# Presentation: 680391

## Enlargement of PCR-based marker resources in oat by surveying  

**              ****genomic-derived SSRs from barley and wheat**
_**Gongshe Hu**_, Robert Campbell, Eric Jackson, and J. Michael Bonman.  USDA-ARS, Small Grains and Potato Germplasm Research Unit, 1691 South 2700 West, Aberdeen, Idaho 83210

**Abstract**
**Simple sequence repeats (SSRs), a relatively new generation of DNA markers, are developing rapidly in most crop species.  SSR’s are advantageous because they are present in large numbers, are evenly dispersed, and are highly polymorphic. Oat lacks of SSR markers due limited investment in developing genomic and genetic resources in *****Avena*****. To search for an alternative means to enlarge the SSRs pool in oat, we surveyed 356 genomic derived SSR markers from wheat and barley chosen to based on even dispersal across different chromosomes. These were tested for amplification and polymorphisms in parental lines of the main mapping populations of oat; TAM O-301, Ogle1040, Kanota156, and Ogle157. Ninety of 210 wheat SSRs (43%) and fifty six of 146 barley SSRs (38%) successfully amplified in oat. At least 66 markers (45% of the amplified markers and 19% of total markers) showed polymorphism between the parental lines of at least one mapping population. The percentage of polymorphic SSRs among the amplified primers is comparable to that for oat-derived SSRs in published data. Work is underway to determine if the polymorphic SSRs from the Ogle X TAM O-301 population can be mapped.  The survey results indicate that genomic SSRs from wheat and barley may be good sources of SSR markers for oat genetics research. The genomic relationships regarding DNA marker amplification provides clues for adapting of other types of DNA markers, such as SNPs, from barley and wheat to oat in the future.**

**Introduction**
**Simple sequence repeats (SSRs) are ideal DNA markers because of their  convenience and rapid performance in the assay (Powell W, 2006;Morgante M, 1993). SSRs have been used in a wide range of applications in genetic and genomic research (Roder MS, 1995;McCouch et al., 2002b;McCouch et al., 2002a;Varshney et al., 2002). However, there are very few SSR markers available in oat. Limitation of the  genomic sequence information for oat has inhibited the large-scale development of SSR markers from oat. **
**The objective of this study is to systemically survey SSR markers from barley and wheat genomes for the possible large-scale use of these SSRs in oat. **
**Materials and Methods**
**DNA extraction.  Four oat cultivars, TAM O-301, Ogle 1040, Kanota156, and Ogle157 (Holland et al.,   2001;Portyanko et al., 2001), were grown in greenhouse in Aberdeen. Leaf tissue was collected at the seedling stage and DNA was extracted using the CTAB protocol.**
**PCR performance. PCR reactions were set up in 96-well plates with a total of  25 uL per well. Each reaction contains 50 to 100 ng of template DNA, 1 ul of each primer pair (10 uM), 2.5 ul of 10X buffer, 1 ul of dNTPs (2.5 mM each), 1 unit of Taq Polymerase (RedTaq, Sigma). The PCR program was as follows: 940C for 3 minutes and followed by 39 cycles of 940C for 30 seconds, 500C for 30 seconds, and 720C for 1 minute, and then 40C for storage.  PCR reactions were analyzed on 6% polyacrylmide gel. Only strong PCR products were used to score polymorphisms between parental lines of each mapping population (TAM301 x Ogle1040 and Kanota x Ogle 157).**
**SSR selection. We selected about 20 SSRs from each chromosome of  barley genome based on the major SSR maps (Li et al., 2003b; Li et al., 2003a; Ramsay et al., 2000). We also selected 10 SSRs from each chromosome of a hexploid wheat map (Somers et al., 2004). The only criteria for SSRs selection was to cover evenly as much of the genome as possible. We surveyed a total of 146 SSRs from barley and 210 from wheat.**

**Results**
**A total  of 146 SSRs from barley and 210 SSRs from wheat were surveyed on 4 oat lines. **
**Fifty-five from SSRs from barley (38%) and 90 SSRs from wheat (43%) amplified repeatable PCR products on oat lines.**
**Sixty-six of the 146 (45%) oat-amplified SSRs showed polymorphisms on the parental lines in either the Ogle-TAM or the Kanota-Ogle mapping populations.**
**Amplification and polymorphism rates were  slightly different  among the SSRs from the sub-genomes of wheat and from different chromosomes.**

**Conclusion****Wheat and barley genomic SSRs are good resources  for development of oat SSR markers.**

**Table 1. Summary of oat-amplifiable SSR markers selected from barley and wheat.**

| 
Species | Markers selected | Markers amplified | Amplification frequency (%) |
| --- | --- | --- | --- |
| Barley | 146 | 55 | 38 |
| Wheat | 210 | 90 | 43 |

**Table 2. Comparison of oat amplification rates of SSR markers from different wheat and barley chromosomes**

| SSR Source |  | CH1 | CH2 | CH 3 | CH 4 | CH 5 | CH 6 | CH 7 | Average |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Barley | amplified | 7/19 | 13/34 | 7/16 | 9/20 | 6/19 | 10/18 | 3/20 | 55/146 |
|  | % | 37 | 38 | 44 | 45 | 32 | 56 | 15 | 38 |
| Wheat A | Amplified | 3/10 | 6/10 | 6/10 | 4/10 | 7/10 | 4/10 | 5/10 | 35/70 |
|  | % | 30 | 60 | 60 | 40 | 70 | 40 | 50 | 50 |
| Wheat B | Amplified | 2/10 | 7/10 | 5/10 | 6/10 | 3/10 | 5/10 | 3/10 | 31/70 |
|  | % | 20 | 70 | 50 | 60 | 30 | 40 | 30 | 44 |
| Wheat D | Amplified | 2/10 | 3/10 | 4/10 | 6/10 | 5/10 | 2/10 | 2/10 | 24/70 |
|  | % | 20 | 30 | 40 | 60 | 50 | 20 | 20 | 34 |

**Table 3. Polymorphic SSR markers for parental lines of two oat mapping populations **

| 
Genome | Polymorphic assay of SSRs on oat lines |  |  |
| --- | --- | --- | --- |
|  | Amplified | Polymorphic | Polymorphic 
frequency (%) |
| Barley | 55 | 27 | 49% |
| Wheat A | 35 | 14 | 40% |
| Wheat B | 31 | 17 | 55% |
| Wheat D | 24 | 8 | 33% |
| Total | 146 | 66 | 45% |

-   1H(5)    2 H(2)   3H(3)     4H(4)    5 H(7)   6H(6)    7H(1)

- GMS021

- Bmac0399
- Bmac0213

- Bmac0063
- EBmac0816
- Bmac0105
- EBmac0501
- EBmac0405
- EBmac0656
- Bmac0344c
- Bmac0347
- Bmac0504
- Bmac0154
- Bmac0382

- Bmag0718
- HVHVA1
- cMWG676

- EBmac0783

- Bmac0314

- HVM36

- EBmag0793
- HVM54
- aMSU21
- EBmac0415
- ABC257
- ABC317
- ABC252
- cMWG694

- Bmac0218

- EBmac0737
- HVHOTR1
- Bmag0381
- Bmag0140
- EBmac0715
- EBmac0640
- HVM63
- EBmac0607
- EBmac0849
- HVM23
- EBmac0558
- Bmac0222
- HvXan

- Bamg0711
- Hv5s
- Bmag0125
- Bmag0692
- cMWG660
- EBmatc0039
- HVCSG23

- Bmag0749

- HVM40
- HvOLE
- EBmac0691
- EBmac0906
- EBmac0775
- EBmac0540
- Bmac0310
- Bmac0181
- Bmac0298
- Bmac0030
- Bmac0577
- Bmac0375
- EBmac0781
- EBmac0658
- EBmac0701
- EBmac0635
- EBmac0679
- HVMLOH1A
- EBmac0788
- WMS6
- Bmac0138b
- HVM67

- EBmac0565b
- EBmac0705
- Bmag0603
- Bmag0136
- Bmac0067
- Bmac0043
- Bmag0122
- HCM60
- Bmag0225
- Bmag0606
- Bmag0013
- Bmag0877
- HVM62
- EBmac0708
- HVM70
- Bmag0853

- Bmac0303
- Bmac0306
- Bmag0387
- Bmag0337
- Bmag0394
- Bmag0323
- EBmatc0040
- Bmac0311
- Bmag0003b
- Bmag0005
- EBmac0518
- EBmac0970
- Bmag0113b
- HvLOX
- GMS061
- EBmatc0003
- EBmac0824
- GMS027
- Bmag0222

- Bmac0316
- Bmag0500
- Bmag0173
- Bmag0219
- EBmac0853
- Bmag0496
- EBmac0674
- EBmac0874
- EBmac0639
- EBmatc0028
- Bmac0018
- HVM31
- Bmag0009
- HVM65
- EBmac0602
- Bmag0613
- Bmac0251
- Bmac0040

**EBmac0713**
**Bmag0021**
**EBmag0794**
**Bmag0206**
**HVM4**
- Bmag0007
**Bmag0516**
**EBmatc0016**
**MWG2301.2**
- Bmac0582
- Bmag0011
**Bmac0031**
**Bmag0369**
**Bmag0385**
**Bmag0507**
**Bmag0120**
**EBmag0757**
**EBmac0565c**
**Bmag0004**
- Bmag0135

**Figure 1.  Distribution of barley SSR markers **
**selected and amplified (red) in oat**

- Wmc51
- Barc8
- Wmc128
- Barc187
- Wmc626
- Cfd48
- Wmc206
- Wmc44
- wmc367
- wmc728

- Gwm11
- Wmc336
- Wmc95
- Barc83
- Barc119
- Wmc183
- Wmc9
- Wmc716
- Wmc59
- barc17

- Gdm33
- Barc149
- Cfd15
- Wmc222
- Gwm337
- Barc169
- Wmc732
- Barc66
- gwm232
- gwm111

- Cfd36
- Wmc382
- Wmc598
- Wmc453
- Wmc296
- Wmc261
- Wmc109
- Cfd86
- Wmc181
- barc76

- 1

- A     B     D

-  2 A   B    D

- Wmc764
- Wmc25
- Wmc213
- Gwm148
- Barc183
- Barc98
- Wmc245
- Barc101
- Wmc175
- wmc356

- Barc124
- Barc90
- Wmc503
- Wmc112
- Wmc470
- Cfd2
- Gwm30
- Cfd73
- Wmc243
- barc59

- 3  A    B    D

- Wmc532
- Barc45
- Wmc505
- Barc67
- Wmc527
- Wmc264
- Wmc559
- Wmc153
- Wmc169
- wmc594

- Wmc430
- Barc87
- Wmc623
- Wmc78
- Wmc625
- Wmc307
- Wmc418
- Cfd283
- Wmc326
- wmc632

- Wmc11
- Wmc43
- Gwm2
- Wmc492
- Barc68
- Cfd71
- Wmc435
- Wmc529
- Wmc552
- barc71

- Wmc516
- Wmc173
- Wmc513
- Wmc468
- Wmc161
- Wmc718
- Wmc232
- barc70
- Wmc776
- wmc219

- 4  A    B    D

- Wmc125
- Barc109
- Barc163
- Cfd22
- Wmc657
- wmc259
- Barc20
- Wmc511
- Wmc710
- wmc617

- Wmc617
- Wmc285
- Wmc89
- Wmc52
- Wmc489
- Gwm165
- Wmc399
- Cfd84
- Wmc622
- wmc74

**Figure 2.  Distribution of wheat SSR markers selected and amplified (red) in oat**

- Barc10
- Wmc47
- gwm109
- Barc197
- Barc141
- Barc40
- Wmc415
- Wmc388
- Wmc96
- wmc727

- Wmc773
- Wmc47
- Wmc149
- Barc4
- Wmc616
- Barc89
- \wmc537
- Wmc75
- Wmc734
- barc59

- Wmc233
- Wmc150
- Wmc318
- Wmc405
- Wmc630
- Wmc289
- Wmc95
- Cfd19
- gdm133
- barc144

- 5 A    B     D

- Gwm334
- Barc206
- Barc23
- Wmc145
- Wmc553
- Wmc179
- Wmc417
- Wmc580
- Wmc621
- wmc254

- Wmc419
- Wmc487
- Cfd13
- Wmc597
- Wmc398
- Wmc397
- Baec127
- Barc24
- Barc178
- barc134

- Barc183
- Barc173
- Cfd132
- Barc54
- Gwm325
- Barc5
- Barc204
- Barc175
- Barc96
- wmc773

- 6   A    B    D

- Wmc158
- Wmc497
- Wmc479
- Wmc283
- Wmc83
- Gwm260
- Wmc422
- Barc195
- Wmc633
- wmc525

- Wmc323
- Gwm68
- Barc85
- Gwm43
- Barc95
- barc176
- Cfa2106
- Wmc517
- Wmc613
- wmc10

- Wmc506
- Gdm145
- Gwm130
- Wmc463
- Gwm44
- Barc128
- Wmc221
- Wmc94
- Gwm37
- wmc166

- 7   A    B     D

**Figure 3. Examples of barley SSRs tested on oat lines**

- T   O1   K   O2    T   O1   K   O2    T    O1   K   O2    T    O1   K    O2

**EBmac674   EBmac0405  GMS021    Bmac213**

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**References**