Characterization of Est-SSR markers related to Colletotrichum fructicola infection in strawberry (Fragaria ×ananassa Duchase)

Abstract

Colletotrichum fructicola causes strawberry anthracnose, which results in considerable economic losses in quality and production in Asia. However, the molecular mechanism of strawberry infection with C. fructicola is still poorly understood. Here, we employed RNA-Seq technology to mine the strawberry transcriptome for EST-SSRs (Expressed Sequence Tag–Simple Sequence Repeats) following C. fructicola infection. Fortunately, we detected 3019 potential EST-SSRs from the transcriptomic data of cv. Jiuxiang. The seven commonly occurring repeat motifs were A/T (278), GAA/TTC (263), GAG/CTC (215), AAG/CTT(188), GGA/TCC (163), AGA/TCT (153) and AGG/CCT (153). We identified 31, 84 and 129 differentially expressed EST-SSR-containing sequences corresponding to 24, 72 and 96 Hpi, while 13 DE-EST-SSRs -containing sequences were differentially expressed and common for all the three time points. Furthermore, we identified allele polymorphisms for some DE-EST-SSRs among several resistant and susceptible strawberry varieties and wild species. The EST-SSRs in our research were related to the reported QTL resistance loci in strawberry which are differentially expressed after C. fructicola infection and have allele polymorphisms in different strawberry varieties and wild species. These provide a clue for utilisation of the EST-SSRs in marker-assisted breeding to improve anthracnose-resistant breeding in future.

Keywords:

• strawberry
• EST-SSR
• polymorphism
• resistance
• transcriptome

Introduction

The cultivated strawberry (Fragaria ×ananassa), also known as ‘the queen of fruits’, is very popular with consumers due to its flavor and nutritional value. Fragaria ×ananassa are generally octoploid with 2n = 8x = 56 chromosomes. Anthracnose caused by Colletotrichum spp. is one of the most devastating diseases for cultivated strawberry [1–3], and can cause major problems in the industry of strawberry cultivation where infections are thought to be spread primarily through latently infected plants [4, 5]. C. fructicola was formerly known as C. gloeosporioides, and is responsible for strawberry anthracnose in China, Korea and Japan [5–8].

In strawberry, some research has been devoted to elaborating the molecular mechanism related to anthracnose resistance. Two class II chitinase genes are cloned, and analyzed in the strawberry plant [9]. The genes that encode 03B3-thionins, peroxidases, chitinases and 03B2-1-3-glucanases are up-regulated during pathogen infection [10], as shown by microscopic investigations of Colletotrichum fragariae infection [11], and proteomic analyses of strawberry leaves infected with Colletotrichum fragariae [12]. The SA- and JA-dependent signaling pathways are activated in strawberry upon interactions with Colletotrichum acutatum [13]. Comparative transcriptomics revealed differential gene expression related to Colletotrichum gloeosporioides resistance in the octoploid strawberry ‘Yanli’ and ‘Benihoppe’ [14].

In our lab, some progress has also been made in strawberry anthracnose research. NB-LRR genes display ecotype-specific responses to C. gloeosporioide in woodland strawberry [15]. Salicylic acid is involved in the interactions between Colletotrichum gloeosporioides and two strawberry varieties [16]. FaNBS-RPW8 fusions are related to C. fructicola infection in strawberry (Fragaria ×ananassa) [17]. In the early strawberry/Colletotrichum fructicola interaction, a rapid quenching of the burst of host salicylic acid is common [18]. Novel fungal pathogenicity is revealed by transcriptome analysis of Colletotrichum fructicola and strawberry [19].

Simple Sequence Repeat (SSR) markers are abundant and related to transcribed sequences in plant genomes [20]. EST-SSRs (Expressed Sequence Tag–Simple Sequence Repeat) markers are also used to explain the genetic diversity, germplasm characterization and population genetics [21]. Traditional methods for SSR development are expensive and labor-intensive by the screening of genomic libraries and sequencing of positive clones. Now, the transcriptomic resources promote SSR markers development expedient. SSRs from GenBank sequences in the species of Fragaria and Rubus have been previously identified using two different data-mining methods [22]. Four hypervariable SSR markers are developed to identify 75 accessions of strawberry cultivars [23]; 96 strawberry cultivars are distinguished by amplifying SSR fragments from 18 SSR primer pairs [24].

However, the EST-SSR markers related to strawberry anthracnose are still largely unknown or poorly explored. In this study, we generated comparative EST-SSR markers at different time periods after inoculation in susceptible cv. Jiuxiang (Fragaria ×ananassa), uncovering new resistance-related genes, EST-SSR markers by comparative transcriptomics analyses. These data deepen our understanding of EST-SSR markers and molecular mechanisms involved in strawberry defense.

Materials and methods

Plant materials and treatments

All the strawberry cultivars used in this work were from Shanghai Academy of Agricultural Sciences (SAAS) resource nursery, and were grown in a multi-span greenhouse at the Zhuanghang field trial station of SAAS.

For RNA-Seq, only the cultivar of ‘Jiuxiang’ was used, as it is comparatively susceptible to C. fructicola. For genotyping, two strawberry cultivars (‘Benihoppe’ and ‘Sweet Charlie’) were used. ‘Benihoppe’ is susceptible to C. fructicola, whereas ‘Sweet Charlie’ is resistant to C. fructicola [14]. Two wild species were used, including F. vesca (‘Yellow wonder’ and ‘Hawaii4’), and F.nilgerrensis (‘Schlecht’).

For each independent C. fructicola inoculation experiment, healthy plants at the sixth compound stage were cultivated from a nursery in pots with sterilized substrate.

Three technical replicates from 15 independent individuals were used (five plants per replicate). Before C. fructicola inoculation, the pots were transferred to a growth chamber (Conviron, Adaptis A1000AR, Winnipeg, Canada) for 2 weeks. The conditions in the growth chamber were set as follows: 12-h-light/12-h-dark cycle, 125 μ mol m^{- 2} s⁻¹ photo flux density, 70% relative humidity (RH) and a constant temperature of 25 °C. After inoculation, the plants were cultivated under continuous darkness. The compound leaves of ‘Jiuxiang’ at the sixth compound leaves stage were collected at 0, 24, 72 and 96 Hpi for RNA-Seq.

Cultivation of C. fructicola

The C. fructicola isolate (cgmcc3.17371) was maintained at the Shanghai Academy of Agricultural Sciences. The plant growth conditions and inoculation methods have been previously described [17].

RNA-Seq

The cultivar Jiuxiang was infected with C. fructicola for RNA-Seq. C. fructicola was inoculated on leaves at the sixth compound leaves stage. We prepared the samples during the process of infection with C. fructicola at 24, 72 and 96 Hpi. RNA-Seq, construction of Illumina library, sequencing and raw reads have been previously described in our group [19].

Mapping reads to the reference genome

Clean reads were preprocessed by removing the adaptor sequences and discarding the empty and low-quality sequences. The reads were then mapped to Fragaria_vesca_ v1.1.a2 [25] using Bowtie 2 (version2.1.0) with the default parameters. Then we analyzed the key DEGs in enriched pathways and EST-SSR in fragaria_vesca_v4.0.a2 [26] with the locus IDs of two nomenclatures between v2.0.a1 and v4.0.a2 (Supplemental Table S1).

Identification Est-SSR markers and primers design

We employed MIcroSAtellite (MISA, v-1.0, http://pgrc.ipk-gatersleben.de/misa/misa.html) for microsatellite mining[27]. In this study, the EST-SSRs were considered to contain motifs of one to six nucleotides in size and a minimum of 5 contiguous repeat units. The parameter was set to the default values (1-10, 2-6, 3-5, 4-5, 5-5 and 6-5). Therefore, the core unit was a single base, repeated 10 times or more; the core unit was two bases, repeated more than 6 times, and so on. The MISA results, Primer3 v2.3.5 (http://www-genome.wi.mit.edu/genome_software/other/primer3.html) was used to design the primer pairs with the default setting, and the size of PCR product ranged from 100 to 280 bp. Six varieties were selected to validate the polymorphism of 160 random SSR markers.

Identification of DE-Est-SSRs

The gene expression levels were calculated using the RPKM method (fragments per kb per million reads). Samtools were used to obtain the RPKM values. The RPKM values were ≥ 0.3. Baggerley’s test and the false discovery rate (FDR) had a significance level of ≤ 0.001, and the p-value was ≤ 0.01. The ratios of RPKM at 24, 72 and 96 Hpi to 0 Hpi ≥ 2 or ≤ 0.5 (fold change >1) were set as the threshold to determine the differentially expressed genes.

DNA extraction, Est-SSR amplification and genotyping

The genotyping of DE-EST-SSRs was done using genomic DNA extracted from strawberry varieties and wild species using the CTAB method. The DNA quality was confirmed using 1% agarose gels, and the quantity was confirmed using a UV–visible Spectrophotometer and a nano spectrophotometer (Implen, Westlake Village, CA, USA), respectively. The working concentration of DNA was adjusted to 50 ng μL⁻¹.

The DE-EST-SSR primers were synthesized by Shanghai Sangon Biological Engineering Technology (Shanghai, China).

Polymerase chain reactions (PCRs) were performed in 20 μL reaction volumes containing 25 ng of template DNA, 10 μL of PrimeSTAR Max DNA Polymerase (TaKaRa, Japan), 1 μL of 10 μmol/L of each primer and 7 μL sterile distilled water. The following PCR program included an initial step at 98 °C for 3 min followed by 32 cycles of 98 °C for 10 s, the appropriate annealing temperature (temperature depended on primers) for 5 s at 55-60 °C and 5 s at 72 °C, and a final extension for 5 min at 72 °C in an Eppendorf Mastercycler PCR instrument. Finally, the products were examined in 1% agarose gels. Amplified products, which showed a band of the expected size, were sequenced for genotyping by Shanghai Sangon Biological Engineering Technology (Shanghai, China).

Results and discussion

Identification and characterization of the Est-SSR markers

In order to develop the strawberry anthracnose EST-SSR markers, we screened all 31,029 mapped genes in this transcriptome to mine EST-derived SSR motifs. In total, 3,019 putative EST-SSRs were detected in 2,497 genes, including 204 genes with one or more than one SSR (Table 1). The major motif types of the identified EST-SSRs were trinucleotides (2298; 76.12%), dinucleotides (363; 12.24%) and mononucleotides (292; 9.68%). We also detected some other motif types (2.2%), such as hexanucleotides, tetranucleotides and pentanucleotides, in this transcriptome (Figure 1A). In general, the number of EST-SSRs detected in each size category (mono-, di-, tri-, hexa-) decreased with increasing repeat number (Figure 1B-E). The top five abundant motifs of the trinucleotide repeats were (GAA/TTC)5 (127; 4.2%), (GAG/CTC)5 (118; 3.9%), (AAG/CTT)5 (117; 3.8%), (GGA/TCC)5 (100; 3.3%) and (AGA/TCT)5 (96; 3.2%). For the dinucleotide repeats, (AG/CT)6 (65; 2.2%) was the most common motif, followed by (GA/TC)6 (54; 1.8%). For the mononucleotide repeats, the most abundant motif was (A/T) > 10 (167; 5.50%), followed by (A/T)10 (111; 3.7%) (Table 2).

Figure 1. The distribution of repeat unit and repeat number for EST-SSRs in cv. Jiuxiang transcriptome. (A) Distribution of repeat units for mono-, di-, tri-, tetra-, penta- and hexa-nucleotide. (B–E) Distribution of the repeat number for mono- (B), di- (C), tri- (D), and hexa-nucleotides (E).

Table 1. Statistic of EST-SSR detected in ‘Jiuxiang’.

Result of EST-SSR searches
Total number of sequences examined	31029
Total number of identified EST-SSRs	3019
Number of EST-SSR-containing sequences	2497
Number of sequences containing more than one EST-SSR	204

Table 2. Frequency of mono-, di- and trinucletide EST-SSR repeat motifs in cv. Jiuxiang.

Repeat motif	Repeat numbers							Total
Mono-nucleotides	5	6	7	8	9	10	>10	292
A/T						111	167	278
C/G						3	11	14
Di-nucleotides								363
AG/CT		65	30	19	10	4	17	145
GA/TC		54	30	8	2	5	28	127
AT/AT		6	8	5	4	5	11	39
TA/TA		11	3	9	3		3	29
TG/CA		4	4		2	1		11
AC/GT		2	2		1		1	6
GT/AC		1				1		6
Tri-nucleotides								2298
GAA/TTC	127	61	26	21	10	3	15	263
GAG/CTC	118	60	20	5	8		4	215
AAG/CTT	117	38	15	8	3	3	4	188
GGA/TCC	100	33	20	2	5	1	2	163
AGA/TCT	96	25	9	9	4	4	6	153
AGG/CCT	92	32	18	5	4		2	153
TGA/TCA	76	30	11	8	2	1	3	131
ATC/GAT	62	21	10	1	3		1	98
CAC/GTG	60	22	8	7				97
ACC/GGT	59	16	12	5				92
CAG/CTG	56	20	8	5	1		1	91
CCA/TGG	43	19	8	6	1	2	1	80
GCT/AGC	44	14	7	2				67
CAA/TTG	37	18	7	2			2	66
CCG/CGG	48	10		3	3			64
CGG/CCG	48	10		3	3			64
ATG/CAT	36	15	7	1	1	1	1	62
CAT/ATG	36	15	7	1	1	1	1	62
GCA/TGC	37	13	3	1				54
AAC/GTT	20	9	9	2	3			43
CGC/GCG	32	5						37
GAC/GTC	30	3	1					34
CGA/TCG	14	9	3	1				21
Total	1393	647	293	147	83	156	281

Within the mono-, di- and trinucleotides, five tandem repeats (1393; 46.14%) were the most frequently observed, followed by those with six tandem repeats (647; 21.43%) and seven tandem repeats (293; 9.7%) (Figure 1, Table 2). Approximately 79% (2,388) of the identified EST-SSRs were 20 bp in length, whereas only 3% (92) of the EST-SSRs were longer than 20 bp. A total of 2,510 SSR-containing sequences were adequate for primer design, whereas the remaining 256 sequences did not qualify for primer design because the sequences flanking the SSRs were too short or inappropriate for primer design.

DE- Est-SSRs after C.fructicola inoculation

In our study, we reported the differentially expressed EST-SSRs (DE-EST-SSRs) after C.fructicola inoculation in strawberry. Interestingly, a total of 31, 84 and 129 differentially expressed EST-SSR-containing sequences were detected in 24 Hpi, 72 Hpi and 96 Hpi compared with 0 Hpi, respectively (Figure 2). Thirteen differentially expressed EST-SSR-containing sequences (named EST-SSR01- EST-SSR13) showed increasing or decreasing expression patterns in all three libraries simultaneously (Figure 2B) in this transcriptome. Furthermore, the expression patterns of six EST-SSR-containing sequences (EST-SSR01, EST-SSR03, EST-SSR06, EST-SSR10, EST-SSR11 and EST-SSR13) were downregulated in susceptible cv. Jiuxiang at 24 Hpi and 72 Hpi, and the reduced expression level was maintained at 96 Hpi. Three EST-SSR-containing sequences (EST-SSR02, EST-SSR09 and EST-SSR12) were downregulated in susceptible cv. Jiuxiang in the three libraries continuously. Two EST-SSR-containing sequences (EST-SSR07 and EST-SSR08) were slightly upregulated at 24 Hpi and then were sharply downregulated at 72 Hpi and 96 Hpi. Two other EST-SSR-containing sequences (EST-SSR04 and EST-SSR05) were stably upregulated in the three libraries simultaneously (Figure 2C). Of the 13 differentially expressed EST-SSR-containing sequences, there were 4 transcription factors. Three differentially expressed EST-SSR transcription factors were MYB family proteins, and one was WRKY DNA-binding protein (Figure 2, Table 3). Previous reports have shown that MYB and WRKY transcription factors can participate in the resistance response against pathogen infection by regulating defense genes [28, 29]. TaWRKY70 was identified within the QTL-2DL region, along with three resistance genes that biosynthesize RRI metabolites to resist Fusarium graminearum infection in wheat[30]. Here, we deduced that the DE-EST-SSR6, 8, 13 and 11, which are encoded by MYB and WRKY DNA-binding protein, might play an important role in strawberry defense against C. fructicola infection. EST-SSR1 belongs to facilitator superfamily, which plays an important role in multidrug resistance in fungi [31]. The EST-SSR04 belonging to GRAS family might play important roles in regulating root and shoot development, which can improve strawberry resistance to abiotic stresses [32]. Ethylene responsive factors (ERFs) are important in regulating plant pathogen resistance, abiotic stress tolerance and plant development [33]. The EST-SSR09 of ERF members have greatly enlarged the ERF protein family and revealed more important roles of ERFs in strawberry resistance. Ethylene responsive and MYB transcription factors within the confidence interval for five of the meta-QTLs are recommended as potential targets for partial resistance to white mold in common bean [34]. In our data, the DE-EST-SSR09 (ERF member) and DE-EST-SSR6, 8 and 13 (MYB family) might provide new clues for the mining of QTL loci of resistance to strawberry anthracnose. Therefore, our research provided some new DE-EST-SSRs resources for strawberry anthracnose resistance.

Figure 2. Expression patterns of DE-EST-SSRs after C. fructicola inoculation. (A) Heatmap of differentially expressed EST-SSRs. (B) Venn diagram of differentially expressed EST-SSRs. (C) Expression trends of 13 differentially expressed EST-SSRs at 24, 72, and 96 Hpi.

Table 3. Information about 13 differentially expressed EST-SSRs in all three libraries (24, 72 and 96 Hpi).

	Product size (bp)	157	208	236	273	260	125	102	172	275	234	192	270	275
	Reverse primer (5′-3′)	GAGCTCGAAT TGGACTGGAG	CATGGAGGAC CAGGAGGAG	AGCTCTTGCAT GGCTTCATT	GAATCCCCAC CGCCTATTAT	CTCCAGAACT GGCAAACACA	ACTGTTTTGC TTCCCCACAC	CAAGCCTAATT GGGGTGAGA	CATCGAAGTTA CCCTCCGAA	TCGAAGAAGTC GTCAGGGAT	ACATGGTGGTT GTTGTGGTG	CGGTCCTGGT CAAAGTTGTT	GCCTCAACTTCA GCGTAAGG	GAGTCAGAAGA GTCGACGGC
	Forwaed primer (5′-3′)	AGGAGGAGGA GGACAAGGAG	ATGATCAGTGAC CACCATGC	TTCGTGTTCG TCTGATTTGG	AGGCTTTTCT CTCTCCGCTC	GAATGGTAGCA AACCTGGGA	TGAGCCCCAAG CCTAAAGTA	AATGGCCCTTC ATTCACAAG	GAGAAAGCGAC AATGGCTTC	ATGGAACCTG CTCTCGACAT	GAGGCTGCAT CTCATTAGCC	CCACTTCTC AGCCGCACA	CTCAGCGGCAA ACTACACAA	GAAGCGGAAGT ACTCGTCCA
	SSR motif	(AGG)9	(TCC)5/6/7	(CTT)5	(ATC)6	(GCA)5	(GCGGCT)5	(AG)6	(GAA)6	(ACC)5(TCC)5	(CCA)7	(TCT)5	(CCA)5	(GCC)5
	Primer no.	EST-SSR01	EST-SSR02	EST-SSR03	EST-SSR04	EST-SSR05	EST-SSR06	EST-SSR07	EST-SSR08	EST-SSR09	EST-SSR10	EST-SSR11	EST-SSR12	EST-SSR13
	Genes function	Facilitator superfamily		Oxidative stress 3	GRAS family TF	Cysteine synthase D1	Myb 1	Fatty alcohol oxidase 3	Myb 6	Ethylene responsive element	DUF3049	WRKY t 33		Myb 70
	Genes number	mrna30825.1-v1.0-hybrid	FvH4_1g01772	FvH4_1g06410	FvH4_1g13400	FvH4_6g03790	FvH4_2g33810	FvH4_4g04030	FvH4_5g19400	FvH4_5g19800	FvH4_6g05230	FvH4_6g10510	FvH4_6g15790	FvH4_2g05210

Note: The genes function represented in boldface indicates that they are transcription factors.

The allele polymorphisms of DE-Est-SSRs in strawberry varieties and wild species

We detected DE-EST-SSRs polymorphisms by genomic PCRs and genome sequencing. Among the 13 DE-EST-SSRs, six DE-EST-SSRs showed polymorphisms in different cultivars and wild species. In detail, we analyzed the DE-EST-SSRs polymorphisms in the susceptible cultivar ‘Benihoppe’, resistant cultivar ‘Sweet Charlie’ compared with wild species varieties ‘Yellow Wonder’, ‘Hawaii4’ and ‘Schlecht’. Our previous experiment proved that F.vesca is more susceptible than F. nilgerrensis to C. fructicola (unpublished data). Fortunately, our results showed that 6 bases of the EST-SSR04 allele were deleted in resistant ‘Sweet Charlie’ compared with susceptible ‘Benihoppe’. Interestingly, this phenomenon of 6 deleted bases in the EST-SSR04 allele was also consistent in relatively resistant ‘Schlecht’ variety and relatively susceptible ‘Yellow Wonder’ and ‘Hawaii4’ varieties (Figure 3A). There were nucleotide substitution variations within the EST-SSR13 allele between ‘Sweet Charlie’ and ‘Benihoppe’ (Figure 3B). However, regardless of the EST-SSR04 allele or EST-SSR 13 allele, there was very high homology within wild species, compared with ‘Benihoppe’ and ‘Sweet Charlie’ cultivars (Figure 3). Meanwhile, we also found that other DE-EST-SSRs alleles were also polymorphic in different varieties. For EST-SSR06, there was no ‘GCGGCT’ SSR locus in ‘Benihoppe’, and mutated (GCGGCT)2 SSR allele in ‘Sweet Charlie’ cultivar (Supplemental Figure S1A). The genotype of the EST-SSR07 locus was (AG)6 in ‘Hawaii4’ and (AG)7 in other varieties (Supplemental Figure S1B). (GAA)6 SSR locus in EST-SSR08 varied in ‘Schlecht’ and ‘Benihoppe’ (Supplemental Figure S1C). For EST-SSR09, there was no (ACC)5(TCC)5 SSR allele in ‘Sweet Charlie’, and mutated (ACC)4(TCC)5 SSR allele in ‘Benihoppe’ cultivar (Supplemental Figure S1D). Thus, the DE-EST-SSRs showed polymorphism in SSR alleles in different varieties and wild species.

Figure 3. Genotyping of EST-SSR04 and EST-SSR13 in different cultivars and species by PCR and PCR products sequencing. The same EST-SSR with differential SSR genotypes but the same amplicon length. The nucleic acid bases of SSR loci are shown in different colors and the nucleotide substitution variations are shown in black.

The DE-Est-SSRs related to strawberry anthracnose resistance QTL loci

One of anthracnose resistance QTL loci contains SCAR markers, which are linked to Rca2 anthracnose resistance gene [35]. FaRCg1 is a quantitative trait locus conferring resistance to Colletotrichum crown rot caused by Colletotrichum gloeosporioides in octoploid strawberry [36]. FaRCa1 is a major subgenome-specific locus conferring resistance to Colletotrichum acutatum in strawberry, and is effective against European isolates from pathogenicity group 2 [37]. We found that the two published strawberry anthracnose resistance QTLs of FaRCg1 and FaRCa1 are on LG 6B from 66-72 cM to 55-56 cM on LG 6B (Supplemental Table S2). Furthermore, we found that published strawberry resistance QTLs are largely located in chromosome 1, 2, 4, 5 and 6 in strawberry (Fragaria ×ananassa) for strawberry anthracnose, powdery mildew and Verticillium dahliae resistance [38, 39] (Supplemental Table S2). In our data, 4 of the 13 DE-EST-SSRs were located in chromosome 6, which might be one of the main adjacent QTLs for strawberry anthracnose resistance related to FaRCa1 and FaRCg1. Also, the DE-EST-SSR6 correspond to strawberry powdery mildew QTL resistance locus FaRPa2A, with a contiguous distance in chromosome 2 from 25245976-25246956 to 20692727 20699757 in Fragaria ×ananassa genome (Supplemental Table S2). In summary, the candidate DE-EST-SSRs, which were located in the vicinity of strawberry resistance QTLs, might be related to resistance in more than one phenotyping event.

Furthermore, we have analyzed the transcriptome of C. fructicola and strawberry under C. fructicola inoculation [19]. A recent report on strawberry anthracnose pathogen Colletotrichum fructicola showed that the assembly of C. fructicola genome contains 18143 predicted genes with 58.0 Mb [40]. In this research, we have focused on the DE-EST-SSRs after C.fructicola inoculation. These will provide an important cue for understanding the mechanism of fungal pathogenicity and the system of strawberry multilayered defense.

Conclusions

Marker-assisted breeding has not been widely utilized in cultivated strawberry (heterologous octoploid) breeding. In this study, we investigated the EST-SSR in the transcriptome under C. fructicola inoculation using the Illumina sequencing technology. A total of 3019 specific EST-SSRs were identified in cv. Jiuxiang. Also, there were 31, 84 and 129 differentially expressed EST-SSR genes at 24, 72 and 96 Hpi respectively, and 13 candidate DE-EST-SSRs were presented at all the three studied time points. The allele polymorphisms of candidate DE-EST-SSRs were analyzed among the typical resistant and susceptible strawberry varieties and wild species. Four of the 13 DE-EST-SSRs were simultaneously positioned on chromosome 6, and were located in the vicinity of FaRCg1, FaRCa1 and FaRPa2A. The EST-SSRs in our research might be related to resistance loci in strawberry, and provide clues for the development of resistance-related SSR markers in cultivated strawberry.

Disclosure statement

No potential conflcit of interest was reported by the author(s).

Additional information

Funding

This work was supported by funds from Shanghai Municipal Agricultural Commission (Hu Nong Ke Zhong Zi-2017-2-1), National Natural Science Foundation of China (31601731), and Science and Technology Innovation Support Project of Shanghai Academy of Agricultural Sciences (202002).