Induced and primed defence responses of Fragaria vesca to Botrytis cinerea infection

Strawberry is a high-value crop that suffers huge losses from diseases such as grey mould caused by the necrotrophic fungal pathogen Botrytis cinerea. Pesticides are heavily used to protect the strawberry crop, which raises environmental and human health concerns and promotes the evolution of pesticide resistant strains. Upregulating or priming the plants’ defences may be a more environmentally sustainable way of increasing disease resistance. Using Fragaria vesca as a model for the commercially grown octaploid strawberry Fragaria × ananassa, we investigated the transcriptional reprogramming of strawberry upon B. cinerea infection and the effectiveness of four priming chemicals in protecting strawberry against grey mould. First, we found that the transcriptional reprogramming of strawberry upon B. cinerea infection overlapped substantially with the transcriptome responses induced by Phytophthora cactorum (Toljamo et al., 2016), including the genes involved in jasmonic acid (JA), salicylic acid (SA), ethylene (ET) and terpenoid pathways. Furthermore, we investigated the effectiveness of previously identified priming chemicals in protecting strawberry against B. cinerea. The level of upregulated or primed resistance depended on the priming chemical itself (β-aminobutyric acid (BABA), methyl jasmonate (MeJ), (R)-β-homoserine (RBH), prohexadione-calcium (ProCa)) and the application method used (foliar spray, soil drench, seed treatment). Overall, RBH effectively primed strawberry defences against B. cinerea, whereas BABA and ProCa were not effective and MeJ showed mixed effects. Our results not only identify ways to effectively upregulate or prime strawberry defences against B. cinerea, but also provide novel insights about strawberry defences that may be applied in future crop protection schemes.


Summary
Strawberry is a high-value crop that suffers huge losses from diseases such as grey mould caused by the necrotrophic fungal pathogen Botrytis cinerea. Pesticides are heavily used to protect the strawberry crop, which raises environmental and human health concerns and promotes the evolution of pesticide resistant strains. Upregulating or priming the plants' defences may be a more environmentally sustainable way of increasing disease resistance.
Using Fragaria vesca as a model for the commercially grown octaploid strawberry Fragaria × ananassa, we investigated the transcriptional reprogramming of strawberry upon B. cinerea infection and the effectiveness of four priming chemicals in protecting strawberry against grey mould. First, we found that the transcriptional reprogramming of strawberry upon B.
cinerea infection overlapped substantially with the transcriptome responses induced by Phytophthora cactorum (Toljamo et al., 2016), including the genes involved in jasmonic acid (JA), salicylic acid (SA), ethylene (ET) and terpenoid pathways. Furthermore, we investigated the effectiveness of previously identified priming chemicals in protecting strawberry against B. cinerea. The level of upregulated or primed resistance depended on the priming chemical itself (β-aminobutyric acid (BABA), methyl jasmonate (MeJ), (R)-β-homoserine (RBH), prohexadione-calcium (ProCa)) and the application method used (foliar spray, soil drench, seed treatment). Overall, RBH effectively primed strawberry defences against B. cinerea, whereas BABA and ProCa were not effective and MeJ showed mixed effects. Our results not only identify ways to effectively upregulate or prime strawberry defences against B. cinerea, but also provide novel insights about strawberry defences that may be applied in future crop protection schemes. treatment or cold storage. Cold treatment and pesticide application can affect plant physiology and may possibly have a priming-like effect. For example, cold treatment is known to induce epigenetic modifications and molecular memories that may be inheritable across generations (Yang, Howard and Dean, 2014;Berry and Dean, 2015;Dubin et al., 2015;Tao et al., 2017;Yang et al., 2017). The use of pesticides also defy the purpose of investigating induced plant defences, as defence priming agents should serve as an alternative to pesticide treatment.
Understanding the transcriptional reprogramming of F. vesca upon pathogen infection will identify key genes and pathways that help in devising appropriate agronomic solutions. In addition, exploiting the knowledge of pathogen induced transcriptional responses to choose appropriate defence priming chemicals will be a valuable method for crop protection schemes.

Botrytis cinerea induces major transcriptional reprogramming in Fragaria vesca
To provide baseline information about transcripts involved in F. vesca defenses we studied the transcriptional reprogramming in F. vesca infected by B. cinerea using RNA-seq analysis of leaves from five infected and five mock-treated plants harvested 24 hours after infection.
An average of 26,176,008 reads were generated per sample (median: 25,291,981) and the data were submitted to GenBank as XXX. Out of 28,588 mapped transcripts, 7003 were significantly differentially expressed in infected plants (p-value < 0.05) (Table S1), representing 24% of the F. vesca transcriptome. Among these differentially expressed genes (DEGs), 3314 were downregulated and 3689 were upregulated after infection, indicating a major transcriptional response upon B. cinerea infection. As expected, many upregulated DEGs were involved in defence responsive hormone biosynthetic pathways. Examples include genes encoding phenylalanine ammonia-lyase (PAL) involved in the SA pathway, 12oxophytodienoate reductase (12OR) involved in the JA pathway, and 1-aminocyclopropane-1carboxylate oxidase (ACCox) involved in ethylene (ET) biosynthesis (Table S1). In addition, many genes involved in the biosynthesis of defence metabolites such as terpenoids, flavonoids and major allergens were upregulated, suggesting that several defence systems were activated upon pathogen attack (Table S1). Interestingly, we also observed upregulation of the genes encoding gibberellin precursor ent-copalyl diphosphate synthase (ECDS) and the DELLA protein RGL1 that acts as a repressor of gibberellin signalling (Table S2). To confirm the differential gene expression observed in the RNA-seq data, qPCR analysis of selected genes was performed (Table 1). The qPCR gene expression results showed similar regulation as the RNA-seq data, thereby confirming the RNA-seq results ( Figure 1).

Botrytis cinerea and Phytophthora cactorum induce very similar transcriptomic responses in Fragaria vesca
To test if the transcriptomic response of F. vesca varies with the pathogen's lifestyle and the plant organ that is infected we compared our RNA-seq data with previously published data from F. vesca roots infected with the oomycete hemibiotroph Phytophthora cactorum (Toljamo et al., 2016). In contrast, B. cinerea infects above-ground plant parts and is generally known to have a necrotrophic lifestyle, possibly with a short initial biotrophic phase (van Kan, Shaw and Grant-Downton, 2014;Veloso and Kan, 2018). Our comparison of DEGs in leaf and root tissues infected by B. cinerea (Table S1) or P. cactorum (Toljamo et al., 2016) respectively revealed great similarities in gene expression patterns. This prompted us to make a more detailed transcriptome comparison of strawberry's responses to B. cinerea and P. cactorum infecting shoots and roots, respectively (Table S3). The overall similarity was visualised in a scatter plot comparing the transcriptional responses to the two pathogens.

Effects of defence-priming agents on Fragaria vesca resistance to Botrytis cinerea
The RNA-seq data showed upregulation of several defence related genes upon B. cinerea infection, including genes involved in SA, JA and ET signalling and synthesis of secondary metabolites (Table S2). As the priming agents BABA, MeJ and RBH normally function by inducing these defence responsive pathways, we investigated if BABA, MeJ and RBH also activated such defence pathways in F. vesca and increased resistance against B. cinerea. We treated plants with potential defence priming agents two days prior to fungal inoculation. Soildrench treatment with RBH induced resistance in F. vesca, as the relative amounts of B. cinerea was less than half in the leaves of RBH treated plants compared to the control ( Figure   3). However, BABA did not increase resistance, but on the contrary seemed to induce susceptibility or decrease resistance, as relative B. cinerea amounts were about twice as high in leaves of BABA treated plants as compared to the control (Figure 3). In Arabidopsis, BABA treatment has been found to confer protection against both B. cinerea (Zimmerli, Métraux and Mauch-Mani, 2001) and abiotic stresses like salt and drought (Jakab et al., 2005). To verify our observation with BABA-soil drench, we tested different concentrations of BABA and observed that plants had increasing amounts of B. cinerea infection with increasing BABA concentrations ( Figure S3). BABA treatment combined with drought stress reduced the relative water content (RWC) of F. vesca in a BABA-concentration dependent manner ( Figure S4), indicating that BABA increased susceptibility to both types of stress, biotic and abiotic.
Spray treatment of BABA, MeJ and RBH all decreased B. cinerea infection but only MeJ treatment was statistically significant, indicating that these chemicals primed or induced resistance in F. vesca (Figure 3b). Extensive growth of B. cinerea on potato dextrose agar plates with high concentrations of these chemicals suggests that the results in planta were not due to direct effects of the chemicals on B. cinerea growth and survival ( Figure S5).
To test the effect of putative defence priming agents under the influence of a plant protection chemical, F. vesca plants were treated with an insecticide Imidacloprid (Confidor) two weeks before treatment with priming agent. As shown in Figure 3c

Gibberellic acid and ProCa influence resistance against Botrytis cinerea
The RNA-seq data showed that the gibberellin precursor encoding gene ECDS was

Seed priming for induced resistance against Botrytis cinerea
We then studied the effect of seed priming on germination, vegetative growth and resistance against B. cinerea. Seeds of F. vesca treated overnight with BABA, MeJ and RBH did not show differences in germination rates as compared to control treatment ( Figure 6a). While treatment with BABA and MeJ had no significant effect on early growth of seedlings compared with control, RBH-treated seeds showed higher petiole lengths, indicating a positive effect of RBH on early seedling growth (Figure 6b). Plants grown from seeds treated with BABA and MeJ were more susceptible to B. cinerea infection than control treatment whereas plants from RBH treated seeds were more resistant to infection, indicating induced or primed-resistance following seed treatments with RBH ( Figure 6c). Seedlings from MeJtreated seeds showed some temporary growth inhibition 12 days after germination ( Figure   S7), but this effect was not significant in the longer term (Figure 6b, d). Interestingly, RBH seed treated and infected seedlings grew significantly better than mock-inoculated seedlings during the six days of infection period (Figure 6d).

RBH primes specific defence genes
To understand the molecular details of the observed priming effect of RBH seed treatments, gene expression analysis of the four chosen genes were performed with and without infection.
The genes PR4, BG2-3, 4CL, ECDS, from different pathways were chosen from our RNA-seq data and show upregulation after infection. As shown in Figure 7, PR4 is only slightly upregulated upon RBH treatment, but highly upregulated after RBH treatment and B. cinerea infection, a signature of priming. Seed treatments with BABA did not have effects on PR4 and BG2-3 but 4CL and ECDS were slightly upregulated upon BABA treatment alone and induced upon infection. These observations indicate that priming by RBH and BABA have some overlaps and some differences that is important for revealing resistance mechanisms against B. cinerea. The resistance induced by RBH alone suggests that the specific genes/pathways primed by RBH are important for resistance against B. cinerea.

Discussion
To effectively address problems related to plant diseases it is important to understand how plants respond to pathogens. Botrytis cinerea is an important plant pathogen with a very broad host range. We found that B. cinerea induces large transcriptomic responses in F. vesca that include genes functioning in major hormone signalling and secondary metabolite pathways (Table S1). Interestingly, we observed many similarities in the transcriptomic responses of F. vesca leaves infected by B. cinerea (Fv-Bc) and F. vesca roots infected by P. cactorum (Fv-Pc) (Toljamo et al., 2016). Thus, the recognition mechanisms, downstream signalling and genes that F. vesca uses to resist infection by these two pathogens appear to be similar. In cinerea) revealed interesting insights ( Figure S1b). Although, there are similarities in the regulation of homologous genes, we observed some interesting differences. One prominent difference was the expression of a key resistance gene, Pathogenesis-Related 1 (PR1), that is highly induced upon B. cinerea (Ferrari et al., 2007) and Pseudomonas syringae pv tomato DC 3000 (Pst DC 3000) infection in Arabidopsis (Zimmerli et al., 2000), whereas the corresponding F. vesca ortholog (FvPR1.1) is downregulated upon infection by B. cinerea (Table S2) and P. cactorum (Toljamo et al., 2016). Whether these differences stem from the plant's own responses or if the pathogen manipulates the host's immune responses differently is also an interesting question that needs further investigations.
Genes involved in well-known defence responsive pathways such as jasmonic acid (JA), salicylic acid (SA), ethylene (ET) and terpenes were upregulated upon B. cinerea infection.
Defence priming agents such as BABA, MeJ and RBH impart resistance by inducing the defence responsive pathways such as SA, JA and ET. Primarily using BABA and RBH, we explored the effects of different treatment methods in imparting resistance against B. cinerea.
Soil-drench treatment of BABA induces susceptibility and spray treatment of the BABA induces resistance against B. cinerea (Figure 3a-d and Figure S3). As the infection assays are performed in leaves, it follows that BABA induces resistance to the treated plant part and not the parts distant to the treatments, reflecting the local resistance rather than systemic resistance. Drought experiments using BABA soil-drenched plants indicate that BABA decreases the ability to retain water in the leaves, also following similarities in responses as infection by B. cinerea. These observations were consistent between F. vesca Hawaii-4 and F.
vesca Alexandria ruling out the genotype specific differences. Whereas for RBH, both soildrench and spray treatments imparted resistance, with soil-drench being most effective, probably reflecting that systemic resistance is more effective than local resistance. RBH imparts resistance by cell-wall reinforcement and inducing JA and ET pathways in Arabidopsis (Buswell et al., 2018). However, if the mechanisms of RBH-induced resistance (RBH-IR) in F. vesca are similar to that in Arabidopsis needs further investigations.
Understanding if RBH-IR is active for several weeks after soil-drench or spray treatment will hold great potential for agricultural applications.
A report in Arabidopsis (Zimmerli et al., 2000) indicates the formation of necroses after spray treatment of BABA, that can induce systemic acquired resistance (SAR) pathway. Such induction of SAR pathway can mask the primary effect of BABA, forming the reason for not using the spray treatment in Arabidopsis (Zimmerli et al., 2000). However, no visible necroses was observed in F. vesca plants upon spray treatment of chemicals or the mock treatment. Also, there are reports of spray treating strawberry plants with different chemicals to study induced resistance (Terry and Joyce, 2000;Eikemo, Stensvand and Tronsmo, 2003;Hukkanen et al., 2007;Landi et al., 2017;Saavedra et al., 2017). We therefore consider the observed effects to be the plant's induced responses to priming agent treatments.
The experiment with an insecticide imidacloprid showed similar effects of BABA and RBH as in the experiments with untreated plants, however with reduced effectiveness, possibly indicating the interference of imidacloprid with the functions of priming chemicals. Based on this observation, we argue that using chemical control agents for growth and maintenance of plants interferes with the studied plant's responses and may provide less reliable results, especially when studying effectiveness of defence priming agents. These observations therefore serve as a cautionary note to the researchers studying the effects of priming agents and emphasizes the importance of using clean, healthy and non-chemical treated plants in studying plant responses to defence priming agents.
Our experiments with ProCa provided some interesting insights. ProCa acts by blocking the dioxygenases that require 2-oxoglutaric acid as a co-substrate in gibberellin biosynthesis. Due to this action, the plants treated with ProCa accumulate less bioactive gibberellins that are required for shoot growth, thereby display shoot growth inhibition (Rademacher, 2004;McGrath et al., 2009) Arabidopsis, it is observed that a quadruple knockout mutant of DELLA genes (quadruple-DELLA mutant), lacking four out of the five DELLAs, is resistant to the hemibiotroph Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000) and susceptible to the necrotrophic fungus Alternaria brassicicola. This is because the quadruple-DELLA mutant displayed attenuated induction of a JA responsive gene PDF1.2 that is essential for JA mediated resistance against necrotrophs (Navarro et al., 2008). In rice, a necrotrophic fungus Gibberella fujikuroi (Fusarium moniliforme) causes bakanae or foolish-seedling disease leading to elongated seedlings and slender leaves causing drastic reduction in crop yields (YABUTA and T., 1938). Although it is known that GA produced by G. fujikuroi is responsible for the observed symptoms in rice, the mechanism of its virulence action is unknown. The authors (Navarro et al., 2008) propose that G. fujikuroi triggers the degradation of DELLAs by secreting GA into the plant, thereby disabling JA-mediated resistance against necrotrophs. On the contrary, exogenous application of GA decreased resistance against the hemibiotrophs, Magnaporthe oryzae (Mo) and Xanthomonas oryzae pv. oryzae (Xoo) in rice (Yang et al., 2008;Qin et al., 2013;De Bruyne, Höfte and De Vleesschauwer, 2014).
Our experiments indicate that spray application of GA 3 induces resistance in F. vesca to the necrotrophic fungus B. cinerea, contrary to the observations in Arabidopsis and rice. In addition, B. cinerea infection induces upregulation of a gene encoding ent-copalyl diphosphate synthase (ECDS, ent-kaurene synthase A) involved in the biosynthesis of diterpenoid compounds and gibberellin (Table S1). Interestingly, ECDS is also upregulated in F. vesca upon infection by a hemibiotrophic oomycete Phytophthora cactorum (Toljamo et al., 2016), pointing out to the similarities in infection strategies and/or F. vesca's responses between two different pathogens in the same host.
Treatment of F. vesca seeds with the priming chemicals BABA, MeJ or RBH did not affect seed germination and growth of the seedlings (Figure 6a-c), however only RBH treatment induced resistance against B. cinerea infection (Figure 6d). Interestingly, induced resistance against B. cinerea was partially consistent with that reported for tomato seed treatment.
Tomato plants from seeds treated with JA are resistant against B. cinerea whereas plants from seeds treated with BABA are susceptible (Worrall et al., 2012). Also, consistent with the tomato seed treatment study (Worrall et al., 2012), we observed inhibition of seedling growth 12 days after germination upon MeJ treatment ( Figure S6), which was not significant in the long term (Figure 6b, d).
The observation that seed treatments with RBH improves the ability of F. vesca to resist B. cinerea infection indicates that a priming signal is transmitted from the seed to the adult plant, reflecting molecular memory. Gene expression experiments (Figure 7) reveal that BG2-3, 4CL and ECDS are induced by both BABA and RBH seed treatments whereas PR4 is only induced upon RBH treatment, clearly differentiating between the mechanisms of two different priming agents. Based on our results (Figure 6c), it is reasonable to assume that BABA and MeJ might also induce molecular memory that is functionally different from that of RBH induced memory in imparting resistance against B. cinerea. The difference in functionalities of BABA and RBH are also true for the soil-drench experiments, where we observed induced susceptibility with BABA and induced resistance with RBH (Figure 3a and Figure S3). As soil-drench BABA treatment induces broad-spectrum resistance against pathogens with different lifestyles in Arabidopsis (Zimmerli et al., 2000;Zimmerli et al., 2001) The plants were about 10-12 cm tall from the soil surface at the start of the experiments, bearing 6-10 trifoliate leaves. For the imidacloprid (Confidor) experiment, F. vesca 'Hawaii-4' seeds were germinated in greenhouse conditions and imidacloprid was administered to the plants through water two weeks before application of the priming agent.

Treatment with defence priming chemicals
Solutions of defence priming chemicals were prepared as follows:  (Yoder, Miller and Byers, 1999;McGrath et al., 2009;Spinelli et al., 2010). For seed treatments, seeds were surface sterilized and incubated overnight with rotation in 0.3 mM BABA, 0.3 mM MeJ or 0.5 mM RBH. Seeds were then washed twice with water and germinated on Petri dishes with moist filter paper.
The germinated seedlings were transferred to pots 12 days after seed treatment.

Botrytis cinerea inoculum preparation and infection
Botrytis cinerea isolate Bc101 was grown on potato dextrose agar plates in darkness. Spores on four-to five-week-old plates were harvested using liquid potato dextrose broth (PDB) media. The spore suspension was passed through a sterile 70 µM nylon mesh and centrifuged at 3000g for 1 min, before the spore pellet was re-suspended in fresh PDB media. Spores were counted using a haemocytometer and the suspension was adjusted to a concentration of 10 6 spores mL -1 . The spore suspension was supplemented with 0.02% Tween-20 and sprayed on plants with a hand sprayer. PDB media supplemented with 0.02% Tween-20 was used for mock inoculations. Sprayed plants were placed in a tray, covered with transparent polypropylene bags to maintain high humidity, and incubated in a plant growth room until symptoms appeared. Necrotic lesions on leaves usually started appearing 4-5 days after spore spraying. When clear symptoms were observed, all the leaves were harvested for DNA extraction and qPCR quantification of B. cinerea colonization.

Genomic DNA based quantification method for Botrytis cinerea infected leaves
To establish a working quantification method for B. cinerea infection of F. vesca, leaves were drop inoculated with B. cinerea spore suspension and incubated under high humidity to promote fungal infection. We detected clear symptoms of infection, in the form of necrotic lesions on leaves, 5 days after infection ( Figure S2a). As expected, B. cinerea quantification using genome-specific primers for B. cinerea (Bc3.1F and Bc3.1R) and F. vesca (EF1αF and EF1αR) revealed that infected leaves contained much more B. cinerea than mock treated controls, indicating fungal disease progression in the necrotic lesions ( Figure S2b).

Drought treatments
BABA-treated F. vesca plants were water deprived by withholding water from the day of BABA treatment. Leaves were harvested over a 10-day interval (0D, 3D, 6D, 8D and 10D) after BABA treatment to determine their relative water content (RWC). RWC was calculated using the formula RWC = (FW -DW) x 100/(SW -DW) (So et al., 2014), where FW = fresh weight, SW = water saturated weight, and DW = dry weight of the leaves. Quantification of B. cinerea was performed using genomic DNA isolated from infected strawberry tissues. C T values from B. cinerea specific genomic DNA primers (Bc3F and Bc3R, Table 2) were normalised against F. vesca specific EF1α primers to obtain the relative B. cinerea levels in the tissue. For RNA-seq validation using qPCR, EF1α was used as the housekeeping control gene and relative expression levels of genes were determined using the Δ C T method (Pfaffl, 2001).  (Benjamini and Hochberg, 1995).

Relative Growth Rate (RGR) assay
Young petioles on 8-to 12-week-old F. vesca plants were marked with plastic tags and petiole length was measured before and after treatments with priming chemicals or fungal infection. The following formula was used to calculate RGR: RGR = (ln h 2 -ln h 1 )/t 2 -t 1 , where ln = the natural logarithm, h2 and h 1 = petiole length (in cm) at time t 2 and t 1 respectively (Buswell et al., 2018).

Acknowledgements:
Botrytis cinerea Bc101 was provided by Gunn Mari Strømeng, NIBIO. We thank Jurriaan Ton for useful discussions and advice. This work was funded by a Toppforsk grant (249958/F20) from the Norwegian Research Council.  The dots in quadrants I and III represent genes that respond similarly to both the pathogens, while the dots in quadrants II and IV represent the genes that respond oppositely to both pathogens. 'r' represents the correlation coefficient and 'R 2 ' the coefficient of determination (b) Venn diagram showing numbers of DEGs common between B. cinerea and P. cactorum infection.

Figure 3: Effects of priming chemicals on Fragaria vesca resistance against Botrytis cinerea. (a)
Amount of Botrytis cinerea quantified by qPCR after treatment with priming agents by soil-drenching (b) spraying. (c-d) plants treated with Imidacloprid before priming agent treatments. 'n' represents number of biological replicates. Error bars indicate standard error. Student's t-test was performed for statistical significance. 'ns' represents not statistically significant.

Figure 4: Effect of priming chemicals on relative growth rate (RGR) of Fragaria vesca.
RGR of soil-drenched (a) and spray treated (b) plants subsequently infected with Botrytis spores or mock control. 'n' represents number of biological replicates. Error bars indicate standard error. Student's t-test was used for statistical significance. 'ns' represents not statistically significant.    The dots in quadrants I and III represent genes that respond similarly to both the pathogens, while the dots in II and IV quadrants represent the genes that respond oppositely to both pathogens. 'r' represents the correlation coefficient and 'R 2 ' the coefficient of determination.         r  i  m  e  r  S  e  q  u  e  n  c  e  (  5  '  -3  '  )  G  e  n  e  I  D  s  R  e  f  e  r  e  n  c  e   1  F  v  E  F  1  a  R  T  F  G  C  C  C  A  T  G  G  T  T  G  T  T  G  A  A  A  C  T  T  T   F  v  H  4  _  7  g  2  0  0  5  0  .  1   (  S  i  l  v  a   e  t  a l . T  T  G  T  C  A  A  T  G  C  F  v  H  4  _  5  g  3  2  6  3  0  .  1   4  2  F  v  1  2  O  R  2  -R  T  R  T  C  G  G  T  A  C  A  A  G  A  A  A  T  T  G  G  A  G  C  C  T  G   4  3  F  v  A  C  C  O  X  1  -R  T  F  G  A  G  G  T  T  C  C  A  A  C  C  A  A  C  T  A  T  G  A  C  A  G  G   F  v  H  4  _  5  g  1  9  2  9  0  .  1   4  4  F  v  A  C  C  O  X  1  -R  T  R  C  A  G  T  T  A  C  T  C  C  A  G  C  A  T  C  A  A  C  A  A  G  A  C  C   4  5  F  v  E  C  D  S  -R  T  F  T  T  C  T  T  C  C  C  T  C  A  A  T  C  T  G  C  A  A  T  G  C  G   F  v  H  4  _  2  g  2  3  4  4  0 . 1 e t a l .