Impaginato 321 Adv. Hort. Sci., 2019 33(3): 321-326 DOI: 10.13128/ahs-23369 Efficiency of AFLP markers to detect genetic variation in Phthorimaea operculella (Lepidoptera: Gelechiidae) offspring irradiated males I. Idris (*), A. Shoaib Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), P.O. Box 6091, Damascus, Syria. Key words: AFLP technique, IST technique, Phthorimaea operculella. Abstract: AFLP technique was used to evaluate the genetic variation among nor- mal and partially sterilized potato tuber moth males. Mating experiments were carried out to obtain partially sterilized males and their descending offspring. Then, 316 AFLP bands were amplified using eight primer combinations of which 33.8 were polymorphic 85.5%, which varied from 68.57% to 100%. The UPGMA dendrogram generated for the AFLP data revealed that irradiated and unirradi- ated male samples were clustered into two groups, and the offspring of F1 and F2 of unirradiated parents were clustered into one group. Moreover, the progeny of F1 and F2 of irradiated parents clustered into three groups. No specific DNA marker could identify the irradiated males; however, there was a clear genetic variability between examined individuals. Thus, the AFLP technique could be uti- lized to study genetic variations among individuals of the same line. The AFLP markers could enhance the monitoring system of mass-released insects program when inherited sterility technique is applied against potato tuber moth. 1. Introduction The potato tuber moth Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae) is a cosmopolitan pest on potato crop, causing an annual yield reduction of 50 to 100% in some country around the world (Ahmed et al., 2013). Insecticides are widely used to control this pest, but these methods have many drawbacks like high cost, nonselective and environ- mentally unfriendly. Moreover, insects could develop resistance to insec- ticides (Harba and Idris, 2018). Therefore, more environmentally friendly methods are required. The inherited sterility technique (IST) was suggest- ed as an alternative control method to compact P. operculella (Makee and Saour, 1997; Larraín et al., 2009). Because of no-practical methods are available to separate the adult moths by gender, the males and females are mass-reared, irradiated with low sterilizing doses of gamma radiation, then released within the targeted area (Eyidozehi et al., 2015). Moths irradiated with low doses live longer, stronger fliers and mate more frequently than moths irradiated with higher radiation doses (*) Corresponding author: eidris1972@yahoo.com Citation: IDRIS I., SHOAIB A., 2019 - Efficiency of AFLP markers to detect genetic variation in Phthorimaea operculella (Lepidoptera: Gelechiidae) offspring irradiated males. - Adv. Hort. Sci., 33(3): 321-326 Copyright: © 2019 Idris I., Shoaib A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 5 June 2018 Accepted for publication 22 March 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(3): 321-326 322 (Vreysen et al., 2016). However, a dose of 400 Gy induced almost 90% sterility in irradiated males while, a complete sterility in P. operculella females was achieved by 200 Gy dose (Makee and Saour, 2004). Furthermore, the costs of using IST program are likely to be more acceptable in terms of mone- tary expenditures and efficacy, as reported by Edgington and Alphey (2017), when they released dominant-lethal strain Aedes aegypti (L.) (Diptera: Culicidae) mosquitoes. The cost-effective improve- ments to the IST programs are required by applying modern genetic methods (Leftwich et al., 2018). RAPD, AFLP, microsatellites and ESTs are popular DNA marker systems used in insect genetic research (Singh et al., 2017). They are used as monitoring sys- tems of insects mass-release programs to improve the application of IST against insects (Oliva et al., 2012; Edgington and Alphey, 2018). In this study, AFLP technique was employed to investigate the genetic variation among the offspring of partially sterilized males of P. operculella. 2. Materials and Methods Inherited sterility experiment P. operculella insects used in this study were obtained from our laboratory stock cultures. They were reared on wax coated potato slices, maintained at a constant temperature of 25±1˚C, with 70±5% rel- ative humidity, and 12 hour light-darkness cycle as described by Makee and Saour (2004). Fifty couples of females and males were placed in 350 ml transparent plastic boxes with filter papers as an oviposition site. A 10% sucrose solution was provided as food source. Both females and males were kept together until death. The eggs were removed daily, counted, and left until hatching. From the 50 reared couples only two were chosen depending on their fecundity (num- ber of eggs per female), and fertility (percentage egg hatch). All the newly hatched larvae of two couples choosing were reared on small-waxed potato pieces, and the pupae were collected. The couple, with most pupae, was chosen to be the first family for tracking to the F1 and F2 progeny. Males were divided into two groups, the first male group was used as a control (♂ N x N ♀), and the second group was irradiated with a 150 Gy in a gamma cell supplied with a Co-60 source rounded the cylindrical (15x25 cm2) irradiation cham- ber (Isslcdo-vatel Gamma Irradiator, Techsnabexport Co. Ltd. USR). The average dose rate at the time of irradiation was approximately 40.12 Gy/min with a factor of homogeny (max:min dose ratio) of about 1.05 and the absorbed dose was calibrated with Fricke solution. During this treatment, adult females were kept individually in small plastic tubes inside the irradiation source. The second males group was indi- vidually mated with normal virgin females (♂ T x N ♀). All F1 and F2 generations were reared on small waxed potato pieces as mentioned above. Fecundity and fertility of the F1 and F2 generation were record- ed. Adult male parents were kept for DNA extraction and AFLP analysis. DNA extraction and AFLP analysis Six DNA isolation protocols of P. operculella males from adult stage were used to obtain a good quality and quantity of DNA for AFLP analysis (M1: Beye and Raeder, 1993; M2: Blanchetot, 1991; M3: Favia et al., 1994; M4: Harrison et al., 1987; M5: Marchant, 1988; M6: Moeller et al., 1992) (Reineke et al., 1998). The M5-modified protocol was the most appropriate to produce a high quality and quantity of DNA from one adult moth. From each adult moth of 4-5 mg, an 8 to 12 µg pure genomic DNA was obtained. The AFLP protocol was carried out as reported by Shoaib et al. (2008). DNA from all samples was digested with EcoR1 and MseI restriction enzymes (0.125 U/µl). Selective amplification reactions were performed using eight primer combinations and the amplified fragments were separated by gel elec- trophoresis. The sequences of eight primers combi- nations and adapters used in this study are presented in Table 1. AFLP data analysis for each primer pair, the numbers of polymorphic and monomorphic bands were determined. Each gel from the AFLP experiments was scored as presence (1) or absence (0) of a specific band for every sample. Percentage of polymorphism was calculated as the proportion of polymorphic bands over the total number of bands. Allelic polymorphic information content (PIC) was calculated using the formula of Botstein et al. (1980). Data for all the 8 primer combinations were used to estimate the genetic distances among analyzed indi- viduals on the basis of the number of shared amplifi- cation products by using the Nei and Li, (1979) method. A dendrogram was generated using the Un- weighted pair group of arithmetic means (UPGMA) by Statsoft program (2003). 3. Results The data revealed that the first and the second Idris and Shoaib ‐ Genetic variations of Potato tuber moths by AFLP 323 couples were the best. The fecundity and fertility of the two couples were (111/103) and (95/88) (total eggs/ hatched eggs), respectively (Table 2). The first couple (89 pupae, no. of males and females 37 ♂/ 35 ♀) was selected to be the first family. Table 2, 3 show the F1 and F2 generations of irradiated and unirradiat- ed males that resulted from seventeen males of this family, which were irradiated with 150 Gy dose and seven males were kept as a control. Table 2, 3 show the families of irradiated (T) and unirradiated (N) males which were selected based on the fecundity and fertility of F1 and F2 generations, and presenting in a marker (*). All purified genomic DNA of P. oper‐ culella samples submitted to AFLP analysis (Table 4). Eight primer pairs successfully amplified DNA frag- ments from the genomic of 17 samples. However, 316 fragments were scored with an average of 85.5% polymorphic bands per primer combination. The per- centage of polymorphism detected by individual primer combination ranged from 68.57% for E-AAG/ M-CTA primer combination to 100% for E-AAC / M- CTG primer combination (Table 5). The ratio of num- ber of fragments produced by primer pairs were 39.5. The UPGMA dendrogram generated for the AFLP data shows that irradiated and unirradiated males samples were clustered into two groups. Hence, the offspring of F1, and F2 of unirradiated parent clus- tered into one group. While, the progeny of F1 and F2 of irradiated parent clustered into three groups. The first group include female parent, the second include the male parent, and the third one include all F2 prog- eny that were produced from irradiated male parents (Fig. 1). Table 1 - Sequences of oligonucleotide adapters and primers used in the pre amplification step and the selective AFLP primers combina- tions Table 2 - Inherited sterility technique experiments and the fami- lies of F1 generations selected for AFLP analysis * Families selected for AFLP analysis. Name Reaction Code Sequence EcoRI adapter Ligation 5¢-AATTGGTACGCAGTCTAC3¢ 3¢- CCATGCGTCAGATGCTC-5¢ MseI adapter Ligation 5¢-TACTCAGGACTCAT-3¢ 3¢-GAGTCCTGAGTAGCAG-5¢ EcoRI Preamplification E 5¢-GACTGCGTACCAATTC3¢ MseI M 5¢-GATGAGTCCTGAGTAA3¢ EcoRI +A Selective amplification E-A 5¢-GACTGCGTACCAATTCA-3¢ EcoRI +G E-G 5¢-GACTGCGTACCAATTCG-3¢ EcoRI+ C E-C 5¢-GACTGCGTACCAATTCC-3¢ EcoRI+ T E-T 5¢-GACTGCGTACCAATTCT-3¢ MseI + C M-C 5¢-GATGAGTCCTGAGTAAC-3' MseI + T M-T 5¢-GATGAGTCCTGAGTAAT-3' MseI + A M-A 5¢-GATGAGTCCTGAGTAAA-3' MseI + G M-G 5¢-GATGAGTCCTGAGTAAG-3' * Families selected for AFLP analysis. Tow couples were chosen from 50 No. of families No. of eggs Eggs hatching No. of pupea *1 111 103 89 2 95 88 77 Irradiated F1 males (♂ N/ ♀ N) No. of families No. of eggs Eggs hatching No. of ♂\♀ 1 41 25 1\6 2 Death - - 3 4 0 0/0 4 29 14 2\1 5 11 2 1\1\ 6 48 37 8\1 7 Death - - 8 6 3 1\1 9 48 20 5\1 10 38 26 14\1 11 3 3 2\1 12 204 140 45\12 *13 131 70 22\8 14 25 4 2\1 15 5 3 0\1 *16 206 146 61\17 *17 45 14 6\2 Unirradiated F1 males (♂ N/ ♀ N) *18 127 67 8\11 19 Death - - 20 34 18 2\5 21 26 19 0\0 22 23 5 0\0 *23 138 92 38\47 *24 153 153 22\18 Adv. Hort. Sci., 2019 33(3): 321-326 324 4. Discussion and Conclusions Potato tuber moth, like most of Lepidoptera moths, when exposed to substerilizing doses of gamma rays undergo several physiological, biochemi- cal and genetic changes (Makee and Saour, 2004; Hallman et al., 2013, Sachdev et al., 2017 ). However, some of the DNA damages due to irradiated male parents are inherited by their progeny (Steinitz et al., 2015). Although, inherited sterility did not occur in P. operculella females but infertility of irradiated males and females is irreversible (Makee and Saour, 1997, 1999; Idris et al., 2019). Thus, the sterility in F1 proge- ny was more than in its irradiated male parents when IST applied against P. operculella (Makee and Saour, Fig. 1 - UPGMA dendrogram showing genetic relationships among 17 DNA samples of unirradiation and irradiation of P. operculella. Samples are: 1. Female, 2. Male, 3-4. F1 irradiated males, 5-6. F1 unirradiated males, 8-12. F2 unirradiated males, 7. Mix DNA samples of F2 irradiated males 8-12, 18-14, 13. Mix DNA samples of F2 irradiated males 14-18. Table 3 - F1 progressed studied families and the families of F2 generations selected for AFLP analysis * Families selected for AFLP analysis. The F1 progressed studied families No. of F1 families No. of couples studies No. of couples sustained 12 6 1 13 1 0 *16 8 6 18 6 4 *23 11 8 24 5 2 Irradiated F2 males No. of F1 families No. of cross No. of eggs Eggs hatching *16 2 2 0 3 52 0 4 7 0 5 7 0 7 7 0 8 3 0 12 6 23 0 Unirradiated F2 males No. of F1 families No. of cross No. of eggs Eggs hatching 18 1 17 12 2 11 45 3 1 0 6 8 0 *23 1 23 1 2 35 29 3 44 35 4 28 11 7 153 63 8 8 7 9 166 65 11 58 27 24 3 80 37 5 121 67 Table 4 - DNA samples for AFLP analysis Extraction from No. of Samples Female 1 Male 2 Irradiated males of F1 3-apr Unirradiated males of F1 5-giu Mix samples of DNA 8-9-11-12 7 Unirradiated males of F2 8-9-11-12 Mix samples of DNA 14-15-16-17-18 13 Irradiated males of F2 14-15-16-17-18 Table 5 - Percent polymorphism, band numbers and poly- morphic bands produced by eight primer combina- tions No. Primers combination Total No. of bands Polymorphic bands Polymorphism % 1 E-ACT x M-CTG 49 46 93.87 2 E-AAG x M-CTA 35 24 68.57 3 E-ACG x M-CAC 51 42 82.35 4 E-ACG x M-CTA 47 42 89.36 5 E-ACA x M-CAT 45 36 80 6 E-AAC x M-CAC 41 34 82.92 7 E-AGG x M-CTC 24 23 95.83 8 E-AAC x M-CTG 24 24 100 Total 316 271 Average 39.5 33.8 85.5 Idris and Shoaib ‐ Genetic variations of Potato tuber moths by AFLP 325 References AHMED A.A.I., HASHEM M.Y., MOHAMED S.M., KHALIL S.S., 2013 - Protection of potato crop against Phthorimaea operculella (Zeller) infestation using frass extract of two noctuid insect pests under laboratory and storage simulation conditions . - Arch. 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