Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay
H.X. Zhao1, Z.J. Li1, S.W. Hu 2,4, G. L. Sun3, J.J. Chang2, Z.H.Zhang1
1 College of life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, P.R. China.
2College of Agronomy, Northwest A&F University, Yangling, Shaanxi, 712100, P.R. China.
4 3 Department of Biology, Saint Mary’s University, Halifax, Nova Scotia, B3H 3C3, Canada
4Corresponding author: S.W. Hu
College of Agronomy, Northwest A&F University, Yangling, Shaanxi, 712100, P.R. China.
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Abstract: Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In the presentthis study, we report a simple multiplex PCR method was developed, which canto distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseedBrassica napus by one PCR reaction. Using this method, cCytoplasm type for of 35 F1-hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F1-hybrids are the napNap, and 25 are the pol Pol cytoplasm type which is consistent with the information provided by the breeders. Among Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam, and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that all these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method.. The multiplex PCR assay methodresults of this study can be applied to will be an important guide on CMS "three-line" breeding, the selection and validation of hybrid rapeseed with existing resources in Rapeseed. , and it is more accurate and fast compared with the conventional test-crossing method. By using this method, 35 F1-hybrids in Region Test Field of Shaanxi Province in 2007-2008 and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested for their cytoplasm type. The results indicated that, ten of 35 F1-hybrids were identified to be nap and 25 to be pol cytoplasm type. This result is consistent with the information provided by the breeder. Among 140 accessions tested, 100 accessions (71.4%) possess nap cytoplasm, 21 (15%) Cam cytoplasm, and 19 (13.6%) Pol cytoplasm. All 19 accessions with Pol cytoplasm came from China. Pedigree analysis indicated that all these accessions with Pol cytoplasm were some restorers, including Shaan 2C, Huiyehui, 220,etc. or derived from hybrids with Pol CMS as female parent. Our molecular results is are consistent with thatthose of the previous investigations by the classical test-crossing method in the case of the for some same accessions included in our this experiment and the previous investigations. The results of this study will be an important guide on CMS "three-line" breeding and the selection and validation of hybrid rapeseed with existing resources in Rapeseed.
Key words: Brassica napus L.; Cytoplasm types; identification; a multiplex PCR assay
Introduction
Cytoplasmic male sterility (CMS) is a widespread, maternally inherited trait of higher plants that results in pollen abortion due to defects in mitochondrial function. A lot of investigations revealed that CMS-specific factors should be present in CMS mitochondria(mitochondria (Hanson 1991;Hanson; Hanson and Bentolia 2004;Sota; Sota and Kinya 2008). The Ccompleted sequences of the circular master mitochondrial genomes of Arabidopsis (Unseld et al.1997), sugar beet (Kubo et al. 2000), rice( Notsu et al. 2002)，rapeseed (Handa 2003) and tobacco(Sugiyama et al.2005) have been reported. At present, theresulted in the identification of At present, key molecules involved in CMS-associated genes are identified fromare identified fromin various plant species (Sota and Kinya, 2008). The eExpression of these CMS-associated genes can often be influenced by specific nuclear genes, named as restorers of fertility (Rf), that which suppress male sterility and allow for normal pollen development (Sota and Kinya 2008).
SThe utilization of the heterosis is the most important approach in F1 hybrid production to increase rapeseed yield, and CMS has been widely used as an efficient pollination control system in rapeseed hybrid production. In Brassica napusrapeseed L., several CMS lines have been discovered and used in rapeseed F1 hybrid production, including alloplasmic type the Ogura cytoplasm of radish, i.e. Ogu CMS, and endogenous cytoplasms such as napNap, pol Pol, and Shaan 2A cytoplasms found within species. The CMS-associated genes in these CMS lines have been identified and characterized. In Ogu CMS, the presence of aA 19-kDa protein, ORF138, has been reported to bewas correlated with the Ogu CMS (Bonhomme et al.1992; Grelon et al. 1994 ). The expression of Brassica In pol Pol CMS is due to, the presence of orf224/apt6 locus in mitochondrial DNA ( mtDNA ) and its expression of the orf224/apt6 gene region is highly correlated with the pol Pol CMS trait (Singh and Brown, 1991；; L’Homme and Brown et al. 1993), ). while While The nap Nap CMS is associated with the expression of orf222/nad5c/orf139 region in the mtDNA of male-sterile nap Nap (L’Homme et al. 1997) cytoplasm mtDNA,. However, these mtDNA segments were missing missed from the mitochondrial genometDNA of fertile cam Cam cytoplasm mitochondrial genome (L’Homme et al. 1997). These CMS lines have been widely used in rapeseed production.
The utilization of the heterosis is the most important approach in F1 hybrid production to increase rapeseed yield, and CMS has been widely used as an efficient pollination control system in rapeseed hybrid production. The discovery and utilization of pol Pol CMS lines has greatly stimulated the rapeseed research and production. During the period 2000 to 2005, there were 217 varieties registered in China, 170 of the 217 varieties (78.3%) were hybrid varieties, 47(21.7%) were open pollination varieties, and further 107 of the 170 (69.2%)hybrids were CMS hybrids ( Fu,2008).(delete these sentence) However, prevailing usage of the limited resource of CMS linesresources could cause problems for substantial development of rapeseed production. Thus, However, dominant usage of the limited resource of CMS lines will cause problems for substantial development of rapeseed production. In the 1970s maize hybrids generated using T cytoplasm suffered an epidemic of Southern Corn leaf Blight（Pringle and Lonsdale 1989；Levings 1993），forcing recognition of the cytoplasmic uniformity and genetic vulnerability of major food crops (Anonymous 1972). Today, some of our most economically important crops are still too genetically uniform and vulnerable to major epidemics. The rapeseed may be a case in point. So, the discovery and utilization of new cytoplasm resource has been the focus of rapeseed breeders. Development of efficient methodThe identification of cytoplasm type of rapeseed is becoming th to identify CMS types is e most important basic task work for rapeseed breeding.
At present, tThe most common classical methods for identification of ifying cytoplasmic type in Brassica napusrapeseed are is test-crossing. Test-crossing is a classical method for cytoplasmic type identification. By this classical method,which requires 3 to 4 years, are required to determine a cytoplasm type of B.napusrapeseed accession due to Brassic napusitrapeseed is a biennial plant and 3 to 4 years are required to determine a cytoplasm type by test-crossingsthis classical method. Shiga (1976, 1978) identified the cytoplasmic type of 162 rapeseed materials from Japan and Europe. Hu et al. (1992) tested the cytoplasmic type of 43 rapeseed materials from 8 countries. Two shortages of this method for identification of cytoplasmic type are time consuming and labor costing. (delete this sentences) Compared with the test-crossing method, RFLP (rRestriction fragment length polymorphism analysis of mtDNA is a significantlya faster method method to distinguish the different cytoplasm types (Yang et al. (1998) identified 9 different CMS lines by RFLP and of mtDNA fragment polymorphism analysis,; with four restriction endonucleases of EcoR I, HindIII , Pst I and Sal I , respectively, the results of identification were consistent with the data using test-crossing. Wei et al. (2005) completely distinguished 5 CMS lines from each other, i.e. NCa, pol, nap, ogu and tour in Brassica napus by RFLP analysis of the mtDNA, and demonstrated the NCa CMS hold a new type of sterile cytoplasm different from pol, nap, ogu and tour CMS, but. not suitable in high-throughput genotyping Because of the requirement of large amounts of purified mtDNA, appropriate restriction endonucleases and suitable probes in southern hybridization., RFLP analyses of mtDNA is hardly suitable in high-throughput genotyping.
In contrast, PCR-based markers developed from specific gene sequences would is provide rapid, accurate and efficient and high-throughput data tool for determination ofdetermining cytoplasm type in breeding materials. Previous reports elucidated different CMS lines in Brassica napus having specific chimeric genes associated with the CMS, such as orf224/atp6 in pol, and orf222/nad5c/0rf139 in nap cytoplasm(delete this sentences ). The sequences of these CMS-associated genes inin the polpPol and nNap cytoplasm and the complete sequence of nap Nap mitochondrial genome have beenare available in GenBank(GenBank (Singh and Brown 1991;L’Homme; L’Homme and Brownet al. 1993; L’Homme et al. 1997;Handa; Handa 2003), thus, . Wei et al (2005) designed 22 pairs of PCR-primer to amplify the specific fragments in order to distinguish a new cytoplasm type NCa CMS lines from ogu, pol and nap cytoplasm, based on the sequences of Orf138, orf222/nad5c, and Orf224/atp6 as well as the mtDNA sequence of Arabidopsis thaliana, but the result did not successfully distinguish the five CMS lines from each other. The possible reason might be the shortage of knowledge about CMS-associated gene in NCa CMS lines. (delete this sentences) Iit is essentialThe urgent event is toprovide an opportunity to do much more work on the development of PCR markers based on these genes sequences based on gene sequences for rapidly identification ofying cytoplasm in rapeseedBrassica napus. breeding, with the rapidly increased rapidly increased information on the sequences of CMS-associated genes rapidly increased.
Multiplex PCR is a variant of PCR which enabling enables simultaneous amplification of many target genes interested in one reaction by using more than onemultiple pairs of primers (Chamberlain et al. 1998). Multiplex PCR would further reduce costs and increase the efficiency of marker-assisted selection ( MAS ) in plant breeding. This technique has been developed inused for identification ofidentifying HMW-GS genes Three examples of multiplex PCR for wheat HMW-GS(Ahmad 2000; Ma et al. 2003; Moczulski and Salmanowicz 2003 ), and one for waxy protein genes (Nakamura et al. 2002 ), in wheat and one for genes ω-secalin, Glu-B1-2a, Glu-D1-1d,Glu-A3d,Glu-B3 and pinb-D1b in wheat ( Zhang et al. 2008 ) have been reported., and in distinguishing of Kim et al.(2009) developed a molecular marker( multiplex PCR or not??) for distinguishing among three cytoplasm types in onion (Allium cepa L.)(Kim et al. 2009).To the best of our knowledge, mMultiplex PCR assay for identification identifying cytoplasmic types in rapeseedBrassica napus breeding programme has not been developed yet.
The objectives of this study were to: (i1) develop a multiplex PCR assay for identification ofying cytoplasm type in rapeseedBrassica napus L., ( ii2) evaluate the accuracy of the multiplex PCR assay by detecting comparing the results of cytoplasm type of 35 F1 hybrids cytoplasm types and making comparisonngwith the results with the information provided by the breeders, (iii3) test the cytoplasm types of 140 open pollinated varieties or breeding lines usually used in rapeseed breeding programs by the multiplex PCR assay, and 4) reveal the distribution of the cytoplasm types in order tofor efficiently utilize utilization of these materials in rapeseed heterosis breeding programme.
Materials and methods
Plant materials
Two accessions of each cytoplasm type Nap, Pol and Cam, were used as materials for DNA isolation and molecular marker development. An One Ogu CMS accession and an one improved Ogu CMS accession named as( Ogu-NWSUAF CMS) （(Chang et al., 2010 impressed）) wasere also included (Table 1) in the materials. The seeds were germinated and cultured in trays in dark condition to get yellow seedlings for mtDNA and gDNA isolation. All these 8 accessions for molecular marker development and their cytoplasm type were shown in table 1. Each of the all 8 accessions were germinated and cultured in trays in dark condition to get yellow seedlings for mtDNA isolation.
Cytoplasm types of 35Thirty-five F1 hybrids in Region Test Field of Shaanxi Province in 2007-2008 (Table S2), and 140 rapeseed accessions from different countries (93 open pollinated varieties and 47 breeding lines) (Table S2) were used to detect their cytoplasm type bycharacterized using the molecular markermethod developed in this study. Ten single plants were randomly chosen from each F1-hybrids for molecular analyses.
One hundred and forty rapeseed accessions from different countries, consisting ofincluding 93 open pollinated varieties and 47 breeding lines, were used to identify their cytoplasm type by the molecular markermethod developed in the presentis investigation (Table S3). These All rapeseed accessions open pollinated varieties and breeding lines were sown in the experimental field of Northwestern A & F University, Yangling, Shaanxi, P.R. China in 2007-2008. Ten individual plants of three-leaf stage randomly chosen from each hybrid/accession were used for total gDNA isolation.
Ten individual plants of each variety or line were randomly selected for molecular analyses.
Mitochondrial DNA and Genomic DNA extraction
SSHighly purified mtDNA of the 8 accessions was extracted according to the method reported by Wang et al. (2008). Total genomic DNA (gDNA) of each accession all accessions was extracted from the leaves of three-leaf stage rapeseed seedling following the protocol described by Edawards et al. (1991).
Highly purified mitochondrial DNA (mtDNA) of the eight accessions used for molecular marker development was extracted from yellow seedling plants cultured in dark conditions, according to the method reported by Wang et al. (2008).
sof the fourto theihereisa nuclear male sterility gene MS2Bnap specific primer in rapeseed (Hu et al. 2006) PCR amplification using single primer set
Four pairs of gene (in mtDNA) specific primers previously described by Wen et al. (2005), and a pair of gene (in gDNA) specific primer reported by Hu et al. (2006) were synthesized in Shanghai Sangon Biological Engineering Technology & Service Co. Ltd (http: (Table 4). PCR amplification with single primer pair was performed using 50 ng of gDNA or mtDNA in a final volume of 20 μul, containing 0.5 uM of each primer, 150 uM of each dNTP, and 0.25 units of Taq DNA polymerase (Tiangen biotech. Co., Beijing, China). PCR was carried out with initial denaturation step at 94 ℃ for 2 min, followed by 35 cycles of 94 ℃ for 1 min, 54℃ for 1 min and 72 ℃ for 2 min, and a final extension at 72 ℃ for 10 min.
The PCR-products were separated by flatbed electrophoresis using 1.5% agarose gels in 1X 1x TAE buffer. The bands were stained with ethidum bromide and visualized under UV light.
Multiplex PCR
Multiplex PCR was developed for a simultaneous identification amplification of genes Orf138, Orf224, and Orf222/nad5c targeting the oguOgu, pol Pol and Nap cytoplasm, respectively. The reaction components contained 50 ng of mtDNA/gDNA, 150 uM of each dNTP, 0.25 units of Taq DNA polymerase, and 0.15 uM of each primer for genes Orf138, Orf224 , and Orf222/nad5c,, respectively, in total volume of 20 μul. The PCR amplification programme and PCR-products electrophoresis condition of the multiplex PCR was the same as that for the above-mentioned single primer set.
Results
The test of purification of MtDNA mtDNA and gDNA isolated from rapeseedBrassica napus L.
In order to make comparison of the PCR-products using gDNA as template with those using purified mtDNA as template, highly purified mtDNA is essential. To test if the mtDNA isolated from each known accession is in this study mixed with gDNA, nuclear ( )we amplified a gene MS2Bnap existing in gDNA，,related to nuclear male sterility in Brassica napus L. according to Hu et al. (2006), with the gene-specific primer (primer 5 in table 4)，using gDNA and mtDNA samples as templates, respectively. The patterns of PCR-products were shown in (Fig.1a). showed It could be seen that there were no PCR-products obtained when usingfor mtDNA as templates, indicating that the mtDNA isolated in this study was free from gDNA.
Moreover, to check if the total gDNA samples are mixed with mtDNA, PCR amplification of mtDNA orf139, a gene existing only in mtDNA, was performed with using the gene specific primer (primer 5 in (table 4)). The Fig. 1b showed that the target fragment of orf139 were amplified in all the mtDNA as well as the gDNA samples, suggesting that the gDNA isolated in the present investigation contained some mtDNA and could be used to amplify the gene specific for to mtDNA. This would be certified in the following part.
PCR amplification using single primer set for detection odetectingf orf224, orf222, and orf138 in different cytoplasm types of rapeseedBrassica napus L.
To get molecular marker specific for to each of cytoplasm typeO Nap,P pol, ogu in rapeseed Brassica napus L., mt DNA isolated from each of the 8 accessions (Table1) were used to perform PCR with ( )each set of primers specific for gene orf224, orf222, orf138 respectively (Table 4). The patterns of PCR products were shown in Fig.2a, 2b and 2c, respectively. A 465-bp band appeared in the accession with the Ogu cytoplasm or Ogu-NWSUAF cytoplasm (Fig. 2a). A 1102-bp PCR product was generateddisplayed in the two accessions with Nap cytoplasm and the accession with Ogu-NWSUAF cytoplasm (Fig.2b), while aA 747-bp fragment amplified in the two accessions with pol Pol cytoplasm type indicated the presence of gene orf224 (Fig.2a2c), while a 465-bp band appeared in the accession with ogu cytoplasm or improved ogu, i.e.ogu-NWSUAF cytoplasm (Fig. 2c2a). A 1102-bp PCR product was generated in the two accessions with Nap cytoplasm and the accession with ogu-NWSUAF cytoplasm（Change et al.2009Fig.2b）, was indicative of gene orf222 . All these results indicated that Tthe 465-, 1102- 747-, 465- and 747-1102-bp fragment were was molecular marker specific for to the Ogu, Nappol, Ogu and Pol Nap cytoplasm types , respectively.