Barley Genetics Newsletter 37:21-23 (2007)

PCR-based markers

targeting barley putative grain yield and quality QTLs regions

Isabel A. del Blanco\*, Deric A. Schmierer, Andris Kleinhofs and Steven
E. Ullrich

Departament of Crop and Soil Sciences, Washington State University,
Pullman, WA, 99164-6420

\*e-mail: [**mailto:blanco@wsu.edu**](mailto:blanco@wsu.edu)

Abstract

Eight restriction fragment length polymorphism (RFLP) and two genes
delimiting, or included in, chromosome fragments containing putative
QTLs for grain yield and quality were sequenced and converted to
PCR-markers. Eight markers were co-dominant between two-rowed barley
cultivars Harrington and Baronesse after digestion with restriction
enzymes. Three were dominant-recessive after designing specific primers
exploiting single nucleotides polymorphisms (SNPs) between those two
cultivars.

#### Introduction

Two chromosomal regions from Baronesse have been reported as containing
putative grain yield QTLs, one on chromosome 2(2HL) (between markers
ABG461C and MWG699) and the other on chromosome 3(3HL) (between markers
MWG571A and MWG961) (Schmierer et al., 2004). Subsequent analysis of
Harrington/Baronesse derived inbred lines suggested other additional
regions as candidates for grain yield QTLs. These regions are on
chromosome 7(5HL) (between markers ABC717 and ABC718) and on the
telomeric region of the short arm of chromosome 2(2HS) (ABG058).

Since RFLP methodology needs large amounts of DNA and entails a complex
procedure with radioactively labeled probes and Southern blotting, which
requires several days to produce results, we converted RFLP targeting
those putative QTL regions to PCR-markers: cleaved amplified polymorphic
sites (CAPS) and SNPs. The conversion of RFLP clones to PCR-based
markers rendered a much simpler technique that facilitated the screening
of large numbers of genotypes at the seedling stage, since it requires a
small amount of DNA.

#### Materials and Methods

Genomic DNA extraction was modified from Edwards et al. (1991); the
modification added an extra-step of chloroform-isoamyl alcohol (24:1)
extraction. CAPS were developed from RFLP clones. Primers were designed
from the cloned sequences and used to amplify genomic DNA from the
parental cultivars Harrington and Baronesse. If no fragment length
polymorphism was observed, the fragments from both parents were
sequenced to discover SNPs. These SNPs were analyzed to identify
restriction enzymes that could be used to develop CAPS, or to design
cultivar-specific primers. CAPS markers were also developed for gene
*Dhn1* (Choi et al., 2000) and a candidate gene for seed dormancy and/or
pre-harvest sprouting, *GA20-oxidase* (Li et al., 2004). Reactions for
SNPs were set under stringent conditions (annealing temperatures \~ Tm)
and short cycles (annealing ≤ 15 s). Extensions were 1min or 2min
depending on product size.

Primer3 software (Rozen and Skaletsky, 2000) assisted the process of
primer design. PCR products were visualized on 1% or 2%, depending on
the fragment size, agarose gel under UV light. PCR products were
purified with Exonuclease I (Exo-SAP-IT, UBS, Cleveland, OH). Sequencing
reactions were performed on an Applied Biosystems 3100 Genetic Analyzer
(Perkin Elmer Applied Biosystem Division, Foster City, CA) with the ABI
PRISM Big Dye Terminator v3.1 cycle sequencing kit. Products were
confirmed by sequencing in both directions. Analysis of sequences to
find restriction sites and/or SNPs was done with the tools provided by
the San Diego Super Computer Center (SDSC,
[http://workbench.sdsc.edu](http://workbench.sdsc.edu/)).

#### Results and Discussion

Details of developed PCR markers are listed in Tables 1 (CAPS) and 2
(SNPs). CAPS marker MWG699 yielded small fragments difficult to
visualize after digestion with enzyme *Taq1,* even when 3% UltraPure
Agarose-1000 was used to run PCR products. To avoid this problem,
cultivar-specific markers were developed taking advantage of SNPs
between the parental genotypes. The two SNP sites are different than the
*TaqI* restriction site, for this reason they are considered two
different markers for locus MWG699.

**Table 1.** Summary of developed CAPS markers, their location, primers
and restriction enzymes

+----+----------+---+-----------------------------------+------------+
| ** | *        | * | **Forward primer**                | **Enzyme** |
| Ch | *Locus** | * |                                   |            |
| r. |          | B | **Reverse primer**                |            |
| ** |          | i |                                   |            |
|    |          | n |                                   |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+
| *  | **M      | * | **5'-GTATCGTCAACACGGCAGCGT-3'**   | *          |
| *3 | WG571A** | * |                                   | **Bam*HI** |
| (3 |          | 0 | **5'-TACCTGTCAGAAGTGCAGTACC-3'**  |            |
| H) |          | 0 |                                   |            |
| ** |          | 9 |                                   |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+
| *  | **       | * | *                                 | ***Bsg*I** |
| *3 | MWG961** | * | *5'-TCAACTCCAGCCTTCACACACAAC-3'** |            |
| (3 |          | 0 |                                   |            |
| H) |          | 1 | *                                 |            |
| ** |          | 2 | *5'-AAGACGAAGGAGACGTTGTTCATG-3'** |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+
| *  | **       | * | **                                | **         |
| *2 | MWG699** | * | 5'-ACCCACTGGGTTTGATACTACAAAG-3'** | *Taq*I\*** |
| (2 |          | 0 |                                   |            |
| H) |          | 1 | **                                |            |
| ** |          | 0 | 5'-GTGATGTTATTGGTGACTTGAACTC-3'** |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+
| *  | **M      | * | **                                | *          |
| *1 | WG851A** | * | 5'-CAAGAACTCCATTCCAATGTACCTG-3'** | **Hae*III, |
| (7 |          | 0 |                                   | *Msp*I**   |
| H) |          | 0 | **                                |            |
| ** |          | 1 | 5'-TACTTCCAGATCCATGACAAGCTAC-3'** |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+
| *  | ***Dhn   | * | *                                 | ***Taq*I,  |
| *7 | 1\*\**** | * | *5'-TCACTGTTCGTACTTCGTAGCACC-3'** | *Hpy*CH4   |
| (5 |          | 0 |                                   | IV**       |
| H) |          | 1 | **5'-TCCGCAGTTGCTCCTCCAAT-3'**    |            |
| ** |          | 1 |                                   |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+
| *  | **       | * | **                                | *          |
| *7 | ABC309** | * | 5'-CAGAGATACCACTGGGATTCTAAAC-3'** | **Hinf*I** |
| (5 |          | 0 |                                   |            |
| H) |          | 1 | **5'-CGAAAACCCTAGGAGAGCTAATC-3'** |            |
| ** |          | 5 |                                   |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+
| *  | **M      | * | **                                | ***Alu*I** |
| *7 | WG2249** | * | 5'-AGCCATGCCGGTCTTGTCAAGAAAG-3'** |            |
| (5 |          | 0 |                                   |            |
| H) |          | 1 | **                                |            |
| ** |          | 5 | 5'-ATGCATCTGATCCCTGGAGAAGAAC-3'** |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+
| *  | **       | * | **                                | ***AvaI*** |
| *7 | *GA20-ox | * | 5'-GTCCATCATGCGCCTCAACTACTAC-3'** |            |
| (5 | idase*** | 0 |                                   |            |
| H) |          | 1 | **                                |            |
| ** |          | 5 | 5'-TAGCAAATCTTGCCATCCATCCATG-3'** |            |
|    |          | * |                                   |            |
|    |          | * |                                   |            |
+----+----------+---+-----------------------------------+------------+

\* Restriction enzyme (TaqI) previously reported by Tanno et al. (2002)
in a different population

\*\* Primers from Choi et al. (TAG 101:350-354)

**Table 2**. Cultivar-specific PCR primers developed exploiting
nucleotide polymorphisms between Harrington (H) and Baronesse (B)
cultivars

+------+------------+------+------------------------------------------+
| **Ch | **Locus**  | **B  | **Forward primer**                       |
| r.** |            | in** |                                          |
|      |            |      | **Reverse primer**                       |
+------+------------+------+------------------------------------------+
| *    | #####      | **0  | **5'-ATGGCTATCGCTTGACCAA-3'**            |
| *2(2 | MWG699-H\* | 10** |                                          |
| H)** |            |      | **5'-GTGATGTTATTGGTGACTTGAACTC-3'**      |
+------+------------+------+------------------------------------------+
| *    | **MW       | **0  | ### 5'-ATGGCTATCGTTTGACCAG-3'            |
| *2(2 | G699-B\*** | 10** |                                          |
| H)** |            |      | **5'-GTGATGTTATTGGTGACTTGAACTC-3'**      |
+------+------------+------+------------------------------------------+
| *    | **         | **0  | **5'-TCTAGGCTTGCATTTGTCTACAAAG-3'**      |
| *2(2 | ABG058-H** | 01** |                                          |
| H)** |            |      | **5'-ATGCTGCTTCGCTGTCTACAATAAC-3'**      |
+------+------------+------+------------------------------------------+
| *    | **         | **0  | ### 5'-CAATAATCTCTCTTGCCATCATGCC-3'      |
| *2(2 | ABG058-B** | 01** |                                          |
| H)** |            |      | **5'-ATGCTGCTTCGCTGTCTACAATAAC-3'**      |
+------+------------+------+------------------------------------------+
| *    | **AB       | **0  | ### 5'-AACCAAGGCTACCAAGGTAATCCTG-3'      |
| *7(5 | C717-H\*** | 09** |                                          |
| H)** |            |      | **5'-CTCGTACTAACTTCCTACATGGCAA-3'**      |
+------+------------+------+------------------------------------------+
| *    | **AB       | **0  | ### 5'-AACCAAGGCTACCAAGGTAATCCTG-3'      |
| *7(5 | C717-B\*** | 09** |                                          |
| H)** |            |      | **5'-CTCGTACTAACTTCCTACATGGCAC-3'**      |
+------+------------+------+------------------------------------------+

\*Note: These primers require stringent conditions when running PCR

#### References

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