Korea, India and etc. Because of exhibiting obvious heterosis, most cabbage cultivars released for market are F1 hybrids produced via self-incompatible lines or male-sterile lines. The production of hybrid seed using male-sterile lines is a reliable and economical choice for the efficient utilization of hetReceived 5 December, 2011
erosis in cabbage. The dominant genic male sterility (DGMS) gene CDMs399-3, also called as Ms-cd1 (Wang et al. 2005), was identified as a spontaneous mutant in an early spring cabbage line 79-399-3 in 1979 (Fang et al. 1997) and has been utilized well in cabbage commercial hybrid seed production in China (Fang et al. 2004). To date, the following aspects of the DGMS controlled by CDMs399-3 have been elucidated: (i) The development of male sterility is associated with abnormal degeneration of the cellulose cell wall and failure of microspore separation from tetrads during anther devel-
Accepted 28 February, 2012
Correspondence ZHUANG Mu, Tel: +86-10-82108756, Fax: +86-10-62174123, E-mail: [email protected]
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60277-4
A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage
opment (Fang et al. 1997; Lou et al. 2007); (ii) the male sterility in 79-399-3 is sensitive to temperature below 15°C, and can develop a few viable pollen grains in certain genetic background. Thus, homozygous malesterile line can be developed by selfing heterozygous male-sterile plants (Fang et al. 1997); (iii) the identification of the homozygous male-sterile plants from an F2 population, derived from selfing a heterozygous malesterile plant with a few viable pollen grains, is a timeconsuming and laborious process. The normal process is as follows: maintain all the male-sterile plants including homozygous male-sterile plants (1/4 of the F2 population) and heterozygous male-sterile plants (1/2 of the F2 population) by tissue culture while all the malesterile plants are test-crossing with a male-fertile tester (CDms399-3/CDms399-3), then cultivate each testcross plant with a population not less than 12 progeny plants to flower in next spring after vernalization, investigate the male-sterility segregating ratio of testcross progeny, finally obtain the homozygous genotype based on that testcross progeny showed 100% male-sterile (Fang et al. 2004). A reduction of the number of plants that need to be test-crossed, vernalized, and maintained by tissue culture will greatly reduce the cost of the development of superior male-sterile lines and facilitate the hybrid seed production. Molecular markers closely linked to the gene CDMs399-3 can make marker-assisted selection (MAS) feasible at the young-seedling stage. Based on bulked segregant analysis (BAS) method, A RAPD marker OPT11900 linked to the CDMs399-3 gene with the distance of 7.48 cM was identified (Wang et al. 1998), and converted into a sequence characterized amplified region (SCAR) marker (Wang et al. 2000b) and extended random primer amplified region (ERPAR) marker (Wang et al. 2000a). Using two backcross populations with the recurrent male parents of two flat-headed autumn cabbage inbred lines, a RFLP marker pBN11 linked to the CDMs399-3 gene was identified (Liu et al. 2003). Due to the limitation of RFLP technology, pBN11 has not been applied to MAS in cabbage yet. Three amplified fragment length polymorphism (AFLP) markers and three SCAR markers that linked to CDMs399-3 were mapped onto linkage group 9, corresponding to chromosome 3 of B. oleracea (Wang et al. 2005). 14 sequence-related amplified polymorphism (SRAP) markers and one simple sequence repeat (SSR) marker linked to CDMs399-3 were iden-
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tified using a segregating BC4 population with an inbred line of Chinese kale (B. oleracea var. alboglabra) as the recurrent male parent (Zhang et al. 2011). Most of the previous markers linked to CDMs399-3 were dominant markers, and no marker had been identified in a fertility-segregated population of spring cabbage line. SSRs are easy, high-efficient, co-dominant and reliable PCR-based markers. Because they are located in transcribed region of the genome, expressed sequence tag-simple sequence repeats (EST-SSR) can be developed rapidly and inexpensively from the EST database by data mining, and they could increase the efficiency of MAS (Gupta et al. 2004). Based on the development of EST-SSR in our lab (Chen et al. 2010), here we reported the identification of an EST-SSR marker linked to CDMs399-3 and use of this marker to rapidly obtain the homozygous male sterile plants of an earlymatured spring cabbage line 397 with a round head.
RESULTS Fertility survey of the two populations For the F2 segregating population P921, 56 plants were male-sterile and 17 were male-fertile. The segregation ratio of male sterility and male fertility was consistent with the expected ratio of 3:1 in a χ2 test (χ2=0.114 and P>0.05). Out of the 56 male-sterile plants of population P921, 7 plants were homozygous and the other 49 plants were heterozygous, indicating a distorted segregation ratio of CDMs399-3 may be resulted from competition among gametes for preferential fertilization (Lyttle 1991). The F2 segregating population P1170 consisted of 7 homozygous male-sterile plants, 9 heterozygous male-sterile plants and 4 fertile plants, which were consistent with 1:2:1 segregation (χ 2=1.1 and P>0.05). The results confirmed that the deduction that the male sterility in B. oleracea var. capitata L. is controlled by one single dominant gene (Fang et al. 1997).
Identification of SSR markers linked to the gene CDMs399-3 978 EST-SSR and 395 SSR primer pairs were screened to identify polymorphisms between the homozygous
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male sterile bulk and the male fertile bulk. The primer pairs that produced polymorphic bands were tested for a second time. Only one EST-SSR marker BoE332 (Appendix) was identified as a candidate marker possibly linked to the DGMS gene CDMs399-3. Scoring of BoE332 in bulks showed that only one approximate 270 bp band could be amplified in homozygous male-sterile plants, and one approximate 290 bp band in male-fertile plants. 73 plants of population P921 were screened with BoE332. BoE332 in population P921 showed that 8 of 73 plants were homozygous male-sterile, 48 of 73 plants were heterozygous male-sterile and 17 of 73 plants were male-fertile (Fig. 1). Compared with the results of the field investigation of male fertility, 2 of 56 male-sterile plants and 2 of 17 male-fertile plants tested by marker BoE332 were not consistent with the fertility results of field investigation. The linkage distance was calculated by scoring BoE332 for all 73 plants of the P921 population that had been identified phenotype for male fertility. The co-dominant marker BoE332 closely flanked the gene CDMs399-3 at 3.6 cM.
The MAS of CDMs399-3 using marker BoE332 Due to the marker BoE332 was not 100% linked to the
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gene CDMs399-3, the homozygous male-sterile plants identified using BoE332 might include heterozygous ones. Therefore, to further identify the real homozygous male-sterile plants, 56 male-sterile plants of population P921 were test-crossed, no less than 12 plants of each test-crossing population were cultivated, marker BoE332 was applied to check the genotypes of the 56 test-crossing populations before field evaluation, and the fertility investigation at flowering were carried out in greenhouse. The male-sterile plant was scored as homozygous male-sterile if all the test crossing plants showed both approximate 270 and 290 bp amplification bands (Fig. 2-A), while the male-sterile plant was scored as heterozygous if at least one of the test crossing plants showed only one approximate 290 bp band (Fig. 2-B). Based on the testing of marker BoE332, 7 homozygous male-sterile plants in population P921 were obtained finally. The result was 100% coincided with that of the fertility investigation. The MAS of homozygous male-sterile plants using marker BoE332 was established: firstly screen the plants of F2 population segregating in fertility; secondly screen no less than 12 plants for each test-crossing population derived from the male-sterile plants of F2 population; finally determine the results by combining results of the two steps.
Fig. 1 PCR products of the partial plants of F2 population P921 with EST-SSR marker BoE332. M, 100 bp DNA ladder; S, homozygous male-sterile bulk; F, male-fertile bulk; a, homozygous male-sterile plant in population P921; b, male-fertile plant in population P921; h, heterozygous male-sterile plant in population P921. *, the recombinant plant.
Fig. 2 PCR products of test crossing plants derived from homozygous male-sterile plant (A) and heterozygous male-sterile plant (B) with EST-SSR marker BoE332. M, 100 bp DNA ladder; S, homozygous male-sterile plant; F, male-fertile plant; b, male-fertile plant of test crossing population; h, heterozygous male-sterile plant of test crossing population.
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A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage
To further test the utility of BoE332, the same procedure was applied to F2 population P1170. The PCR screening results using BoE332 marker indicated that 5 plants were homozygous male-sterile, 11 plants were heterozygous male-sterile and 4 were male-fertile. The 16 male-sterile plants of population P1170 were then followed by test-crossing and male-sterile genotypic status was determined using BoE332 screening. 7 homozygous male-sterile plants were identified, which were coincided 100% with the field observation. The results indicated that marker BoE332 was valid for cabbage inbred line 397.
DISCUSSION Cabbage inbred line 397, a key parent material growing in spring with excellent desired characters such as round head, early maturity and good quality, has been successfully converted into several DGMS lines. These lines has been applied in hybrid seed production of several cultivars in china, such as Zhonggan17, Zhonggan18 and Zhonggan21, which have been released in almost all the areas of China (Fang et al. 2004, 2007; Yang et al. 2004). For cultivar improvement in cabbage, especially for key parent inbred lines as well as key line 397, more novel homozygous male-sterile lines were needed. Because the conventional selection is relatively long procedure, the availability of inexpensive, rapid, and reliable markers linked to CDMs399-3 would greatly improve the efficiency of cabbage breeding programs via MAS. The previously obtained markers linked to CDMs399-3 such as RAPD marker OPT11900 and ERPAR marker EPT11900 were dominant markers and identified from segregating populations of flat cabbage or other B. oleracea crops. They could not assist selection of the homozygous male-sterile plants in spring cabbage lines (Wang et al. 1998, 2000b). Because CDMs399-3 was mutated from the spring cabbage line 79-399-3, the high similarity of its genetic basis made development of practical marker linked to it in spring cabbage lines much difficult. The only previously reported SSR marker was 8C0909, which was linked to the MS-cd1 gene (the other name of CDMs399-3) with a distance of 2.06 cM in a kale population (Zhang et al. 2011). However, this marker showed no polymorphism in the populations we tested. After screening 978 EST-SSRs
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and 395 SSRs, we only obtained one co-dominant marker BoE332 that linked to the CDMs399-3 with a distance of 3.6 cM, indicating that the polymorphism surrounding the male sterility gene is very low in the genetic background of 397. To our knowledge, this is the first SSR maker identified in early spring round cabbage. Even though molecular markers linked to genic male sterility (GMS) have been reported in Brassica family (Lu et al. 2004; Lei et al. 2007; He et al. 2008; Feng et al. 2009; Zhang et al. 2011), there is little information of practical utilization in MAS. BoE332 has potential to be practically used to select homozygous male-sterile lines in fertility segregating population of cabbage key line 397. For example, using marker BoE332, 19 of 67 plants from a F2 population P1171 segregating in male fertility derived from cabbage line 397 were selected as homozygous male-sterile at first, 16 of 67 plants were identified finally as homozygous male-sterile combining the result of test-crossing populations of each male sterile plant (data not shown), and the MAS result was coincided with the field investigation during the flowering period. Therefore, although the linkage distance of BoE332 to CDMs399-3 is 3.6 cM, BoE332 was effective for assisted selection of homozygous male-sterile plants for spring cabbage line 397. SSR marker BoE332 might not be applied directly in MAS of the DGMS transferring of the other cabbage inbred lines (data not shown). The utilization of marker BoE332 in MAS was probably limited to its distance linked to the gene CDMs399-3. In order to obtain more closely linked markers, it might be necessary to employ PCR walking and screen much molecular markers such as SNP and InDel based on the latest advances of genome sequencing and resequencing in B. oleracea var. capitata in which our lab is participating. The molecular markers linked to the gene CDMs399-3 have been mapped onto linkage group O9, corresponding to chromosome 3 of B. oleracea and the region corresponds to chromosome 5 in Arabidopsis thaliana (Wang et al. 2005). In this study, the sequences of the PCR amplification bands of BoE332 correspond also to chromosome 5 in Arabidopsis thaliana (data not shown). Up to now, the DGMS system has been an effective hybrid seed production system in B. oleracea. However, very little is known regarding the molecular basis of the DGMS trait, which limits its wide appli-
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cation to hybrid breeding. Based on the information of genome sequencing and resequencing in cabbage, cloning and characterization of CDMs399-3 gene in the future will greatly improve the development of homozygous male-sterile lines and further its application in hybrid breeding.
CONCLUSION In this study, we obtained a co-dominant EST-SSR marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the spring cabbage line 397 (Brassica oleracea var. capitata L.). BoE332 has potential to be practically used to MAS of homozygous male-sterile lines in fertility segregating population of cabbage key line 397.
CHEN Chen et al.
DNA isolation and SSR analysis Genomic DNA was isolated from fresh leaves of all the plants using a cetyltrimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1990) with minor modifications. The quality and concentration of extracted DNA were tested by using 1% agarose gel electrophoresis. BSA method was employed to identify SSR markers linked to the gene CDMs399-3. 978 EST-SSRs, designed from the published EST sequence of B. oleracea in NCBI (Appendix), and 395 SSRs (http://www.brassica.info) were used for SSR analysis. PCR amplification was carried out in a volume of 20 µL, containing 1 U Taq polymerase, 4 pmol of each EST-SSR or SSR primer, 200 µmol L-1 dNTPs, 1×PCR buffer (contains 1.5 mmol L-1 MgCl2) and 50 ng genomic DNA as the template.
MATERIALS AND METHODS Plant materials and populations 4 homozygous male-sterile plants and 5 male-fertile plants from an F2 segregating population P725 were used to form bulks (Michelmore et al. 1991) for screening EST-SSR and SSR markers. The F2 population P725 was generated by selfing a temperature sensitive heterozygous male-sterile plant from eight generations of backcrossing the early spring cabbage line 397 into the original male-sterile line 79-399-3. 73 plants of the F2 population P921 derived from selfing a temperature sensitive heterozygous male-sterile plant of the population P725, were used to obtain the ESTSSR marker linked to the DGMS gene CDMs399-3. 20 plants from another F2 population P1170 derived from selfing a different temperature sensitive male-sterile plant of the population P725 was used to further test the utilization of the obtained marker linked to the gene CDMs399-3 (Fig. 3). All the male-sterile plants of the population P921 and P1170 were maintained by tissue culture, and were test-crossed with the fertile plant, to determine whether male-sterile plants were homozygous or not. All plants were grown in the open field, and transplanted in the greenhouse of Institute of Vegetables and FLowers, Chinese Academy of Agricultural Sciences, Beijing, China, and cultivation management was conducted normally. Male fertility was investigated in both F2 populations and test cross populations in the spring. During the flowering period, fertility was characterized based on the observation of pollen grains in anthers of open flowers at least three times (Fang et al. 1997; Wang et al. 2000a).
Fig. 3 Segregation population of male fertility in cabbage. MsMs, homozygous male-sterile genotype; Msms, heterozygous malesterile genotype; msms, male-fertile genotype; Ts1 and Ts2 and Ts3, different temperature sensitive male-sterile plants; Bulk-1, homozygous male-sterile bulk; Bulk-2, male-fertile bulk.
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A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage
All the reagents were bought from Beijing Biomed Co., Ltd. (Bejing, China). The PCR profile was as follows: 94°C for 4 min, followed by 35 cycles at 94°C for 0.5 min, appropriate annealing temperature for 0.5 min, 72°C for 1 min, ending with a final extension step of 7 min at 72°C. The PCR products were separated on a 8% polyacrylamide gel (acrylamide:bis=29:1) and visualized with silver staining as described by Panaud et al. (1996).
Linkage analysis Polymorphic SSR marker bands were scored as homozygous sterile (a), fertile (b) and heterozygous sterile (h) for all samples. Linkage distance analysis was performed with Joinmap 4.0, with a minimum LOD score of 3 (Lander et al. 1987).
Acknowledgements We thank Prof. Elizabeth Earle, Department of Plant Breeding and Genetics, Cornell University, USA, for constructive advice on writing the manuscript. This research was supported by the National Science and Technology Ministry of China (2008BADB1B02 and 2009BADB8B03), the Core Research Budget of the Non-profit Governmental Research Institution (ICS, CAAS) (1610032011011), the China Agriculture Research System (CARS-25), and the National High Technology Research and Development Program of China (863 Program, 2012AA100101). This work was conducted at the Key Laboratory of Horticultural Crop Biology and Germplasm Innovation of the Ministry of Agriculture, China. Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm
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