Funding for RosBREED: Combining disease resistance with horticultural quality in new rosaceous cultivars is provided by the Specialty Crop Research Initiative Competitive Grant 2014-51181-22378 of the USDA’s National Institute of Food and Agriculture.

Development of the Simple Sequence Repeat Marker PruG4RS for the Differentiation of Cherry Rootstocks

Kristen Andersen, Audrey Sebolt, Travis Stegmeir, Amy Iezzoni Michigan State University

Summary The use of clonally propagated Prunus sp. rootstocks in sweet cherry production is increasing as these rootstocks provide reduced tree size and precocity. Numerous clonally propagated cherry rootstocks are in

commercial production, such as the GiSelA® series, or under test, such as the Michigan State University (MSU) series (Fig. 1). Many of these dwarfing cherry rootstocks are difficult to differentiate based on morphology

alone, therefore DNA markers that differentiate rootstocks are an important tool to verify identity and prevent mix-ups among these rootstocks during the vegetative propagation stage. The simple sequence repeat (SSR)

marker PceGA59 was previously determined to uniquely distinguish the commercially available GiSelA® rootstocks (Struss et al. 2002)1 (Table 1, Fig. 2).

A targeted approach was used to develop a second SSR that was capable of providing differentiation of the MSU rootstock selections. The approach used was based on the ability to obtain genome-wide SNP (Single

Nucleotide Polymorphism) data using the Illumina Infinium® cherry SNP array (Peace et al. 2012)2. An analysis of genome-wide SNP data for the rootstocks resulted in the identification of a genomic region on linkage group

4 that was likely to differ among the MSU rootstocks (Fig. 4). Using the peach genome sequence, two SSR markers were designed to target this region (Fig. 5). One of these SSR markers, termed PruG4RS, successfully

differentiated the MSU rootstocks (Table 2, Fig. 3). The development of PruG4RS and its combined use with PceGA59 has successfully circumvented the limitations of each individual marker and proven effective for use as

a “quality control” DNA diagnostic tool for the commercial GiSelA® rootstocks as well as the MSU breeding program rootstock selections. The efficient generation of this informative SSR marker illustrates the successful use

of genome-wide SNP data to predict genomic regions most likely to differentiate specific plant materials.

SSR marker Primer Sequence (5’ - 3’)

PceGA59_F TGAACCCCTCTACAAATTTTCC

PceGA59_R GACTGTAGAACCCAAAAGAACG

PruG4RS_F TCAGAAAAGAAATTGCAACGGG

PruG4RS_R CTTAGTGGTCTAGTCTGCATGC

Figure 2. Visualization of the SSR marker PceGA59 on silver-stained polyacrylamide gels.

Figure 3. Visualization of the SSR marker PruG4RS on a silver-stained polyacrylamide gel.

1Struss D, Boritzki M, Karle R, Iezzoni AF. 2002. Microsatellite markers differentiate eight Giessen cherry rootstocks. HortScience 37(1):191-193. 2Peace C, Bassil N, Main D, Ficklin S, Rosyara UR, Stegmeir T, Sebolt A, Gilmore B, Lawley C, Mockler TC, Bryant DW, Iezzoni A. 2012. Development and evaluation of a genome-wide 6K SNP array for diploid sweet cherry and tetraploid sour cherry. PLoS ONE 7(12): e48305. 3Verde I, Abbott AG, Scalabrin S, Jung S, Shu S, Marroni F, Zhebentyayeva T, Dettori MT, Grimwood J, Cattonaro F, Zuccolo A, Rossini L, Jenkins J, Vendramin E, Meisel LA, Decroocq V, Sosinski B, Prochnik S, Mitros T, Policriti A, Cipriani G, Dondini L, Ficklin S, Goodstein

DM, Xuan P, Del Fabbro C, Aramini V, Copetti D, Gonzalez S, Horner DS, Falchi R, Lucas S, Mica E, Maldonado J, Lazzari B, Bielenberg D, Pirona R, Miculan M, Barakat A, Testolin R, Stella A, Tartarini S, Tonutti P, Aru´s P, Orellana A,Wells C, Main D, Vizzotto G, Silva H,

Salamini F, Schmutz J, Morgante M, Rokhsar DS (2013) The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nat Genet 45:487–494. 4Edge-Garza D, Rowland T, Haendiges S, Peace C. 2014. A high-throughput and cost-efficient DNA extraction protocol for the tree fruit crops apple, sweet cherry, and peach relying on silica beads during tissue sampling. Molecular Breeding 34:2225-2228.

Implementing PruG4RS in Applied DNA Diagnostics

The availability of genome-wide genetic data enables the development of diagnostic markers, like

PruG4RS, which significantly increases the efficiency of rootstock production as well as providing growers

with confidence that their plant materials are genetically verified. The development of custom SSR markers

through the analysis of genome-wide data provides a practical approach to meeting specified needs within a

breeding program or commercial production, be it DNA diagnostics or other characteristics of importance.

Table 2. SSR markers were developed as follows: primer pairs were designed using Primer3web

(bioinfo.ut.ee/primer3/) with product size limited to 150-250 bp, and primers selected on either side of the

repeat region with a GC clamp. The primers were then compared to the peach genome v1.0 scaffolds using

NCBI BLAST. Primer sequences which co-located to multiple scaffolds with at least 75% sequence similarity

were no longer considered. Those primer sequences determined to be unique were selected and tested for

their adequacy in clearly distinguishing the rootstock selections of interest. The primer sequence that was

determined to be the most informative and of the highest quality in uniquely identifying the rootstock selections

was selected and named PruG4RS. GC clamps were added to the primer pair sequences of PceGA59.

The forward and reverse primer sequences of the SSR markers PceGA59 and PruG4RS are listed in the

table below. In conducting DNA diagnostic tests of the rootstocks of interest DNA is extracted from fresh,

young leaf tissue using the silica bead method developed by Edge-Garza et al. (2014)4. A touchdown PCR is

used for both primers with the conditions as follows: 94 °C for 5 min followed by 9 cycles of 94 °C for 30 s, 60

°C for 45 s (-1 °C per cycle), 72 °C for 1 min and then 24 cycles of 94°C for 30 s, 55 °C for 45 s, 72 °C for 1

min with an elongation step of 72 °C for 5 min. The PCR fragments are separated in a 6% polyacrylamide gel

and visualized with silver staining.

Primers Gi 3 Gi 5 Gi 6 Gi 12 CASS CLARE CLINTON CRAWFORD LAKE

PceGA59 189 194 226 226 226 226 194 194 226

(Struss, 2002)1 186 186 189 186 194 194 189 189 194

182 182 182 182 186 - 186 182 189

- - - - - - 182 - -

PruG4RS 200 200 200 200 182 198 200 200 196

192 192 192 190 172 182 196 192 190

- - - - - 172 192 172 172

- - - - - - 172 - -

Figure 1. Rooted liners of the five MSU cherry rootstocks CASS, CLARE, CLINTON, CRAWFORD, and LAKE.

Table 1. Fragment sizes (bp) of the amplification products for the SSR markers PceGA59 and PruG4RS

tested on the four GiSelA® (Gi) and five MSU rootstocks.

*The rootstock BENZIE has been included as a representation of the second generation of the rootstock LAKE.

Figure 4. Genome-wide SNP data for the MSU rootstock selections of interest was obtained using the

RosBREED Illumina Infinium® cherry SNP array (Peace et al., 2012)2. Analysis of this data identified a region

of linkage group 4 centered on the SNP ss490546237 that appeared to be exceptionally different among the

rootstocks. This region was chosen as the target for SSR marker development.

The GenomeStudio cluster plot revealed differences in the SNP marker ss490546237 for the MSU

rootstock selections. Genotypes are called for each sample (dot) by their signal intensity (Norm R) and allele

frequency (Norm Theta) relative to canonical cluster positions (dark shading) for the SNP marker (red = AA,

purple = AB, blue = BB. In the below cluster plot CASS, CLARE, CRAWFORD, LAKE, and Gi6 cluster together

in the homozygous class BB; Gi5 and Prunus canescens (the common ancestor of two of the five MSU

rootstock selections) cluster together in the homozygous class AB, and CLINTON is classified as a no-call.

Figure 5. A total of 100,000 base pairs (bp) of DNA sequence located up and down stream of the SNP marker

ss490546237 were obtained from the peach genome v1.0 (Verde, 2013)3, available at the Genome Database

for Rosaceae (www.rosaceae.org). This sequence was then searched for SSRs ranging from 2-5 bp repeat

length and having a minimum of 6 repeats. Multiple SSRs meeting these conditions were identified, so those

with the largest number of tandem repeats were selected.

The below GBrowse image of the peach genome sequence illustrates the position of the cherry SNP

marker ss490546237 and the adjacent sequence used to develop PruG4RS. This alignment is from the

Genome Database for Rosaceae (rosaceae.org).

Gi5

LA

KE

CA

SS

C

LA

RE

BE

NZ

IE*

CLIN

TO

N

CR

AW

FO

RD

Gi6

PceGA59

194

186

226

189

182

BE

NZ

IE*

PruG4RS

CA

SS

CL

AR

E

CL

INT

ON

CR

AW

FO

RD

LA

KE

Gi5

Gi6

198 200

192 196

182

172

CASS CLARE CLINTON CRAWFORD LAKE

Prunus canescens

Gi 5

Gi 6

CASS

CLINTON

CLARE, LAKE

CRAWFORD

AA BB AB

S-locus

190