Biochemical Systematics and Ecology 57 (2014) 227e230

Contents lists available at ScienceDirect

Biochemical Systematics and Ecology

journal homepage: www.elsevier.com/locate/biochemsyseco

The development of 204 novel EST-SSRs and their use

for genetic diversity analyses in cultivated alfalfa
Qiang Zhou, Tianlong Chen, Yanrong Wang, Zhipeng Liu*
State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou
730020, China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 2 July 2014
Accepted 24 August 2014
Available online 16 September 2014

Cultivated alfalfa (Medicago sativa L.) is one of the most important forage legume in the
world. Here, we report the development of 204 novel polymorphic expressed sequence tag
simple sequence repeat (EST-SSRs) markers from transcript sequences via global Illumina
sequencing for cultivated alfalfa. Among the synthesized 750 pairs of primers, 204 EST-SSR
primer pairs showed polymorphisms among 10 alfalfa accessions (including ve individual
plants in each accession) generating a total of 1222 alleles, with the number of alleles per
locus, the observed heterozygosity (Ho), the corrected heterozygosity (He) and the Shannon-Wiener diversity index (H'c) averaging at 5.99, 0.73, 0.71 and 1.14, respectively. Of the
204 novel EST-SSRs in alfalfa, 120 can be in silico mapped onto the eight Medicago truncatula chromosomes (Mt3.5.2). Considering the high polymorphism, these EST-SSRs can be
applied to assess genetic diversity, population structure, relatedness, evolution, linkage
mapping and cultivar protection of cultivated alfalfa to facilitate alfalfa breeding programs.
2014 Elsevier Ltd. All rights reserved.

Keywords:
EST-SSRs
Alfalfa
Genetic diversity
Medicago sativa

1. Introduction
Cultivated alfalfa (Medicago sativa L.) is one of the most important forage legume in the world and the third most valuable
crop in the United States. In addition to being a valuable forage crop for ruminants, alfalfa possesses considerable potential as
a biofuel crop for a sustainable feedstock for ethanol production. This species is a perennial, cross-pollinated and autotetraploid (2n 4x 32) plant with a genome size of 800e900 Mb (Choi et al., 2004). Extensive efforts have been made to
develop alfalfa transcriptome data, including 454 sequencing (Han et al., 2011) and Illumina sequencing (Li et al., 2012; Liu
et al., 2013; Postnikova et al., 2013; Yang et al., 2011), which have facilitated the development of transcript-based molecular markers for alfalfa, such as expressed sequence tag simple sequence repeat markers (EST-SSRs).
SSR markers are very useful for a spectrum of genetic and breeding because of their codominant nature, abundance in
genomes, high reproducibility, hyperpolymorphism and high rates of transferability. EST-SSRs always exhibit lower levels of
polymorphism than the genomic sequence-based SSRs. However, they are predicted to possess advantages like easy access,
presence in gene-rich regions and high level of transferability to related species, and they can often be used as anchor markers
for comparative mapping and evolutionary studies (Zhou et al., 2014). Despite the growing availability of single nucleotide
polymorphism (SNP) markers, SSRs are still the most used molecular markers by many breeders that might not have access to
expensive SNP-genotyping platforms for their species (Tyrka et al., 2008).

* Corresponding author. Tel.: 86 9318914051.

E-mail address: lzp@lzu.edu.cn (Z. Liu).
http://dx.doi.org/10.1016/j.bse.2014.08.023
0305-1978/ 2014 Elsevier Ltd. All rights reserved.

228

Q. Zhou et al. / Biochemical Systematics and Ecology 57 (2014) 227e230

Extensive studies have reported the usage of SSR markers in alfalfa (Diwan et al., 1997; Eujayl et al., 2004; Robins
et al., 2008; Sakiroglu et al., 2010; Sledge et al., 2005). But all of them were derived from Medicago truncatula. To
date, 61 polymorphic genomic SSRs (He et al., 2009), 27 EST-SSRs conrmed by individual alfalfa plants in each
accession (Liu et al., 2013), and 401 EST-SSRs conrmed by mixing plants in each accession have been developed (Wang
et al., 2013, 2014), which is still very insufcient for genetic research when compared with other plants, such as 1281
polymorphic EST-SSR markers available for peanut (Arachis hypogaea L.) (Koilkonda et al., 2012) and 2240 polymorphic
SSR markers for rice (Oryza sativa L.; McCouch et al., 2002). Moreover, considering the autotetraploid trait of alfalfa, it is
more precise to validate the polymorphism of EST-SSRs by the DNA of individual plants instead of mixing plants in each
accession.
Based on our previous study, here, we investigated the sequence identity and presence of EST-SSRs in the effective
unigenes on a large scale. Our objective was to increase the number of EST-SSR markers for cultivated alfalfa by further mining
the current unigene resources for polymorphisms. An additional 750 EST-SSR primer pairs were designed and synthesized,
and 204 novel EST-SSR primers showed polymorphisms using 50 individual plants. Totally, 120 polymorphic EST-SSR loci
were in silico mapped to the M. truncatula chromosomes.
2. Materials and methods
2.1. EST-SSRs detection and primer design
EST-SSRs were detected in 40,433 alfalfa unigenes with the simple sequence repeat identication tool (SSRIT) program.
Only unigenes longer than 1 kb were included in the EST-SSR detection. To avoid possible duplicates of published EST-SSRs
(Liu et al., 2013; Wang et al., 2013, 2014), a local BLASTX was performed using the published EST-SSR sequences against the
1494 SSR-containing unigene sequences (E-value <10E-10). The parameters were adjusted to identify perfect di-, tri-, tetra-,
penta- and hexa-nucleotide motifs with a minimum of 6, 5, 5, 5 and 5 repeats, respectively. The EST-SSR primers were
designed using BatchPrimer3. The primer design parameters were set as follows: length range, 18e23 nucleotides with 21 as
optimum; PCR product size range, 100-250 bp; optimum annealing temperature, 55  C; and GC content, 40e60%, with an
optimum content of 50%.
After EST-SSR identication, 750 non-redundant EST-SSR primer pairs were newly designed and synthesized. Five
different plants, one plant from each accession (StarQueen, AmeriStand IV, Longdong I, Xinjiangdaye and Qingyang; Table S1),
were employed for optimizing Mg2 concentration and melting temperatures (Tm) for polymerase chain reaction (PCR) and
for investigating the sequence identity between the primers and assembled unigenes.
2.2. Plant materials and EST-SSRs amplication
A total of 10 alfalfa accessions, including AmeriStand IV, Ladak DL, AmeriStand III, Resis, Longdong I, Algonquin, Xinjiangdaye, StarQueen, Qingyang, and Longdong III were selected for polymorphism analyses with the EST-SSRs (Table S1). To
accurately validate the polymorphisms of EST-SSRs, young leaves of ve individual plants in each accession were used to
extract genomic DNA via a modied cetyltrimethylammonium bromide (CTAB) method (Liu et al., 2013). PCR amplications
were conducted in a nal volume of 10 mL containing 40 ng template DNA, 1 PCR buffer, 2.0 mM MgCl2, 2.5 mM dNTPs, 4 mM
each primer and 0.8 U Taq polymerase (TaKaRa, Kyoto, Japan). The PCR reactions were performed using the following conditions: 4 min at 94  C, 35 cycles of 30 s at 94  C, 35 s at the annealing temperature (Table 1 and Table S2) and 1 min at 72  C,
with a nal extension step of 5 min at 72  C. The PCR products were subjected to electrophoresis on an 8.0% non-denaturing
polyacrylamide gels and stained by ethidium bromide (Liu et al., 2013). The PCR product sizes were identied by comparison
with the DL500 DNA maker (TaKaRa, Kyoto, Japan).
2.3. Diversity analysis
The observed heterozygosity (Ho) was calculated as previously shown (Liu et al., 2007), and the corrected heterozygosity
(He, corrected for sample size) and ShannoneWiener diversity index (H'c, corrected for sample size) were analysed using the
ATETRA 1.2.a software program. Only specic bands that could be unambiguously scored across all individual plants were
used in this study. A clustering analysis was used to generate a dendrogram using the unweighted pair-group method with
arithmetic mean (UPGMA) and Nei's unbiased genetic distance with NTSYSPC 2.0 software package (Nei, 1978).

Table 1
Characterization of 204 EST-SSRs in 10 alfalfa accessions.
Primer number

Number of alleles standard derivation

Ho standard derivation

He standard derivation

H'c standard derivation

204

5.99 1.53

0.73 0.19

0.71 0.09

1.14 0.19

Note: Ho, observed heterozygosity; He, corrected heterozygosity; H'c, ShannoneWiener diversity index.

Q. Zhou et al. / Biochemical Systematics and Ecology 57 (2014) 227e230

229

2.4. In silico mapping of EST-SSRs

The unigenes sequences containing simple sequence repeats were searched using Blat against the genome sequences of M.
truncatula (Mt3.5.2), including a threshold of 95% identity and 90% coverage (Han et al., 2011). The in silico map was drawn
with MapChart 2.0 as described previously (Liu et al., 2013).
3. Results and discussion
Among the 750 designed and synthesized primer pairs, 610 primer pairs showed successful PCR amplication in ve
alfalfa individuals. The remaining 140 primers failed to generate PCR products at various Mg2 concentrations and annealing
temperatures, which may have been due to the amplication with genomic DNA, the location of the primer across splice sites,
large introns, a chimeric primer or poor-quality sequences. Among the 610 working primer pairs, 512 amplied PCR products
at the expected sizes, 47 primer pairs resulted in larger product sizes than expected, indicating that there may be an intron or
transposon insertion within the amplicons, and PCR products of the other 51 primer pairs were smaller than expected,
suggesting a deletion within the genomic sequence, a lack of primer specicity or the possibility of assembly errors. Of the 512
primer pairs, 308 presented only one band or several bands without polymorphism, which might result from the primer
design or the homozygosity of the loci in alfalfa germplasm. The other 204 PCR amplications resulted in more than one band
and contained polymorphisms, which might be collected with high heterozygosity of the autotetraploid alfalfa germplasms.
An estimation of the number of polymorphisms in the 204 EST-SSR loci using the ATETRA 1.2.a software program indicated
that the number of alleles per locus (Van et al., 2010), Ho, He and H'c were 4e14 (mean 5.99), 0.40e1.00 (mean 0.73),
0.25e0.89 (mean 0.71) and 0.11e1.62 (mean 1.14), respectively. And a total of 1222 scored alleles were generated (Table 1,
Table S2 and Fig. 1). This result showed a high level of polymorphism in alfalfa, as suggested previously (Liu et al., 2007; Wang
et al., 2013, 2014). The estimates of polymorphisms are comparable to those reported for 27 EST-SSRs with the same statistical
method in alfalfa (mean alleles per locus, Ho, He and H'c of 6.11, 0.64, 0.64 and 1.23, respectively; Liu et al., 2013). This result
may be due to the autotetraploidy and cross-pollination in this species.
Different locations of EST-SSR repeats among unigene sequences may result in different putative functions. SSR variations
in the coding regions should be subjected to much stronger selective pressure than variations in the 50 -UTR (untranslated
regions) and 30 -UTR (Zhou et al., 2014). In the present study, 204 EST-SSR variations were all found in the coding regions.
Meanwhile, 28 of the unigenes did not yield close homology to known proteins and were not annotated (Table S2).
In addition, the available M. truncatula genome sequence (Mt3.5.2) was used as a scaffold to align the alfalfa unigenes
containing EST-SSRs. Under stringent conditions using Blat, an alfalfa in silico map was constructed (Fig. S1). The map units
provided in Fig. S1 represent the base pair length of each chromosome instead of the centimorgans of chromosomes. For
example, 473.7 on the chromosome 4 means about 47,370,000 bp in the chromosome 4. Among the 204 developed novel ESTSSRs in this study, 120 EST-SSRs can be mapped to Mt3.5.2. Among the eight chromosomes, chromosome 4 (25) and 6 (5)
showed the highest and the lowest number of EST-SSR loci on the Mt3.5.2 chromosomes, respectively. This result may be due
to the length of the chromosomes because chromosome 4 (47,531,895 bp) and 6 (23,177,108 bp) were the longest and shortest
chromosomes on the Mt3.5.2 pseudomolecules in M. truncatula (Han et al., 2011).

Fig. 1. EST-SSR marker variations at the Ms-227, Ms-703, Ms-402 and Ms-413 loci of 10 alfalfa accessions. Each accession includes ve individual plants. The
corresponding detailed information from the 10 alfalfa accessions is displayed in Table S1. The letter M denotes the molecular markers, which are 200 bp and
150 bp (top to bottom).

230

Q. Zhou et al. / Biochemical Systematics and Ecology 57 (2014) 227e230

The dendrogram showed that the 10 alfalfa accessions fell into cluster A and cluster B (Fig. S2). The cluster B comprised
three landraces, including Longdong I, Longdong III and Xinjiangdaye, suggesting that the three accessions may share some
genetic background. The cluster A contained seven accessions such as AmeriStand IV, Resis, Ladak DL, Qingyang, Algonquin,
AmeriStand III and StarQueen, which were introduced from different countries. The results showed no clear relationship
between the clustering pattern and geographical distance, suggesting that the use of a greater number of accessions from
close geographical locations and more individual plants per accessions will be essential to verify the genetic diversity of alfalfa
in future studies. Furthermore, the alfalfa accessions are synthetic populations developed through either phenotypic
recurrent selection or multiple hybridizations, which also partly explained why different geographical accessions did not
form separate clusters.
Acknowledgments
This research was supported by the National Basic Research Program of China (2014CB138704), the National Natural
Science Foundation of China (31272492) and the Regional Test for National Forage Variety in China e Alfalfa DUS test (NCF
[2013] No. 49).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2014.08.023.
References
Choi, H.K., Mun, J.H., Kim, D.J., Zhu, H.Y., Baek, J.M., Mudge, J., Roe, B., Ellis, N., Doyle, J., Kiss, G.B., Young, N.D., Cook, D.R., 2004. Estimating genome
conservation between crop and model legume species. Proced. Natl. Acad. Sci. U.S.A. 101, 15289e15294. http://dx.doi.org/10.1073/pnas.0402251101.
Diwan, N., Bhaqwat, A.A., Bauchan, G.B., Cregan, P.B., 1997. Simple sequence repeats DNA markers in alfalfa and perennial and annual Medicago species.
Genome 40, 887e895. http://dx.doi.org/10.1139/g97-115.
Eujayl, I., Sledge, M.K., Wang, L., May, G.D., Chekhovskiy, K., Zwonitzer, J.C., Mian, M.A., 2004. Medicago truncatula EST-SSRs reveal cross-species genetic
markers for Medicago spp. Theor. Appl. Genet. 108, 414e422. http://dx.doi.org/10.1007/s00122-003-1450-6.
Han, Y.H., Kang, Y., Torres-Jerez, I., Cheung, F., Town, C.D., Zhao, P.X., Udvardi, M.K., Monteros, M.J., 2011. Genome-wide SNP discovery in tetraploid alfalfa
using 454 sequencing and high resolution melting analysis. BMC Genomics 12 (350). http://dx.doi.org/10.1186/1471-2164-12-350.
He, C., Xia, Z.L., Campbell, T.A., Bauchan, G.R., 2009. Development and characterization of SSR markers and their use to assess genetic relationships among
alfalfa germplasms. Crop Sci. 49, 2176e2186. http://dx.doi.org/10.2135/cropsci2007.04.0456.
Koilkonda, P., Sato, S., Tabata, S., Shirasawa, K., Hirakawa, H., Sakai, H., Sasamoto, S., Watanabe, A., Wada, T., Kishida, Y., Tsuruoka, H., Fujishiro, T., Yamada, M.,
Kohara, M., Suzuki, S., Hasegawa, M., Kiyoshima, H., Isobe, S., 2012. Large-scale development of expressed sequence tag-derived simple sequence repeat
markers and diversity analysis in Arachis spp. Mol. Breed. 30, 125e138. http://dx.doi.org/10.1007/s11032-011-9604-8.
Li, X.H., Acharya, A., Farmer, A.D., Crow, J.A., Bharti, A.K., Kramer, R.S., Wei, Y.L., Han, Y.H., Gou, J.Q., May, G.D., Monteros, M.J., Brummer, E.C., 2012. Prevalence
of single nucleotide polymorphism among 27 diverse alfalfa genotypes as assessed by transcriptome sequencing. BMC Genomics 13 (568). http://dx.doi.
org/10.1186/1471-2164-13-568.
Liu, Z.P., Chen, T.L., Ma, L.C., Zhao, Z.G., Zhao, P.X., Nan, Z.B., Wang, Y.R., 2013. Global transcriptome sequencing using the illumina platform and the
development of EST-SSR markers in autotetraploid alfalfa. PLoS One 8, e83549. http://dx.doi.org/10.1371/journal.pone.0083549.
Liu, Z.P., Liu, G.S., Yang, Q.C., 2007. A novel statistical method for assessing SSR variation in autotetraploid alfalfa (Medicago sativa L.). Genet. Mol. Biol. 30,
385e391. http://dx.doi.org/10.1590/S1415-47572007000300015.
McCouch, S.R., Teytelman, L., Xu, Y.B., Lobos, K.B., Clare, K., Walton, M., Fu, B.Y., Maghirang, R., Li, Z.K., Xing, Y.Z., Zhang, Q.F., Kono, I., Yano, M., Fjellstrom, R.,
DeClerck, G., Schneider, D., Cartinhour, S., Ware, D., Stein, L., 2002. Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.). DNA
Res. 9, 199e207. http://dx.doi.org/10.1093/dnares/9.6.199.
Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583e590.
Postnikova, O.A., Shao, J., Nemchinov, L.G., 2013. Analysis of the alfalfa root transcriptome in response to salinity stress. Plant Cell. Physiol. 54, 1041e1055.
http://dx.doi.org/10.1093/pcp/pct056.
Robins, J.G., Hansen, J.L., Viands, D.R., Brummer, E.C., 2008. Genetic mapping of persistence in tetraploid alfalfa. Crop Sci. 48, 1780e1786. http://dx.doi.org/
10.2135/cropsci2008.02.0101.
Sakiroglu, M., Doyle, J.J., Brummer, E.C., 2010. Inferring population structure and genetic diversity of broad range of wild diploid alfalfa (Medicago sativa L.)
accessions using SSR markers. Theor. Appl. Genet. 121, 403e415. http://dx.doi.org/10.1007/s00122-010-1319-4.
Sledge, M.K., Ray, I.M., Jiang, G., 2005. An expressed sequence tag SSR map of tetraploid alfalfa (Medicago sativa L.). Theor. Appl. Genet. 111, 980e992. http://
dx.doi.org/10.1007/s00122-005-0038-8.
Tyrka, M., Perovic, D., Wardynska, A., Ordon, F., 2008. A new diagnostic SSR marker for selection of the Rym4/Rym5 locus in barley breeding. J. Appl. Genet.
49, 127e134. http://dx.doi.org/10.1007/BF03195605.
Van, Puyvelde.K., Van, Geert.A., Triest, L., 2010. ATETRA, a new software program to analyse tetraploid microsatellite data: comparison with TETRA and
TETRASAT. Mol. Ecol. Resour. 10, 331e334. http://dx.doi.org/10.1111/j.1755-0998.2009.02748.x.
Wang, Z., Yan, H.W., Fu, X.N., Li, X.H., Gao, H.W., 2013. Development of simple sequence repeat markers and diversity analysis in alfalfa (Medicago sativa L.).
Mol. Biol. Rep. 40, 3291e3298. http://dx.doi.org/10.1007/s11033-012-2404-3.
Wang, Z., Yu, G.H., Shi, B.B., Wang, X.M., Qiang, H.P., Gao, H.W., 2014. Development and characterization of simple sequence repeat (SSR) markers based on
RNA sequencing of Medicago sativa and in silico mapping onto the M. truncatula genome. PLoS One 9, e92029. http://dx.doi.org/10.1371/journal.pone.
0092029.
Yang, S.S., Tu, Z.J., Cheung, F., Xu, W.W., Lamb, J.F.S., Jung, H.J.G., Vance, C.P., Gronwald, J.W., 2011. Using RNA-Seq for gene identication, polymorphism
detection and transcript proling in two alfalfa genotypes with divergent cell wall composition in stems. BMC Genomics 12 (199). http://dx.doi.org/10.
1186/1471-2164-12-199.
Zhou, C.P., He, X.D., Li, F.G., Weng, Q.J., Yu, X.L., Wang, Y., Li, M., Shi, J.S., Gan, S.M., 2014. Development of 240 novel EST-SSRs in Eucalyptus L'Herit. Mol. Breed.
33, 221e225. http://dx.doi.org/10.1007/s11032-013-9923-z.