Development of genomic SSR and potential EST-SSR markers in Bupleurum chinense DC

Nineteen genomic SSR markers were developed using inter-simple sequence repeat (ISSR)suppression PCR technique in Bupleurum chinense DC., a widely used Chinese medicinal plant. A total of 126 alleles were detected across 22 individual plants of B. chinense DC. f. octoradiatum (Bunge) Shan et Sheh, with an average of 3 13 alleles per locus. The observed heterozygosity (HO) and the expected heterozygosity (HE) values ranged from 0.23 to 1.00 and from 0.29 to 0.92, respectively. Nine loci deviated from Hardy-Weinberg equilibrium (HWE) (P < 0.05) and eight pairs of loci showed significant linkage disequilibrium (LD) (Fisher’s exact test, P < 0.01). The species transferability of these genomic SSR markers was also detected in seven other Bupleurum species. Eight SSR markers were successfully amplified in all tested species. In addition, forty four EST-SSRs which can be amplified with expected sizes were identified from a B. chinense root cDNA library. The genomic SSR markers and potential EST-SSR markers developed in the present study should be useful for genetic diversity and molecular marker assistant selection breeding research in Bupleurum species.


INTRODUCTION
Radix bupleuri (Chaihu; Chinese Thorowax Root), sourced from the dried roots of Bupleurum species (Umbelliferae family), has been used in indications such as common cold with fever, alternate chills and fever such as malaria, distending pain in the chest and hypochondriac regions, menstrual disorders, prolapse of the uterus, prolapse of the rectum with the action to release both exterior and interior, soothe the liver and elevate yang (Chinese Pharmacopoeia Commission, 2005).It is widely used in China, Japan, Korea and other south Asian countries.In China, although Bupleurum chinense DC. and B. scorzonerifolium Willd.are the two official R. bupleuri source species (Chinese Pharmacopoeia Commission, 2005), other Bupleurum species can be often found in the medicinal materials markets of some Chinese districts, such as B. yinchowense Shan et Y. Li, B. smithii Wolff, B. smithii Wolff.Var.parvifolia Shan et Y. Li, B. marginatum Wall. ex DC., B. bicaule Helm. and B. scorzonerifolium Willd. var. angustissimum (Franch.)Huang (Song, 2002).Many studies have shown the active component, such as saikosaponins, volatile oils and polysaccharides, varied remarkably between different Bupleurum species and also regional populations of the same species (Zhang et al., 2006;Shon et al., 1997;Shon et al., 2008;Chen et al., 2006).So, to use the species correctly demands the authentication of the R. bupleuri sourced species and learning their phylogenetic relationship.The great difficulty on distinguishing Bupleurum species through morphologic appearance has been noticed (Neves and Watson, 2004;Yang et al., 2007).A few attempts had been done with molecular methods to identify species and detect geographic variations, such as RFLP (Restriction Fragment Length Polymorphism) (Mizukami et al., 1993), chloroplast DNA restriction site analysis (Matsumoto et al., 2004) and rDNA ITS (Internal Transcribed Spacer) sequence analysis (Yang et al., 2007).
SSR is the kind of preferred marker rather than several others such as RAPD, ISSR and AFLP.Recently, they have been developed and used for authentication and population structure analysis in some medicinal plants (Kumar et al., 2007;Guan et al., 2008).To develop SSR markers in Bupleurum will be valuable for studies on genetic diversity and phylogenetic relationship of the genus Bupleurum.It will also be of significance to authenticate and promote the proper use of medicinal Bupleurum species.
As wild resources sharply shrinking, several Bupleurum species have been cultivated since 1970's in China.Nowadays about one third of R. bupleuri circulating in Chinese medicinal materials markets was derived from cultivation.B. chinense is the most universal species being cultivated in China.In Taiwan, the species of B. kaoi, B. falcatum and B. chinense and in Japan and Korea, the species of B. falcatum are commonly cultivated and pharmacologically studied as the source of R. bupleuri, respectively (Lin et al., 2008;Iida, 2006;Choi et al., 1995).Bupleurum cultivation methods have been researched in details (Minami et al., 1995;Shon et al., 1998;Liu et al., 1991;Kim et al., 1997;Kubo et al., 1993).It is obvious that plants with consistent agronomic traits and high saikosaponins content are the basis of standardization and modernization of the cultivation.To select and breed new cultivars with favorable agronomy traits and active components is a good way.There were some reports about the conventional breeding practices on Bupleurum cultivars.B. falcatum L. cv.Tainung No.1 was a breeding line developed through mass selection from three sources of B. fatcatum in Taiwan (Liu et al., 1989).In Japan, new Bupleurum plants were intended to acquire by crossing between the Japa-nese source plants and the Korean plants (Ohta et al., 2006).To improve cultivated Bupleurum in China, B. chinense DC. cv.Zhongchai No. 1 was mass-selected recently in our laboratory.Molecular marker-assisted selection (MAS) is an important method in modern breed-ing technologies.To develop SSR markers in Bupleurum species will be helpful for the identification of desired Bupleurum germplasm, the analysis of genetic structure of the cultivated population and the bred lines, the genetic map construction and quantitative trait loci (QTL) mapping.
Generally, two methods were used to develop SSR markers.For those organisms with plenty of genomic sequences or ESTs data, primer pairs of genomic and EST-SSR markers usually were developed by sequence database mining.For those organisms with little information of DNA sequences or ESTs, other methods for SSR markers isolation have been exploited.Inter-simple sequence repeat (ISSR)-suppression PCR, reported by Lian et al. (2001) for the first time, was a time and cost saving method for genomic SSR markers isolation and has been used in several plant species and fungi (Tamura et al., 2005;Lian et al., 2003).No SSR markers have been developed in the genus Bupleurum.We previously in silico analyzed and obtained 86 potential EST-SSR loci in 1650 uniESTs from a B. chinense root cDNA library (Sui et al., 2010, in press).Their primer pairs pairs and validities to amplify were reported here.A set of nineteen genomic SSR markers were also developed using ISSR-suppression PCR method.

Plant materials
B. chinense DC. cv.Zhongchai No. 1, which is a mass-selected cultivar of B. chinense, was used to isolate genomic SSR markers and analyze the validity of the EST-SSRs.Twenty two wild individual plants of B. chinense DC. f. octoradiatum (2n = 12), collected from Baihua Mountain, Beijing, were used to analyze variation of the genomic SSR markers.At least two plants of each of seven Bupleurum species collected from Shaanxi, Shanxi, Hebei, Sichuan, Qinghai, Hei Longjiang and Anhui provinces (Table 1) were used to test the species transferability of the genomic SSR markers.Plant materials used in this experiment were identified by Chunsheng Liu, Professor of Beijing University of Chinese Medicines and their specimens were deposited in the Specimen Museum of IMPLAD, CAMS and PUMC.

Development of genomic SSR primer pairs
The ISSR-suppression PCR technique established by Lian et al. (2001) was used to develop genomic SSR markers.A total of 100 different ISSR-PCR amplified fragments were cloned and sequenced to determine one flanking region for each SSR locus.SSR Hunter 1.3 software (Li and Wan, 2005) was used to find SSR loci on the inner sequence of clones and then forward and reverse primers to clone these SSR loci were designed.For SSR loci on the terminal of ISSR-PCR amplified fragments, the other unknown region flanking each locus was determined by the following steps: Genomic DNA was separately digested with one of the restriction enzymes: EcoRV, HaeIII, RsaI, SspI and AluI (New England Biolabs, Beijing).After digestion, fragments were precipitated by alcohol with 1/10 volume of 3 M NaOAc (pH 5.2) and ligated with an adaptor (consisting of two single-stranded oligonucleotides: 5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGG GCTGGT-3′ and 5′-ACCAGCCC-NH2-3′) using DNA Ligase (TaKaRa).The ligation products were diluted 10 times with ddH2O and used as the template of the first PCR.For the second PCR, primers IP1 and its corresponding nested primer IP2 based on the sequence between IP1 and the SSR, were designed from the determined sequences.An adaptor-primers AP1 (5′-CCATCGTAATA CGACTCACTATAGGGC-3′) and the nested primer AP2 (5′-CTATAGGGCACGCGTGGT-3′) were also prepared.The first PCR reaction was conducted using primers IP1 and AP1 with the diluted ligation product.The second PCR reaction was conducted using primers IP2 and AP2 with a 100-fold dilution of the first PCR products.Single-banded fragments were amplified after second PCR and were then cloned and sequenced.Finally a primer (IP3) was designed with the opposite flanking sequence of SSR locus to primers IP1 and IP2.The primer pair, IP1/IP3 or IP2/IP3 was examined as a potential SSR marker.All primers were designed by Primer Premier 5.0 software (Premier Biosoft International).Primer pairs that yielded expected size fragment were then subjected to loci polymorphism analysis.

Polymorphism evaluation of genomic SSR loci
Total DNA was separately isolated from leaves.PCR amplification conditions were: 25 µl reaction mixture containing 1× PCR buffer, 100 ng template DNA, 1.0 U Taq DNA polymerase (TaKaRa), 1.5 mmol/l MgCl2, 0.2 mmol/l of each dNTP, 0.4 µmol/l reverse primer  (Schuelke, 2000)).Amplification was performed by a PCR thermal cycler (Biometra ® T-Gradient 96) with the program of 5 min at 95°C; 30 cycles of 30 s at 95°C, 30 s at the annealing temperature of each primer pair (Table 2) and 2 min at 72°C (additional 8 cycles of 30 s at 95°C, 30 s at 53°C and 2 min at 72°C for fluorescent dye labeled primer), 10 min at 72°C.PCR products with the GeneScan-500 ROX internal-lane size standard (Applied Biosystems) were electrophoresed on ABI 377 (Applied Biosystems).SSR alleles were resolved by GeneScan 3.7.GENEPOP version 3.4 (Raymond and Rousset, 1995) was employed to calculate HO (Observed heterozygosity) and HE (Expected heterozygosity) values and to test LD (Linkage disequilibrium) and HWE (Hardy-Weinberg equilibrium).

Development of EST-SSR markers
The primer pairs intended to amplify 86 potential SSR loci, in silico identified in 1650 uniESTs from a B. chinense root cDNA library (Sui et al., 2010, in press) recently, were designed by Primer Premier 5.0 software.PCR amplification was performed using DNA from leaves of two B. chinense DC. cv.Zhongchai No. 1 individual plants.Their conditions were the same to that of genomic SSR markers with annealing temperatures showed in Table 4.

RESULTS AND DISCUSSION
Nineteen primer pairs which amplified a clear PCR product with the expected size were obtained (Figure 1 and Table 2).All the loci comprised perfect (6), interruptted perfect (4) and compound ( 9) SSRs.Size of the amplified fragments ranged from 132 to 373 bp.A total of 126 alleles were produced, ranging from 3 to 13 alleles with an average of 6.63 per locus.The observed heterozygosity (H O ) value ranged from 0.23 to 1.00, while the expected heterozygosity (H E ) value varied from 0.29 to 0.92.Nine of the nineteen polymorphic loci (labeled in Table 2) departed significantly from the Hardy-Weinberg equilibrium (HWE) (P < 0.05) and eight pairs of loci, BC-ac001 and BC-tc005, BC-ag001 and BC-tc005, BC-tg001 and BC-tc004, BC-at001 and BC-tc004, BC-at006 and BC-ag001, BC-tc001 and BC-at006, BC-tc002 and BC-at006 and BC-ac001 and BC-tc001, showed significant Linkage disequilibrium (LD) (Fisher's exact test, P < 0.01).Sequences which harbored the nineteen genomic SSR markers were registered in GenBank (Accession Nos.EU596392-EU596410).SSR markers developed in one species usually could be transferable to some other species of the same genus or closely related genera (Tang et al., 2006;Konishi et al., 2006).Therefore, to expand the use of these genomic SSR markers, the species transferability was tested in seven other Bupleurum species including wild and cultivated genotypes (Table 3).All loci were successfully amplified in B. longiradiatum var.porphyranthum, B. yinchowense and B. smithii var.parvifolium, eighteen in B. falcatum, seventeen in B. scorzonerifolium Willd., sixteen in B. sibiricum var.jeholense and eight in B. wenchuanense.These results showed most of the the genomic SSR markers had wide species transferability.
To obtain more useful SSR markers, primer pairs were designed and PCR amplifying verified based on our previous result of EST-SSR data mining in a B. chinense In summary, we firstly provide genomic SSR and potential EST-SSR markers of B. chinense and also some species transferability genomic SSR markers of other a "BC-" in the names of loci was dispensed with; "+": Positive amplification yielding specific PCR products of expected size; " -": Negative amplification yielding PCR products of unexpected size or without PCR products.

Table 1 .
Origin of plant materials used in the present study, B. chinense DC. cv.Zhongchai No. 1 used to isolate genomic SSR markers, B. chinense DC. f. octoradiatum (Bunge) Shan et Sheh used to analyze polymorphism of SSR loci and the others to test species transferability of the SSR markers.

Table 2 .
Characteristics of nineteen polymorphic genomic SSR markers, isolated from B. chinense DC. cv.Zhongchai No. 1, analyzed from 22 individual plants of B. chinense DC. f. octoradiatum (Bunge) Shan et Sheh d HE: Expected heterozygosity root cDNA library(Sui et al., 2010, in press).Only 73 of 86 EST-SSR loci were suitable for primer pairs design.PCR products with expected sizes were amplified by 44 primer pairs (approximately 60.3%) (Table4).The others primer pairs either had no clear PCR products or amplified bands without expected sizes.The uniESTs that harbored these EST-SSR markers were registered in GenBank (Accession Nos.FG341848, GR308099-EU308135).

Table 3 .
Cross-amplification of nineteen SSR markers among seven Bupleurum species.