Mismatch Repair Deficiency Increases The Transfer Of Antibiosis And Antixenosis Properties Against Colorado Potato Beetle In The Somatic Hybrids Solanum Tuberosum + Solanum Chacoense

Mismatch repair deficiency increases the transfer of antibiosis and antixenosis properties against Colorado potato beetle in the somatic hybrids Solanum tuberosum + Solanum chacoense

Imola Molnár1, Enikő Besenyei1, Ramona Thieme2, Thomas Thieme3, Adriana Aurori1, Andreea Baricz1, Horia Leonard Banciu1, Elena Rakosy-Tican1

1 Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, “Babes-Bolyai” University, Romania

2 Institute for Breeding Research on Agriculture Crops, Julius Kühn-Institut (JKI), Groß Lüsewitz, Germany

3 BTL Bio-Test Lab GmbH Sagerheide, Germany

Corresponding author: Elena Rákosy-Tican, Cluj-Napoca, Romania, Clinicilor str. 5-7 nr, RO-400006, e-mail: [anonimizat], Telephone: 0040-264-431878, Fax: 0040-264-591906

Abstract

The cultivated potato Solanum tuberosum ranks third in agricultural crops production on global scale but it still suffers great loses because the attack of various pests. The Colorado potato beetle (CPB) has meanwhile become the biggest enemy of the cultivated potato worldwide. The CBP is very voracious and shows high adaptability to biotic and abiotic changes and multiple resistances to several insecticide classes. The best method to control the growth of CPB population would be the use of resistant potato varieties. One of the most effective sources of host-resistance mechanisms to CPB is the natural resistance of the wild species Solanum chacoense. Resistance to CPB is associated with the expression of rare glycoalkaloids, the leptines which have anticholinesterase-type activity. Leptines are only expressed in aerial tissues and not in tubers which is important to consider regarding the potential consumer toxicity.

The aim of this study was to assay the repellence (detection of food preferences with “choice” test i.e. antixenosis) and toxicity (monitoring of larvae viability, development and female fertility, i.e. antibiosis) of S. chacoense, its somatic hybrids (SHs) and progenies with potato against CPB adults and larvae, respectively. Moreover, transgenic lines of S. chacoense, deficient in DNA mismatch repair (MMR) were used to produce SH clones in order to increase homeologous recombination and hence the introgression of wild species DNA into potato crop. The SHs with or without MMR deficiency and BC1s were compared in their resistance against CPB and the presence of specific RAPD marker, OPT20, known to be closely linked to leptine biosynthesis.

Between SHs without MMR one showed high resistant capability against CPB attack. Its BC1 with S. tuberosum were used for further viability analysis and their resistance properties to CPB showed 1:3 inheritance pattern. MMR deficient SHs performed better in resistance analysis. The most of MMR deficient SHs had similar growing inhibitory effect on the larvae as S. chacoense and have intensive repellent effect also. Resistance ability of SHs and BC1s was attributed to leptine biosynthesis, which was confirmed by RAPD analysis.

This is the first report on somatic hybrids and progenies exhibiting both antibiosis and antixenosis effects against CPB. Resistant somatic hybrids represent an important step forward in combating this voracious pest of potato crop.

Keywords: Colorado potato beetle, MMR deficiency, antibiosis, antixenosis, somatic hybrids, S. chacoense

Introduction

Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is the most devastating defoliator pest of cultivated potato. This can be explained by its multivoltine nature. Usually it has two generations per year, but in suitable climate conditions even three or four are possible (Radcliffe and Lagnaoui, 2007). It is not negligible that CPB has high fecundity: female beetles may lay up to 800 eggs in their lives (Alyokhin, 2009). Therefore, if they are left uncontrolled they could cause total loss of potato production (Hare, 1990). In addition to these properties CBP is highly adaptable and has an unusual ability to rapidly evolve insecticide resistance. Nowadays they are resistant to more than 50 chemicals from different classes of insecticides (Alyokhin, 2009; Udalov and Benkovskaya, 2011). Furthermore in some cases it was enough to overcome the new insecticide in a single year of use (Forgash, 1985; Ragsdale and Radcliffe, 2005). As a consequence of this resistance the number of insecticides treatments was increased (Roslavtseva, 2009), which resulted in an increased cost of production and a higher degree of environmental pollution (Udalov and Benkovskaya, 2011). An alternative method to control CPBs would be host plant resistance (Hare 1990), but this is very difficult, almost impossible, with the existing potato cultivars because they do not have adequate genetic variability.

In the last decades the wild Solanum species attracted the attention of potato breeders because they represent a rich reservoir of resistant genes. This could be useful for potato improvement. Introgression of resistant genes from wild Solanum germplasm could be a durable solution for CPB control. One of the most effective sources of host-resistant mechanisms to CPB is the species Solanum chacoense. Some of its accessions are almost immune to CPB attacks (Sinden et al 1986, Deahl et al 1991). Resistance ability of these accessions is associated with their specific glycoalkaloids: the leptines, which are acetylated analogs of α-solanine and α-chaconine. Leptines are only synthesized in aerial tissues of plants (Lorenzen et al 2001, Friedman 2006), therefore they cannot be located in tubers (Deahl et al 1991). Even after exposure of tubers to low and high light treatment the concentration of leptines did not reach a detectable level (Deahl et al 1991). This is important for breeders because high level of tuber glycoalkaloids are toxic to vertebrate organisms (Maga, 1994), therefore breeders are selecting against high glycoalkaloid producing tubers (Coombs et al. 2002).

Leptine glycoalkaloids are considered as phytochemical defensive agents (Cheng, 1995) due to their antifeedant properties (Deahl et al 1991, Sinden et al 1986, Coombs et al 2002). Their poisoning effects are due to the acetyl cholinesterase inhibition (Bushway et al 1987) and the cellular membrane disruption caused by lysis as it was demonstrated for sterol-containing liposomes (Lawson et al 1993). In experiments conducted by Lorenzen et al (2001) CPB larvae fed with S. chacoense leaves were having a lower developmental rate compared to the ones fed with cultivated potato leaves. Similar results are reported by Tai et al (2014) who have concluded that the foliar leptine glycoalkaloids are associated with decreased adult beetle feeding and with increased larvae mortality.

Differences in ploidy levels between cultivated potato and S. chacoense makes it difficult to transfer the valuable resistance genes into cultivated potato with classical hybridization methods. Somatic hybridization via protoplast electrofusion was confirmed to be a useful method for the transfer of multiple resistance genes from wild Solanum species into cultivated potato (Thieme et al, 2010). Cheng et al (1995) first described somatic hybrids between S. chacoense and S. tuberosum that unfortunately produce only a little amount of leptine glycoalkaoids, therefore their resistance against CPB has not brought the expected results (Cheng, 1995).

In our research two types of somatic hybrids were used: the first type obtained after protoplast electrofusion between S. tuberosum and the wild S. chacoense 138 (from Gross Lusewitz Genebank, Germany, unpublished data of Thieme et al); the second type was obtained between S. tuberosum and wild type or transgenic mismatch repair (MMR) deficient, high leptine producer (HL) S. chacoense (accessions PI 458310 from NPGS Sturgeon Bay, USA) (Rakosy-Tican et al 2004, 2016). Mismatch repair (MMR) system is involved in reducing recombination between homeologous sequences, a function known as antirecombination (Harfe and Jinks-Robertson 2000; Schoffield and Hsieh 2003). This makes it more difficult to transfer the resistance genes from wild species into economically important crops. Recently it was found that the loss of msh2 and PMS1 activities in Arabidopsis thaliana led to increase in homeologous somatic (mitotic) recombination between sequences of varying divergence (Li et al 2006, 2009). Mutants of msh2 induced a threefold increase in intrachromosomal recombination between highly diverged sequences (13%) in germinal tissues of A. thaliana (Lafleuriel et al 2007), moreover msh2 was reported to be involved in the suppression of somatic recombination in different A. thaliana ecotypes (Emmanuel et al. 2006).

In order to increase homeologous recombination and hence the transfer of resistance genes from wild Solanum species into cultivated potato, S. chacoense was transformed with Atmsh2 gene in antisense orientation (AS) or the dominant negative mutant sequence (DN), both inducing MMR deficiency (Rakosy-Tican et al. 2004, 2016).

The goals of our present research were to assess antibiosis properties of the leaves of SHs and BC1 progenies, when available, by determining the toxicity on CPB larvae’s growth, viability and fecundity. Never the less, antixenosis (repellent) properties of the same SHs and progenies were evaluated and corroborated to RAPD markers of leptine biosynthesis as described by Bouarte-Medina et al. (2002).

This is the first report on both antibiosis and antixenosis effects of leptines on CPB larvae sustained by RAPD molecular markers in somatic hybrids between potato and S. chacoense. Our results prove the anti-recombination function of transgenic msh2 gene with increased introgression of resistance trait in SHs involving MMR deficient S. chacoense HL parent.

Materials and methods

Plant material and the laboratory bioassay on Colorado potato beetles

The hybridity nature of somatic hybrids was confirmed by simple sequence repeat (SSR) marker analysis (Besenyei et al, personal communication).

In vitro cultured plants of cultivated potato, S. chacoense HL, 22 SHs with or without MMR deficiency and 6 BC1 of non-transgenic S. chacoense 138 and S. tuberosum somatic hybrids were transplanted into pots and were cultivated in greenhouse for 2 months. The mature leaves were used during the analysis.

In the first experiment a laboratory bioassay was conducted to analyze the effect of somatic hybrids on CPB larvae development and in the second, by applying a choice-test, the food preference of adult beetle was determined.

For the laboratory bioassay CPB larvae were reared at 25oC and a 16:8 photoperiod. Twenty-four hours after hatching, the weight of the not yet fed larvae was measured. Using a small fine-tipped paintbrush 25 larvae were transferred on S. chacoense, S. tuberosum, SHs and BC1s leaves. This step was performed according to Horton (1991) experimental design, also suggesting to eliminate variations of egg masses. Every second day the survival rate, developmental stage (Boiteau and LeBlanc, 1992) and larvae weight were recorded. Larvae were considered dead if no movement was observed after being lightly touched with a small paintbrush. The time to the development of different larval stages (L1 – L4), pupae and adults was also recorded. When the survived larvae reached the adult stage their sexes were determined and the occurring developmental abnormalities were observed. Adult beetles were mated and the female fertility was determined from the female’s oviposition ability and from the laid eggs viability rate.

The surviving rate, mean developmental time (number of days calculated from hatching to emergence of adult), mean weights of juveniles, pupae and adult beetles separated by sexes were measured.

The choice test

In the second experiment the adult CPB food preferences were analyzed using SHs, BC1s and the two parents. The adult beetles were grown up on cultivated potato leaves and were starved for 24 hours before the experiment. After starvation they had to choose between one of the parents (S. tuberosum or respective S. chacoense) and one of the SH or BC1 leaves. The two types of weighed leaves (one parent and one SH or BC1) were put into the two sides of a Petri dish and the beetle was placed to the center. The beetle was monitored for 2 hours and each type of choice design (parental lines vs. somatic hybrid lines or BC1s) was repeated three times. After the experiment the not consumed leave parts were weighted and the preference index (Szafranek et al, 2008) was calculated which was used to compare the capacity of repellence of SHs and BC1s vs. each of their parents.

Using this experiment in the first place we wanted to prove that the adult beetles prefer better to consume S. tuberosum leaves than the SH's ones. On the other hand we were curious how they behave when they had to choose between the least preferred food (S. chacoense HL) and SHs leaves.

Statistical analysis

Statistical analysis was performed using Microsoft Excel Statistics and R statistical software (The R foundation). Statistical analyzes was conducted on the survival and mean relative growth rate of larvae fed on different genotypes using multiple comparison with Fisher's protected least significance difference in the general linear model (LSD) according to Coombs et al (2002). In case of the normal data distributions for pair-wise comparison the one-way ANOVA (Tukey test) was used, where homogeneity of variance have been fulfilled. In case where normal distribution criteria was not implemented the Kruskal-Wallis-test was performed. Comparison of consumed leave parts was performed using unpaired Student t-test. In our analysis if the P value fell below 0.05 we interpreted as significant difference between the compared data.

RAPD markers analysis

Genomic DNA was isolated using a modified cetyl-trimethyl-ammonium bromide (CTAB) extraction method described by Lodhi et al (1994, modified by Pop et al 2003).

In this experiment leptine glycoalkaloid producing SHs and BC1s were determined using the RAPD-PCR method with OPT-20 RAPD marker based on Bouarte-Medina et al, 2002 protocol. This marker amplifies a specific 250 bp DNA sequence that proved to be closely linked to or includes one of the leptine production responsible genes (Bouarte-Medina et al, 2002).

Results

A laboratory bioassay was performed to evaluate the resistance capability of somatic hybrids against CPB. This had similar parameters to the naturally occurring conditions: freshly hatched larvae were kept in Petri dishes with continuously available fresh leaves of one of the potato genotypes, natural light conditions and temperature were set as well. Larvae were monitored until they completed their developmental cycle and the emerged adult beetle’s fecundity success was evaluated. Fitness parameters such as survival rate, mean weight of fourth instar larvae and adults, development time until adulthood, fecundity have been used to evaluate the relative suitability of SHs and their descendants against CPB.

The survival rate of CPB larvae fed on S. chacoense (HL and 138), cultivated potato, SH and BC1 were calculated for the first 23 days. During this period more than 50% of larvae (9 out of 13) fed with S. tuberosum leaves achieved the adult stage, while in case of both accessions of S. chacoense (HL and 138) no larvae achieved this stage until day 23 of experiment. S. chacoense HL proved to have stronger negative effect on larval development than S. chacoense 138. All of the larvae fed with S. chacoense HL leaves died before reaching the adult stage, while in case of S. chacoense 138 a few number of larvae transformed into adults (2 female and 3 male), but they were not fertile.

According to the results presented in Fig. 1 two groups of BC1 can be distinguished based on the genotype susceptibility against the CPB attack. Somatic hybrid 1552/1 used as female parent in backcrossing step with S. tuberosum cv. Sonate (♂) show high resistance ability against CPB. Only one larvae survived the 23 days of experiment but it does not reached the adult stage. Backcrossing cultivated potato with highly resistant SH 1552/1 led to one-to-three segregation of resistance property in descendants (Fig. 1). The survival and growing rates of larvae fed with the susceptible BC1 (4 genotypes) was similar to the ones measured in case of cultivated potato (Fisher's LSD, p>0.05). Between the toxic effect on CPB larvae of resistant groups that highly reduce larvae surviving ability and susceptible group significant differences (Kruskal-Wallis test, p<0.05) were observed. The resistance properties of BC1 genotypes (1552/1/2 and 1552/1/4) and SH 1552/1 could be explained by leptine biosynthesis. These BC1s contain the specific DNA sequence recognized by OPT-20 primer like S. chacoense HL and 138, which has been proven to be highly-linked to leptine synthesis (Bouarte-Medina et al 2002). Susceptible BC1 along with S. tuberosum did not show this 250 bp length DNA sequence (Fig. 8a). Resistant BC1s were not as effective as their resistant SH progenitor (Fisher's LSD, p>0.05) but they also greatly reduce, with more than 50% the surviving rate of larvae fed on their leaves. Besides the reduced surviving rate of larvae fed on resistant BC1 leaves, prolonged developmental time was necessary to reach the fourth larval stage (Fig. 2). Only 15 days after hatching they were transformed to fourth instar larvae (more than 50 % of larvae) while larvae fed with susceptible BC1 developed faster, which achieved the fourth larval stage (>50%) after 9-11 days. The mean weight of larvae after 23 days from hatching grown up on resistant genotypes were not significantly lower than in the case of susceptible plants, which suggest that those larvae that survived on resistant plant leaves had not suffered from poisoning (Fig. 3a). After pupation, when larvae emerged to adults, visible dimension differences between adults fed with different types of hybrids were observed. In the case of resistant BC1 the weight of the emerged female beetles were significantly lower (one-way ANOVA, p<0.05) compared to female beetles grown up on cultivated potato leaves. Between male beetles there was no significant differences observed (ANOVA, p=0.23). The reduced weight of the female beetles did not affect their fertility. All of them were capable to reproduce and viable larvae were hatched from their eggs.

Somatic hybrids with MMR deficiency performed better in resistance analysis. (Fig. 4). From twenty SHs analyzed just seven genotypes behaved similarly as potato (Fisher’s LSD, p>0.05). They had not enough toxic effect to reduce by half the larvae survival rate. This indicates the susceptibility to CPB of these genotypes. Larvae fed with susceptible plant leaves developed normally as demonstrated by larval developmental index on day 23 (LDI=55-76), which approximates the LDI value (LDI=80) obtained in case of the parental cultivated potato. Moreover, the larvae fed with susceptible plants reached significantly more the adulthood compared to the resistant plants (one-way ANOVA, p<0.05). However no significant differences were observed between developed adult beetles weight, indifferent to their sexes, compared to adults beetle fed on cultivated potato. (Tukey test, p>0.05). The adult beetles grown up on susceptible plants were fertile. Females laid large amount of eggs, out of them the majority was viable and hatched healthy larvae. In susceptible plant group an equal ratio of the two types of MMR deficient plants were observed: half of them were SHs with Atmsh2 in antisense orientation (AS) and the others were hybrids with dominant negative mutant Atmsh2 gene (DN).

In case of resistant somatic hybrids the survival rate of larvae were less than 50%. More specifically, less than 30% of larvae fed with these plant leaves survived 23 day after hatching. This indicates their resistance against CPB. The toxic effect of these plants against CPB larvae was significantly higher in case of resistant hybrids group than in susceptible one (Kruskal-Wallis test, p<0.05). Larvae fed with resistant plant leaves developed more slowly: 9 day after hatching larvae fed on S. tuberosum leaves transformed to L4 larval stage, while most of the larvae on resistant plants were only in L3 or L2 stage (De.DN11.29, Dk.DN5.11, Dk.AS10.40), moreover in some cases CPBs were only in the first larval stage (Dk.DN5.4, Dk.DN5.7) (Fig. 5, Fig. 6). In order to transform the larvae fed on resistant plants into L4 instar 13-19 day were necessary after hatching. Similar results were obtained after LDI calculations: larvae fed on resistant hybrids had far lower index value (LDI between 0-30) compared to susceptible SHs or cultivated potato. This indicates greater inhibition of larval development and/or increased mortality. In case of resistant hybrids high degree of mean larvae weight differences were observed (Fig. 3b). Some SHs (marked with *) had stronger post-ingesting effects on larvae than the other resistant genotypes. On day 23 the weight of these larvae varied between 35-72 mg, which was much lower than the average weight of larvae fed on potato leaves, 119.78 mg.

Due to decreased viability, significantly reduced number of adults were developed from larvae fed on resistant plants in contrast with susceptible hybrids (one-way ANOVA, p<0.05). In case of some hybrids (Fig. 6) none of the larvae reached the adulthood.

In those cases when larvae transformed to adults and were fed with resistant plants, lower adult beetle weight were observed. In case of female beetles there was significant difference (one-way ANOVA, p<0.05) compared to adults grown up on susceptible plants. The emerged adults had very low fertility rate because several beetles had visible malformations and they were not capable to reproduce (in case of De.DN5.5, Dk.AS10.47). The very small-sized females did not lay eggs (Dk.DN5.11) and weighted around 75 mg, which is almost half compared to beetles grown up on S. tuberosum having 125 mg average weight. In case of Dk.AS10.43 females were able to reproduce and to lay eggs but no larvae were hatched from them. A small number, but not viable larvae (alive for only three days), were hatched from female beetles eggs grown up on Dk.DN11.10 leaves. Only in case of Dk.AS10.13 and Dk.AS10.35 viable eggs were produced.

Between these resistant hybrids both type of MMR deficient plants: DN (7 genotype) and AS (6 genotype) were observed. Three of them (Dk.DN5.4, Dk.DN5.7 and De.DN11.29) had similarly strong toxic effects on CPB larvae, like S. chacoense HL. In case of De.DN11.29 larvae reached the L2 stage but none of them were capable to transform to L3 stage. The last larvae died after 29 days from hatching.

Dk.DN5.4 and Dk.DN5.7 proved to be the most resistant genotypes: no larvae survived until day 23 similarly to larvae fed on S. chacoense HL leaves. On the 13th day when 100% of the larvae fed on S. tuberosum leaves were transformed to L4 stage with an LDI=84, the larvae fed on these two SHs and S. chacoense HL have this value LDI = 1, which indicates an intense inhibition of larval development. These larvae were just in the first larval stage and also an increased mortality could be observed: only two larvae were still alive in case of S. chacoense HL and Dk.DN5.4 and only one survived until 13th day in case of Dk.DN5.7.

Based on RAPD-PCR analysis one can observe that these genotypes’ resistance properties are due to the synthetized leptine glycoalkaloids that have inhibitory effect on CPB larvae development and viability (Fig. 8b). After the laboratory bioassay one can conclude that several somatic hybrids with MMR deficiency and some BC1 progenies have strong antibiosis effects. They are toxic for CPB larvae because they reduced CPB larvae performance (survival, development) and fitness.

Choice test study was performed to determine SHs and BC1s with antixenosis properties. Beetles had to choose between one type of SHs (and BC1) and one type of the parental lines (S. tuberosum or S. chacoense). After this experiment food preference indices were calculated from quantity of consumed leaves. This was pairwise compared. SHs (and BC1s) with S. tuberosum (Fig. 7a) or S. chacoense (Fig. 7b) using Kruskal-Wallis test. Marked genotypes with a and b denote to have significantly different effects (stimulatory or inhibitory) on CPB feeding behavior than their parents. The preference index was calculated: if this value is over 0 and approaches 1 the plant has a phago-stimulant effect, while in case of values below 0 down to -1 indicates deterrent effect of the plants. In case of the first control experiment beetles had to choose between cultivated potato and S. chacoense HL. All beetles chose cultivated potato leaves none of them tasted the resistant S. chacoense HL. From this control test we can conclude that S. tuberosum has phago-stimulant properties, while S. chacoense HL has strong deterrent effect on CPB.

In a second experiment beetles had to choose between cultivated potato and two types of SHs, respectively BC1s. The phago-stimulant effect and deterrent effect of plants can be observed. These results are presented on Fig. 7a. SHs or BC1 with PI value higher than 0 have not got negative influence on the beetles’ feeding behavior. SHs and BC1 marked with a proved to have significantly higher phago-stimulant effect (t. test, p<0.05) than S. tuberosum. In these cases beetles preferred to consume those SHs and BC1 leaves instead of cultivated potato leaves.

SHs and BC1s proven to be resistant in laboratory bioassay having toxic effect on CPB larvae development, had also deterred feeding on adult beetles. In these cases beetles preferred to consume cultivated potato leaves instead of SHs or BC1s. Significant difference between the consumed leave biomass of S. tuberosum and SHs or BC1s marked with b were observed (t. test, p<0.05). This implies a very strong deterrent activity.

On Fig. 7b the preference indices between SHs or BC1 and S. chacoense of adult beetles is illustrated. Obviously SHs or BC1s which had no deterrent effects in previous cases (when beetles had to choose between cultivated potato and SHs), they do not have this property in current experiment either. Their PI indexes are close to value 1. Some SHs (marked with a on Fig. 7b) were not preferred by beetles compared with cultivated potato, but in comparison with S. chacoense significantly higher amount of biomass (t. test, p<0.05) was consumed. There was no significant difference between PI indices of SHs marked with b on Fig. 7b and S. chacoense (t. test, p>0.05). Based on our results one can conclude that 1552/1, Dk.DN5.4, Dk.DN5.7, Dk.DN5.11 and Dk.AS10.43 have the strongest deterrent effect. Beetles preferably chose to die due to starvation instead of consuming those genotypes, which also proved to have the most toxic effect on CPB larvae in the laboratory bioassay.

The repellent effect of SHs could be associated with leptine synthesis because all of these SHs contain the 250 bp length DNA sequence, which is linked to leptine production (Bouarte-Medina et al. 2002). In case of genotypes with phago-stimulant properties the OPT-20 primer does not recognize this specific sequence.

Between deterrent genotypes both SHs with or without MMR deficiency could be observed. Majority of the most resistant SHs belongs to MMR deficient somatic hybrids containing a dominant negative sequence of Atmsh2 gene. This confirms our findings during laboratory analysis. This mutant allele of msh2 gene can compete in a dominant way the four normal alleles of potato parent, allowing the transfer with increased probability of the resistance genes.

Based on our results one can conclude that some SHs with or without MMR deficiency and BC1s have strong antibiosis due to the increased larval mortality and inhibition of larval development and antixenosis properties as well (Table 1). CPB were very reluctant to feed on these resistant genotypes.

RAPD-PCR analysis using OPT-20 primer was effective to distinguish leptine producer genotypes. S. chacoense, which produces high concentration of leptines (Mweetwa et al 2012) and 16 SHs and BC1s showed a specific sequence at around 250 bp levels. This corresponds to the findings of Bouarte-Medina et al. (2002). But in the case of cultivated potato and 11 of SHs and BC1s this sequence was not found (Fig. 8a and b).

Discussion

The high capability of CPB to adapt rapidly against different chemical insecticides encourages breeders to find a different type of control to CPB attacks. The only long-term solution for CPB management would be the development of host plant resistance. S. chacoense attracted the attention of potato breeders because it possesses broad resistance ability against different pathogens (bacteria, viruses, nematodes) (Rakosy et al 2016) and moreover it is highly resistant against Colorado potato beetle (Sinden et al 1986). Using somatic hybridization via protoplast electrofusion makes it possible to introduce the genetic material of the S. chacoense together with leptine glycoalkaloid multiple encoding genes into cultivated potato (Cheng et al, 1995).

The insect resistance of S. chacoense is attributed to their specific steroidal glycoalkaloids: the leptines. Deahl et al (1991) proved that resistance of S. chacoense is resulting from the high quantity of leptines in their foliage. Introgression of leptine glycoalkaloid genes into cultivated potato germplasm might meet the breeders’ expectations. Foliar leptine glycoalkaloids are associated with high level of larval development inhibition and with increased larval mortality (Lorenzen et al 2001, Hollister et al 2001, Tai et al 2014). These observations were also confirmed in our experiment. CPB larvae fed with S. chacoense HL leaves – known to produce the largest amount of leptine glycoalkaloids – developed slower and weaker, none of them reaching the adult stage. S. chacoense HL leaves proved to be toxic to CPB larvae. Differences between antibiosis effects of leptine producing resistant somatic hybrids are caused by the property of some hybrids to have more toxic effect than the others. This could be explained using the results obtained by Kowalski et al (1999). They observed that concentration of leptine I glycoalkaloid highly influences the degree of inhibition and larval growth while development was reduced in dose-dependent manner on artificial diets containing leptine I.

Leptine producer somatic hybrids proved to have long-term effect on CPB, because resistant genotypes not only reduce the viability of CPB larvae, but the survived beetles’ fecundity was also reduced. The majority of survived beetles were not able to produce viable offspring; thereby application of resistant genotypes contributes to significant reduction of CPB population over several generations.

Beside the toxic effect of resistant hybrids most of them also possess antixenosis properties, which positively correlate with leptine producing ability. Somatic hybrids and BCs containing the specific DNA sequence which is closely linked to/includes one of the leptine production responsible gene inhibited CPB development and had deterrent effect on adult beetles. Tingey and Yencho (1994) obtained similar results. They labeled the leptine glycoalkaloids as potent antifeedants based on the reduced feeding rates of adult beetles on leaf disks of S. chacoense. Leptines proved to have also negative behavioral effects on adult and larvae CPB in the field test (Yencho et al 2000).

Among the analyzed SHs MMR deficient plants performed better in resistance analysis against CPB, which implies that deficiency in DNA repair system increases the resistance of SHs. More of the hybrids were as resistant as the wild species suggesting that MMR deficiency increases the transfer of multiple resistance genes involved in leptine biosynthesis. Dk.DN5.4 and Dk.DN5.7 proved to have the highest toxic effect against CPB larvae. Both SHs were produced involving MMR deficient S. chacoense by integration of the dominant negative Atmsh2 gene (Rakosy-Tican et al, 2016). Therefore one can suppose that this type of transformation allows a greater degree of introgression of genetic information from S. chacoense or a greater reduction in anti-recombination activity of the MMR system. The influence of MMR deficiency by increasing homeologous recombination in case of interspecific somatic hybridization opens a new way to higher introgression of wild resistance genes into crop gene pool. Such results are reported in this study for the first time. Moreover, previous analysis of the MMR deficient SHs between S. chacoense and potato, the same 20 hybrids which were analyzed for resistance ability against CPB, revealed that many of them show a 'mutator' phenotype accompanied by its molecular signature i.e. microsatellite instability. For instance the most resistant MMR deficient SHs: Dk.DN5.4, Dk.DN5.7 and Dk.DN5.11 exhibited a 'mutator' phenotype accompanied by MSI. The SH Dk.DN5.4 presented a new phenotype with variable size purple tubers while DkDN5.7 was presenting a stunted growth with early flowering. Among the other resistant genotypes against CPB attack, some also show 'mutator' phenotype, Dk.DN5.11 had large leaves and it is an early flowering genotype. The SH Dk.AS10.40 provided gigantic growth ability but this trait was not accompanied by MSI suggesting that this phenotype manifestation can be caused only by somatic hybridization. Moreover, MMR deficient SHs were selected in vitro for drought tolerance and then both sensitive and tolerant genotypes were transferred on a phenotyping platform (data to be published). The medium drought stress allowed selection of one MMR deficient SH (Dk.AS10.13), which was able to accumulate the same biomass as the control plants after 6 weeks in the greenhouse, although none of the parent lines (S. tuberosum and S. chacoense) possessed drought tolerance abilities. This particular hybrid has both antixenosis and antibiosis effects on CPB but its resistance is not very high: some of the adults were able to lay fertile eggs. One can suggest that MMR deficiency which induced homeologous recombination is responsible for CPB resistance but somatic hybridization and induced mutations by a deficient MMR system might be also responsible for the new traits, like purple tubers, stunted growth, large leaves, early flowering or drought tolerance of these SHs (see Rakosy-Tican et al. 2016). This valuable plant material represented by SHs between S. chacoense and potato with and without MMR will be further used for basic research for better understanding the effects of one transgenic MMR deficient parent on msh2 gene expression, protein heterodimer formation and recombination in meiosis and mitosis along with somatic hybrid ploidy status and phenotype in this important crop. The produced potato SHs and BC1s proved to have an increased level of resistance to all stages of CPB will be used as a source for improving resistance to this pest in a pre-breeding program.

The strategy described in this study for the first time, the use of MMR deficiency in combination with somatic hybridization by protoplast electrofusion for efficient transfer of multiple gene resistance traits might be successfully used to other crop species amenable for protoplast fusion and somatic hybrid regeneration.

Acknowledgements

The funding from Ministry of Education and Research, the national project CNCS PNII-ID-PCE-2011-3-0586 is acknowledged.

E. B. was fund by the financial support of the Sectorial Operational Program for Human Resources Development 2007-2013, co-financed by the European Social Fund, under the project POSDRU/159/1.5/S/133391 – “Doctoral and postdoctoral excellence programs for training highly qualified human resources for research in the fields of Life Sciences, Environment and Earth”.

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Figure and table legends

Figure 1 Survival rate of CPB larvae (0 to 1, the value of 1 means that all larvae survived), fed on S. chacoense (S. chc) 138 and derived somatic hybrid and BC1 progenies with S. tuberosum (S. t); the segregation of the resistant trait in BC1 is indicated.

Figure 2 Required developmental time for larvae to transform into L4 stage fed on different BC1 plants compared with larvae fed on BC1’s parents (S. tuberosum (S. t), S. chacoense (S. chc) 138 and SH 1552/1). L1 to L4 legends indicate the different larval stages.

Figure 3 Mean weight of larvae (mg) on day 23 after hatching, grown up on different genotypes, parents (S. tuberosum (S. t) and S. chacoense (S. chc) 138, respectively HL), somatic hybrids with MMR deficiency (b) or without and its derived BC1s (a). Plant genotypes marked with * on Fig 3b had the strongest post-ingesting effect on larvae.

Figure 4 Survival rate of CPB larvae (0 to 1, the value of 1 means that all larvae survived) fed on MMR deficient SHs and parent lines (S. tuberosum (S.t) and S. chacoense (S. chc) HL)

Figure 5 Massive developmental difference between the same aged larvae fed on resistant (on the left, Dk.DN5.4) and susceptible (on the right, Dk.AS10.5) plant leaves

Figure 6 Required developmental time for larvae to transform into L4 stage fed on MMR deficient SH and their parents (S. tuberosum (S. t), S. chacoense (S. chc) HL). L1 to L4 legends indicate the different larval stages.

Figure 7 Preference indices (mean ± SE) for adult CPBs feeding on SH with or without MMR deficiency and some BC1 descendant compared with S. tuberosum (a) or with S. chacoense (b). Letter a on figure 7a indicates significantly higher phago-stimulant effect of SHs and BC1s than S. tuberosum, while letter b marks plant genotypes with strong deterrent effect (the consumed leaf area were significantly lower than in the case of S. tuberosum). Plants genotypes marked with a on Fig 7b had deterrent effect compared to S. tuberosum, but they had not efficient deterrent activity compared to S. chacoense, while genotypes marked with b was similarly as effective as S. chacoense (no significant differences between S. chacoense and b marked genotypes were observed)

Figure 8 Selection of leptine producing SHs with (b) or without MMR deficiency and BC1 progenies (a) based on OPT-20 RAPD marker linked to leptine biosynthesis (S.t- S. tuberosum, S. chc- S. chacoense)

Table 1 Synthetic presentation of the analysed parent lines (S. tuberosum (S.t), S. chacoense (S.chc) HL and 138), SHs and BC1s resistance ability against CPB – antixenosis and antibiosis effects (++ = very high similarity to S. chacoense; + = disposes resistance properties; – = lack of the analyzed trait)