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In addition, 139 EST-SSR primer pairs with 240 loci were localized on all the 21 .... 100–300 bp, and dimers should be avoided as best as pos- sible. The primer .... SWES255. 1D(230) ... 2B(280), 3B(320), 2D(200), 3B(240) ... 7D(290), 7B(300).
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Progress in Natural Science 18 (2008) 1485–1490 www.elsevier.com/locate/pnsc

Development of SSR markers from ESTs of gramineous species and their chromosome location on wheat Linzhi Li 1, Junjun Wang, Ying Guo, Fangshan Jiang, Yunfeng Xu, Yingying Wang, Haitao Pan, Guanzhu Han, Ruijun Li, Sishen Li * National Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai’an 271018, China Received 11 February 2008; received in revised form 13 May 2008; accepted 26 May 2008

Abstract A total of 407,663 expressed sequence tags (ESTs) of wheat, barley, maize, rice, and sorghum, obtained from GenBank/dbEST, were used to search for simple sequence repeats (SSRs). A total of 10,253 EST-SSRs, which accounted for 2.52% of all the ESTs, were identified. Using Primer Premier 5.0, 1367 EST-SSR primer pairs were designed, of which 715 with high quality were synthesized. The 715 primer pairs were tested on wheat, rice, maize, cotton, and soybean under the same PCR conditions, and the effective primer pairs in the five crops were 500 (69.93%), 383 (53.57%), 452 (63.22%), 357 (49.93%), and 388 (56.27%), respectively. This indicated a high transferability of EST-SSR markers between far-ranging species. In addition, 139 EST-SSR primer pairs with 240 loci were localized on all the 21 wheat chromosomes by using Chinese Spring nulli-tetrasomic lines of wheat. Ó 2008 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved. Keywords: EST-SSR; Wheat; Maize; Rice; Barley; Sorghum; Cotton; Soybean

1. Introduction The genomes of eukaryotes contain iterations of 1 to 6 bp nucleotide motifs known as simple sequence repeats (SSRs) or microsatellites [1,2]. Because of their random distribution in animal and plant genomes, their abundance, polymorphic nature, co-dominance, and Mendelian inheritance [3], SSR markers are useful in linkage mapping [4–9], DNA functional gene tagging [10–12], map-based gene cloning [13,14], parental identification [15], genetic diversity analysis [14,16], and molecular evolutionary studies [17,18]. The traditional methods of development of genomic SSR markers include constructing genomic libraries, isolating and sequencing clones contained putative SSRs, *

Corresponding author. Tel.: +86 538 8242903; fax: +86 538 8242226. E-mail address: [email protected] (S. Li). 1 Present address: Yantai Academy of Agricultural Science, Yantai 265500, China.

followed by designing and testing of flanking primers, which are usually time-consuming and labor-intensive. Expressed sequence tags (ESTs) are sequenced portions of complementary DNA copies of mRNA, and represent part of the transcribed region of the genome. The numbers of EST sequences for important species, present in public databases, have increased exponentially over the past few years (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html). SSR markers based on ESTs are referred to as EST-SSR or eSSR markers. Compared with the traditional genomic SSR markers, EST-SSR markers originate from the transcribed regions of genomes, so that they may be used as the absolute markers of functional genes and are useful in direct identification of allelic genes for important traits. EST-SSRs are highly conserved, and they are transferable between species [19–23]. The development of SSR markers from ESTs is a fast, efficient, low-cost option [24]. The identification of EST-SSR markers has been reported in some important crops, such as wheat

1002-0071/$ - see front matter Ó 2008 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved. doi:10.1016/j.pnsc.2008.05.012

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[20,24–29], rice [30], barley [31,32], cotton [33–36], tomato [37], and other plant species. The objectives of this study were to develop EST-SSR markers from wheat, barley, maize, rice, and sorghum, test their transferability in the five main crops, and locate them on the specific wheat chromosomes using nulli-tetrasomic lines. 2. Materials and methods

buffer, 1.5 mM MgCl2, 0.1 mM dNTPs, 500 nM primers, 1 U Taq polymerase, and 50–60 ng template DNA. Reactions were run on a T1 thermocycler (Biotech Company, Germany). After a denaturation step at 95 °C for 5 min, 35 cycles were followed with denaturation at 95 °C for 1 min, renaturation at an annealing temperature between 50 °C and 65 °C for 45 s, extension for 1 min at 72 °C, and a final extension step for 10 min at 72 °C. The PCR products were detected on 6% polyacrylamide denaturing gels with silver staining.

2.1. Plant materials 3. Results and discussions Rice, wheat, maize, cotton, and soybean are the most important field crops in China, and they are also very important all over the world. We employed these five crops to test the validity of the new developed EST-SSR markers. The EST-SSR primer pairs were screened against three wheat accessions (Chinese Spring, Chuan 35050, and Shannong 483), one variety from each crop of rice (Yujing 6), cotton (Handan 109), and soybean (Hedou13), and one maize self-cross line (9801). Chuan 35050 and Shannong 483 are the parents of a recombinant inbred line (RIL) population [9]. Yujing 6, Handan 109, and Hedou13, and the self-cross line 9811 are famous varieties or self-cross lines in China. The assignment of EST-SSR markers to the specific wheat chromosomes was carried out by using a set of Chinese Spring nulli–tetrasomic lines (N–T lines). 2.2. Mining SSRs from EST databases EST sequences of wheat, barley, maize, rice, and sorghum were obtained from GenBank/dbEST (http:// www.ncbi.nlm.nih.gov/db-EST/index.html) from August 2004 to September 2005. A simple sequence repeat identification tool (SSRIT, http://www.gramene.org/gremene/ searches/ssrtool) program was applied to identify SSRs. The SSRIT program was run online, and the parameters were set for detection of di-, tri-, tetra-, and penta-nucleotide motifs with a minimum of 10, 7, 5, and 4 repeats, respectively. 2.3. Designing of EST-SSR primer pairs Primer Premier 5.0 software was used to design flanking primers according to the following criteria: the length of ESTs should be more than 100 bp and the SSRs were at least 20 bp, primer length 18–22 bp, annealing temperature 40.4–65.4 °C, GC content 30%–70%, PCR product size 100–300 bp, and dimers should be avoided as best as possible. The primer pairs were synthesized by Shanghai Sangon Biological Technology & Services Co., Ltd. 2.4. PCR amplification and electrophoresis detection PCR amplification was carried out in a 20 ll reaction volume described by Chen et al. [28], which contained 1

3.1. Frequency of EST-SSRs A total of 407,663 EST sequences were obtained from GenBank/dbEST, including 53,580, 27,742, 171,461, 127,527, and 27,353 ESTs of wheat, barley, maize, rice, and sorghum, respectively (Table 1). We found 10,253 SSRs in the ESTs using the SSRIT. The frequencies of EST-SSRs in different species ranged from 1.30% to 3.59% with an average of 2.52%. The average density of one EST-SSR was 24.67 kb (Table 1). This frequency is similar to that reported by Holton et al. [19] and Ramesh et al. [38]. Nicot et al. [26], however, found the frequency was 7.4% in wheat, which was much higher than ours. This might be explained by the different strategies or databases used for mining EST-SSRs. The frequencies of di-, tri-, tetra- and penta-nucleotide motifs were different in the five species (Table 2). Tri-nucleotide motifs were the most abundant repeats in four of the crops with 47.43% in wheat, 42.78% in maize, 55.40% in rice, and 41.23% in sorghum. This is in agreement with the previous studies [20,28,31], and can be explained by the suppression of non-trimeric SSRs in coding regions due to the risk of frameshift mutations that may occur when those microsatellites alternate in size of one unit [39]. The least frequent repeats were tetra-nucleotide motifs in wheat and rice and di-nucleotide motifs in barley, maize, and sorghum. In the previous studies, the frequencies of di-, tetra- and penta-nucleotide motifs are considerably different [20,28,31,33]. In barley, the frequencies of di-, tri-, tetra- and penta-nucleotide motifs were approximately from 21.39% to 28.06%, which differed from those obtained by Thiel et al. [31] and other species of this study.

Table 1 EST-SSRs obtained from EST database of gramineous species Source of EST-SSRs

ESTs (No.)

SSRs (No.)

Percentage

SSR (kb)

Wheat Barley Maize Rice Sorghum Total Mean

53,580 27,742 171,461 127,527 27,353 407,663 –

1033 360 3406 4576 878 10,253 –

1.93 1.30 1.99 3.59 3.21 – 2.52

40.13 30.09 34.25 14.59 19.65 – 24.67

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Table 2 The frequency of various nucleotide repeats Motifs

Di-nucleotides Tri-nucleotides Tetra-nucleotides Penta-nucleotides

Wheat

Barley

Maize

Rice

Sorghum

Number

%

Number

%

Number

%

Number

%

Number

%

225 490 126 192

21.78 47.43 12.20 18.59

77 84 101 98

21.39 23.33 28.06 27.22

598 1457 619 732

17.56 42.78 18.17 21.49

1119 2535 379 543

24.45 55.40 8.28 11.87

129 362 219 168

14.69 41.23 24.94 19.13

Table 3 The effective EST-SSR markers in five crops Source of EST-SSRs

EST-SSRs (Total No.)

Effective EST-SSR primer pairs (Number) Wheat

Wheat Barley Maize Rice Sorghum Total

157 49 196 282 31 715

Rice

Maize

Cotton

Soybean

Number

%

Number

%

Number

%

Number

%

Number

%

142 36 119 181 22 500

90.45 73.46 60.71 64.18 70.97 69.93

69 24 105 174 11 383

43.95 48.98 53.57 61.70 35.48 53.57

64 34 147 193 14 452

40.76 69.39 75.00 68.44 45.16 63.22

59 21 118 146 13 357

37.58 42.86 60.20 51.77 41.94 49.93

69 45 105 156 13 388

43.95 91.83 53.57 55.32 41.94 56.27

3.2. Development of EST-SSR markers and their validity in five crops Using the Primer Premier 5.0, 1367 EST-SSR primer pairs were designed from the 10,253 EST-SSR sequences, of which a total of 715 with high quality were synthesized (marker code: SWES251—SWES965) (Table 3). A summary of the 715 EST-SSR markers is available at http:// www.sdwgi.com/sdwgie.asp. The 715 EST-SSR primer pairs were tested on five field crops under the same PCR conditions. The validities of the EST-SSR markers in wheat, rice, maize, cotton, and soybean were 500 (69.93%), 383 (53.57%), 452 (63.22%), 357 (49.93%), and 388 (56.27%), respectively (Table 3, http:// www.sdwgi.com/sdwgie.asp). The highest effective percentages for wheat, rice, and maize were 90.45%, 61.70%, and 75.00%, respectively, where the primers and template sequences were derived from the same species, e.g. wheat primers amplifying wheat DNA (Table 3). Some authors reported that wheat EST-SSR markers were highly transferable across related genera [23–25,31]. Transferability of EST-SSR markers within gramineous species has also been studied [20,25,27,40]. The value of effective percentages in this study was similar to Gupta et al. [25], Yu et al. [21], and Zhang et al. [23,41], but lower than that in Gao et al. [20] and Tang et al. [40]. Although the taxonomic relationship between gramineous species is wide (especially cotton and soybean), the percentages of effective markers for the five crops were approximate. This indicates that the EST-SSR markers are more conserved than SSR markers [19,20,22,27,40], and the EST-SSR markers could be used in more far-ranging species. This is probably due to the fact that EST-SSRs originate from the coding regions that have a higher level of sequence conservation than intergenic regions.

Table 4 The numbers of effective EST-SSR markers in five crops Crops (No.)

Effective primer pairs (No.)

Percentage

5 4 3 2 1

151 157 119 109 123

21.11 21.96 16.64 15.24 17.20

Of the 715 primer pairs, 151 (21.11%), 157 (21.96%), 119 (16.64%), 109 (15.24%), and 23 (17.20%) successfully amplified products from five, four, three, two, and one crop(s), respectively, only 56 (7.83%) were non-effective in the five crops (Table 4). Yu et al. [27] tested 444 ESTSSR primer pairs on genomic DNA of barley, maize, rice and wheat, 368 (82.88%) produced PCR amplification products from at least one species, and 227 (51.13%) amplified DNA from two or more species; a lower success rate than our results. Conversely, Gao et al. [20] reported that 42.71% of the 478 EST-SSR markers amplified successfully in all the of four species (wheat, rice, maize, and soybean); a higher success rate than our results. 3.3. Chromosome localization of EST-SSR markers in wheat The 500 EST-SSR primer pairs that successfully amplified PCR products from wheat were analyzed using a set of Chinese Spring N–T lines. One hundred and thirty-nine primer pairs with 240 loci were distributed on all the 21 wheat chromosomes (Tables 5 and 6, and Fig. 1, http:// www.sdwgi.com/sdwgie.asp). Eighty-six primer pairs were associated with one chromosome, 34 with two chromosomes, 12 with three chromosomes, 5 with four chromosomes, 1 with five chromosomes, and 1 with six

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Table 5 Chromosome locations of EST-SSR markers using Chinese Spring nulli-tetrasomic lines of wheat Primers

Chromosomes (length in bp)

Primers

Chromosomes (length in bp)

Primers

Chromosomes (length in bp)

SWES251 SWES255 SWES257 SWES260 SWES292 SWES294 SWES302 SWES337 SWES340 SWES342 SWES343 SWES345 SWES346 SWES347 SWES353 SWES356 SWES366 SWES376 SWES379 SWES382 SWES384 SWES387 SWES388 SWES393 SWES411 SWES413 SWES415 SWES420 SWES423 SWES426 SWES429 SWES430 SWES437 SWES439 SWES440 SWES442 SWES456 SWES475 SWES480 SWES487 SWES496 SWES498 SWES499 SWES500 SWES505 SWES516 SWES524 SWES527

6DL(310) 1D(230) 3D(147), 5D(450), 7B(530) 1B(310), 1B(120) 6DL(150), 4B(650) 6DL(380), 6DL(165) 7B(217) 6A(500) 2D(280), 7B(230), 6DL(147) 7D(170) 6B(340) 6DL(140), 6DL(100) 3A(220), 1D(402), 1D(404) 6DL(300) 1D(150) 1A(450) 5B(130) 3A(230), 2B(200), 4D(170) 4D(250) 1A(242), 6DS(240) 3A(250) 7A(210) 6DL(500) 7A(230) 1B(404), 3B(285) 3A(290) 4D(292), 6DL(160) 5D(292), 3B(155) 3B(175) 3B(410), 3D(415) 7B(405) 3D(217) 2B(315) 1B(360), 3B(185), 6DL(390) 6A(395), 1D(390) 2A(197), 3B(195), 5B(310) 1A(210) 7B(225), 5D(155), 7D(140) 7B(100) 6DL(217) 6A(320) 1A(400), 2D(245) 1A(360) 7A(290) 1A(300) 1B(110) 1B(180) 3D(215), 1A(180)

SWES528 SWES530 SWES536 SWES539 SWES541 SWES543 SWES544 SWES545 SWES546 SWES547 SWES550 SWES553 SWES555 SWES558 SWES559 SWES560 SWES565

1A(300), 1A(230), 3D(173), 3D(175) 3B(310), 1D(178) 4D(390), 3A(270) 3A(309) 3B(650) 1D(238) 3D(630) 3B(309) 1B(165) 4D(400), 1B(270) 2D(195) 3A(150), 3B(147) 5D(198) 5D(195) 2B(280), 3B(320), 2D(200), 3B(240) 1B(100), 1B(102) 3B(420), 1D(130), 7B(120) 1D(102), 1D(100) 1B(160) 6B(630) 3B(350), 2B(150) 6DS(260) 7B(150) 3B(410), 1B(155), 1D(170), 6DL(245), 1A(175), 3A(380) 1A(210), 1B(195), 1D(185) 3B(200), 6A(180), 6DL(175) 6DS(140) 4D(185) 4D(385) 5D(400), 4D(235) 7D(630) 2B(400) 3A(315), 7A(180) 7D(290), 7B(300) 3A(280) 3B(630), 2D(230), 2B(220), 2B(190) 7D(309), 5D(245) 3B(630), 1A(290), 2B(220) 2D(230), 2B(190) 3A(205) 3A(390), 7D(165), 7B(150), 7A(130) 7A(155), 7B(150), 7D(130) 2D(227), 2B(225) 2D(130), 2D(132) 6DS(242) 5D(165) 1B(125)

SWES662 SWES672 SWES676 SWES695 SWES705 SWES711 SWES727 SWES728 SWES745 SWES751 SWES761 SWES762 SWES767 SWES784 SWES813 SWES823 SWES828 SWES836 SWES858 SWES870 SWES875 SWES893 SWES895 SWES898 SWES899 SWES905 SWES907 SWES908 SWES910 SWES911 SWES916 SWES921 SWES923 SWES924 SWES925 SWES932 SWES934 SWES936 SWES940 SWES942 SWES947 SWES953

4B(250), 4A(245) 3A(200) 7B(100) 6DL(220), 2A(245) 7D(300) 2A(300), 3D(188), 6DS(155), 7B(115) 7D(400) 7D(400) 1D(400), 2D(155), 4D(165) 2B(155), 2B(153) 4D(222), 4D(220) 3B(527), 6A(400), 6A(280) 2D(622) 3A(330), 6A(350), 3A(215) 3A(320), 1D(310) 7B(250), 7A(210) 1A(500) 7A(380) 1A(280), 1B(290), 1D(300) 7B(410), 7B(412) 4D(300), 7B(530) 1A(520) 6A(140) 6A(530) 3A(217), 5D(195) 5A(210) 1B(165) 1B(185) 5A(380), 1B(215) 7B(170) 1A(170), 1B(130) 2D(182) 3B(200) 3D(165) 6A(150) 3D(165) 3A(140), 3D(160) 6DS(450) 7B(235) 6DL(320) 2B(310), 1D(290), 1B(280), 1A(230) 1A(220), 1B(210), 1D(195), 1B(135), 1D(130), 6DL(120) 6A(180), 6DL(175) 7A(235) 5D(260) 4B(125)

SWES567 SWES568 SWES571 SWES576 SWES577 SWES578 SWES579 SWES586 SWES590 SWES595 SWES596 SWES600 SWES601 SWES602 SWES604 SWES608 SWES609 SWES613 SWES618 SWES619 SWES622 SWES625 SWES626 SWES628 SWES631 SWES639 SWES648 SWES650

chromosomes. For all the 240 loci, only one locus was located on chromosome 4A, and two loci on chromosomes 5A, 5B, and 6A, while 24 loci were located on 6D. The chromosomes 1A, 1B, 3A, 3B, and 7B also had more loci than others. Ninety-seven loci were located on the D genome, 81 on the B genome, and 62 on the A genome. The most loci were on group 1 with 58 loci, and the least were on group 5 with 14 loci and group 4 with 16 loci (Table 6). We had located 93 EST-SSR primer pairs and 193 loci on 19 wheat chromosomes using N–T lines previously [28]. Yu et al. [27] located 80 EST-SSR primer pairs (104 loci) on wheat chromosomes. The chromosomal locations of

SWES954 SWES957 SWES960 SWES965

Table 6 Distribution of EST-SSR markers on chromosomes Homologous group

Genome

Total

A

B

D

1 2 3 4 5 6 7

18 3 18 1 2 11 9

22 13 20 3 2 2 19

18 12 11 12 10 24 10

58 28 49 16 14 37 38

Total

62

81

97

240

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Fig. 1. Chromosomal localization of SWES555 using Chinese Spring nulli-tetrasomic lines. (1) N1AT1B, (2) N2AT2B, (3) N3AT3D, (4) N4AT4D, (5) N5AT5D, (6) N6AT6B, (7) N7AT7B, (8) N1BT1D, (9) N2BT2D, (10) N3BT3D, (11) N4BT4A, (12) N5BT5D, (13) N6BT6A, (14) N7BT7A, (15) N1DT1B, (16) N2DT2A, (17) N3DT3A, (18) N4DT4B, (19) N5DT5B, (20) N6DS, (21) N6DL, (22) N7DT7B, (23) Chinese Spring; M, marker.

EST-SSR loci provide a basis for genetic mapping and gene identification. In addition, EST sequences corresponding to 715 ESTSSR markers were analyzed by the BLASTN and BLASTX programs to obtain information of their possible functions (http://www.sdwgi.com/sdwgie.asp). Forty-eight loci from the above EST-SSR markers (SWES251— SWES965) had been previously mapped [9]. 4. Conclusions (1) A sum of 10,253 SSRs were found in the ESTs using 407,663 EST sequences from wheat, barley, maize, rice, and sorghum, and the average frequency of SSRs in the ESTs was 2.52%. (2) A total of 1367 EST-SSR primer pairs were designed, of which 715 high quality primer pairs were synthesized. (3) The effective primer pairs in wheat, rice, maize, cotton, and soybean were 500 (69.93%), 383 (53.57%), 452 (63.22%), 357 (49.93%), and 388 (56.27%), respectively. (4) One hundred and thirty-nine EST-SSR primer pairs with 240 loci were located on all of 21 wheat chromosomes using N–T lines. Acknowledgements This work was supported by the Major State Basic Research Development Program of China (Grant No. 2006CB101700) and the National Natural Science Foundation of China (Grant No. 3057155). References [1] Tautz D, Renz M. Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res 1984;12:4127–38. [2] Tautz D. Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res 1989;7:6463–71. [3] Powell W, Machray GC, Provan J. Polymorphism revealed by simple sequence repeats. Trends Plant Sci 1996;7:215–22. [4] Ro¨der MS, Korzun V, Wandehake K, et al. A microsatellite map of wheat. Genetics 1998;149:2007–23. [5] Paillard S, Schnurbusch T, Winzeler M, et al. An integrative genetic linkage map of winter wheat (Triticum aestivum L.). Theor Appl Genet 2003;107:1235–42. [6] Daryl JS, Peter I, Keith E. A high-density microsatellite consensus map for bread wheat (Triticum aestivum L.). Theor Appl Genet 2004;109:1105–14.

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