Phenotypic and Genotypic Traits Relationship in Iranian Ophiognomonia Leptostyla Populations Using Stepwise Multiple Regression

2nd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2012) June 30-July 1, 2012 Bali

Phenotypic and genotypic traits relationship in Iranian Ophiognomonia leptostyla populations using stepwise multiple regression

Soleiman Jamshidi1

Also, Jamshidi and Salahi (2011) reported some correlations Abstract—Quantitative and qualitative morphological and between morphological and geographic characteristics of the pathological traits relationships with molecular (RAPD and ISSRs) fungus in Northwest of Iran’s populations [14]. Jamshidi and data of Ophionomonia leptostyla populations collected from Iran Zare (2011) found two distinctive clades studying ITS were analyzed with stepwise multiple analysis regression. RAPD sequences between 16 Iranian markers mostley had significant correlation with quantitative traits than qualitatives’. RAPD12 and RAPD211 were significantly with O. leptostyla isolates and correlate then with host quantitative and qualitative traits, respectively. ISSRs had less specification. The smaller clades were collected from the correlation with quantitative morphological traits than RAPDs, but woodland areas on some wild walnuts [9]. The estimates of more with qualitatives’. There were three RAPD and one ISSR genetic and phenotypic parameters are essential in informative markers significantly correlated with disease index and determining the methods of selection to be employed and in day of acervulus formation on leaves as two pathological traits, assessing the genetic gains in performance traits. A genetic promising a specific primer can be used instead of pathogenicity tests. correlation is a description of the relationship between the sets of genes which are responsible for the genetic part of variance Keywords— correlation, Gnomonia leptostyla, Marssoniella in the corresponding traits [3]. Knowledge of relationship is juglandis, Walnut anthracnose, Walnut black spot. valuable when related traits are considered for selection. If genetic correlation among two traits is positive and high, the selection for one trait would result in an improvement of the I. INTRODUCTION other trait [12]. In statistics, stepwise regression includes ALNUT anthracnose or black spot/blotch is one of the regression models in which the choice of predictive variables Wmost important fungal diseases on Persian walnut in is carried out by an automatic procedure6. It is an estimation Europe and Asia [2]. It is widespread in Iran and has been of the linear relationship between a dependent variable and reported from the north, west, northwest [7], and northeast of one or more independent variables or covariates [15]. The this country [1]. The fungal causal agent is Ophiognomonia projective of current study was finding a possible relationship leptostyla (Fr.) Sogonov 2008, with Marssoniella juglandis between quantitative and qualitative morphological and (Lib.) Höhn 1916 as its anamorph [17]. Belisario (1992) and molecular data in O. leptostyla populations collected from Salahi et al. (2007) did not find any polymorphism in O. Iran. leptostyla Italian and Iranian populations using PCR-RFLPs of ITS and 18S rDNA [3, 13]. Belisario (2008) studied about variability of the fungus and found considerable II. MATERIAL AND METHODS morphological and pathological variation in the Italian A. Fungal Materials populations and found some relationships between morphological and environmental and pathological traits [4]. Seventy-five Persian walnut anthracnose infected leaf Jamshidi et al. (2010) reported sexual diversity and some samples were collected from 11 provinces in the northwest of morphological and pathological characteristics in Iranian Iran (Table I) during 2006-08 and the causal agents were population of this fungus [9]. Miyanaji et al. (2010) found identified as O. leptostyla according to Sogonov et al. (2008) genetic variation in Iranian population of the fungus in identification key [17]. The fungus was isolated on oatmeal nrDNA ITS and IGS regions using RFLP-PCR technique [11]. agar growth medium. Salahi and Jamshidi (2010) reported difference interactions between O. leptostyla isolates and walnut genotypes [13]. B. Phenotypic Characterization Investigated characteristics of O. leptostyla were below: 1 Soleiman Jamshidi is with Department of Plant Protection, Miyaneh Quantitative morphological data: Leafy Acervulus Length Branch, Islamic Azad University, Miyaneh, Iran (corresponding author phone: (LAA), Colony Diameter (CD), Leafy Macrconidium Length + 98 (423) 2237040-4; fax: + 98 (423) 2227290; e-mail: s.jamshidi@m- iau.ac.ir). (ML), Media Acervulus Length (CAL), Leafy Macrconidium Length (LML), Media Macrconidium Length (MML), Day of

17 2nd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2012) June 30-July 1, 2012 Bali

TABLE I GEOGRAPHICAL CHARACTERIZATION OF STUDIED O. LEPTOSTYLA ISOLATES Sampling Information Isolate Acronym Collection site Province Latitude Longitude Altitude

Abk Abhar Kahrizak Zanjan 49˚ 04ˊ 36˚ 21ˊ 1673 Abs Abhar, Shanat Zanjan 49˚ 16ˊ 36˚ 13ˊ 1645 Ahr Ahar Azarbaijane Sharghi 47˚ 03ˊ 38˚ 28ˊ 1341 Ajb Ajabshir, Bonalu Azarbaijane Sharghi 45˚ 53ˊ 37˚ 28ˊ 1423 Ajd Ajabshir, Danalu Azarbaijane Sharghi 45˚ 50ˊ 37˚ 29ˊ 1375 Ajm Ajabshir, Mehmandar Azarbaijane Sharghi 45˚ 51ˊ 37˚ 25ˊ 1333 Ard Ardebil Ardebil 48˚ 17ˊ 38˚ 14ˊ 1500 Arf Ardebil, Fuladlu Ardebil 47˚ 17ˊ 38˚ 15ˊ 1530 Aro Ardebil, Oskanlu Ardebil 47˚ 19ˊ 38˚ 16ˊ 1432 Asa Abhar, Sharifabad Zanjan 49˚ 12ˊ 38˚ 08ˊ 1713 Asl Asalem Gilan 48˚ 57ˊ 37˚ 47ˊ 43 Azr Azar shahr Azarbaijane Sharghi 45˚ 58ˊ 37˚ 45ˊ 1390 Bnb Bonab Azarbaijane Sharghi 46˚ 03ˊ 37˚ 20ˊ 1300 Did Divan darreh Kordestan 47˚ 02ˊ 35˚ 54ˊ 1821 Dlh Dalahu Kermanshahan 46˚ 07ˊ 34˚ 17ˊ 2058 Eag Eslamabad’e gharb Kermanshahan 46˚ 31ˊ 34˚ 06ˊ 1514 Fmn Fuman Gilan 49˚ 17ˊ 37˚ 13ˊ 50 Ggt Gogan, Taimurlu Azarbaijane Sharghi 45˚ 54ˊ 37˚ 46ˊ 1961 Glv Gilvan Gilan 49˚ 25ˊ 36˚ 44ˊ 439 Gnj Ganjeh Gilan 48˚ 28ˊ 36˚ 51ˊ 804 Grm Germi Ardebil 48˚ 05ˊ 39˚ 00ˊ 993 Hmd Hamedan Hamedan 48˚ 21ˊ 34˚ 11ˊ 2150 Hsh Hashtrud Azarbaijane Sharghi 47˚ 05ˊ 37˚ 47ˊ 1660 Ile Ilam, Eywan Ilam 46˚ 19ˊ 33˚ 48ˊ 1645 Ilk Ilkhchi Azarbaijane Sharghi 45˚ 58ˊ 37˚ 57ˊ 1352 Ilm Ilam Ilam 46˚ 25ˊ 33˚ 38ˊ 1759 Jlz Jolfa, Zaviye Azarbaijane Sharghi 45˚ 40ˊ 38˚ 53ˊ 879 Jol Jolfa Azarbaijane Sharghi 45˚ 38ˊ 38˚ 56ˊ 720 Khd Khorram darreh Zanjan 49˚ 11ˊ 36˚ 11ˊ 1655 Khf Khoy, Firuragh Azarbaijane Gharbi 44˚ 49ˊ 38˚ 43ˊ 1309 Khm Khoy, Mortezagholi Azarbaijane Gharbi 44˚ 57ˊ 38˚ 32ˊ 1136 Krk Karaj, Kamalabad Tehran 51˚ 38ˊ 35˚ 49ˊ 1681 Krp Paveh Kermanshah 46˚ 21ˊ 35˚ 02ˊ 2210 Kss Khosro shahr Azarbaijane Sharghi 46˚ 02ˊ 37˚ 57ˊ 1357 Lhj Lahijan Gilan 50˚ 00ˊ 37˚ 12ˊ 29 Lhr Taleghan, Lahran Tehran 50˚ 37ˊ 36˚ 11ˊ 1893 Mdb Miandoab Azarbaijane Gharbi 48˚ 90ˊ 36˚ 57ˊ 1292 Mdd Marand, Dizaj olya Azarbaijane Sharghi 45˚ 37ˊ 38˚ 27ˊ 1442 Mdk Marand, Kandloj Azarbaijane Sharghi 45˚ 43ˊ 37˚ 23ˊ 1326 Mdo Marand, Ordaklu Azarbaijane Sharghi 45˚ 41ˊ 37˚ 24ˊ 1353 Mia Miyaneh, Aghkand Azarbaijane Sharghi 48˚ 04ˊ 37˚ 14ˊ 1747 Mib Miyaneh, Balesin Azarbaijane Sharghi 45˚ 35ˊ 37˚ 38ˊ 1237 Mij Miyaneh, Balujeh Azarbaijane Sharghi 47˚ 46ˊ 37˚ 36ˊ 1503 Mir Taleghan, Mir Tehran 50˚ 33ˊ 35˚ 15ˊ 1753

18 2nd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2012) June 30-July 1, 2012 Bali

Sampling Information Isolate Acronym Collection site Province Latitude Longitude Altitude

Mlk Malekan Azarbaijane Sharghi 45˚ 55ˊ 36˚ 26ˊ 1294 Mrd Marand Azarbaijane Sharghi 45˚ 46ˊ 38˚ 25ˊ 1334 Mrg Maragheh Azarbaijane Sharghi 46˚ 14ˊ 37˚ 23ˊ 1449 Mrs Marivan, Seyf Kordestan 46˚ 16ˊ 35˚ 33ˊ 1563 Mrv Marivan Kordestan 46˚ 09ˊ 35˚ 31ˊ 1543 Msh Meshkin shahr Ardebil 47˚ 40ˊ 38˚ 23ˊ 1452 Msl Masuleh Gilan 48˚ 59ˊ 37˚ 09ˊ 1050 Myr Mohammadyar Azarbaijane Gharbi 45˚ 31ˊ 36˚ 57ˊ 1360 Ngd Naghadeh Azarbaijane Gharbi 45˚ 23ˊ 36˚ 57ˊ 1383 Nhv Nahavand Hamedan 48˚ 30ˊ 34˚ 35ˊ 2916 Osk Osku Azarbaijane Sharghi 46˚ 04ˊ 37˚ 55ˊ 1579 Pir Piranshahr Azarbaijane Gharbi 45˚ 07ˊ 36˚ 41ˊ 2505 Prs Pars abad Ardebil 47˚ 55ˊ 39˚ 38ˊ 46 Qza Abyek Qazvin 50˚ 31ˊ 36˚ 02ˊ 1366 Qzm Qazvin Qazvin 49˚ 59ˊ 36˚ 16ˊ 1390 Qzn Nezamabad Qazvin 49˚ 50ˊ 36˚ 16ˊ 1369 Qzs Sharifabad Qazvin 50˚ 07ˊ 36˚ 11ˊ 1264 Rst Rasht Gilan 49˚ 35ˊ 37˚ 16ˊ 2 Shd Shahin dezh Azarbaijane Gharbi 46˚ 33ˊ 36˚ 40ˊ 1838 Shn Taleghan, Sohan Tehran 50˚ 38ˊ 36˚ 12ˊ 1895 Shs Taleghan, Shahrazar Tehran 50˚ 39ˊ 36˚ 14ˊ 2330 Sms Somee-Sara Gilan 48˚ 18ˊ 37˚ 17ˊ 13 Sof Sofyan Azarbaijane Sharghi 45˚ 58ˊ 38˚ 16ˊ 1505 Thl Lavasanat Terhan 46˚ 27ˊ 35˚ 25ˊ 1781 Tls Talesh Gilan 48˚ 54ˊ 37˚ 48ˊ 72 Tof Toyeserkan, Falakeh Hamedan 48˚ 30ˊ 34˚ 47ˊ 1873 Toy Toyserkan Hamedan 48˚ 26ˊ 34˚ 50ˊ 2060 Tst Takestan Qazvin 49˚ 42ˊ 36˚ 30ˊ 1323 Zia Zia abad Qazvin 49˚ 26ˊ 35˚ 59ˊ 1423 Znj Zanjan Zanjan 48˚ 29ˊ 36˚ 39ˊ 1959 Znk Kushkan Zanjan 48˚ 27ˊ 36˚ 41ˊ 1706

Acervulus formation in Media (DAM), Acervulus Density in quantity of DNA were assessed by spectrophotometer Media (ADM), and Mycelium Density in Media (MDM) [7, (Jenway 6305 UV/Visible, USA). Four random RAPD 9]. primers (CinnaGen, Tehran) Including RAPD1 (5-ccg gcc tta Qualitative morphological data: Initial Growth Pattern (IGP), g), RAPD12 (5-cct ggg cct c), RAPD211 (5-gaa gcg cga t), Colony Color (CC), Microconidium Existence (ME), and and RAPD213 (5-cag cga act a) along with ISSR markers Pycnidium Existence (PE) [7, 9]. (CinnaGen, Tehran) including (GTG)5 (5-gtg gtg gtg gtg gtg) Pathological data: Disease Index (DI) and Day of Acervulus and M13 (5-cac agg aaa cag cta tga cc0. The PCR reaction (25 formation in Leaf (DAL) [4]. μl) contained 50 ng of genomic DNA, 12.5 pmol of each primer, 0.3 mM dNTPs) and 1× PCR buffer containing 2 mM

C. Genotypic6B Characterization MgCl2, 1.5 U Taq DNA polymerase (CinnaGen, Tehran). DNA extraction was carried out according to slightly PCR amplification was carried out using Apollo (ATC. 401, modified Liu et al. (2000) method [10]. 10-day old mycelia on ver. 4/88, CLP, Inc. USA) PCR machine. The PCR program 39% PDA (potato dextrose agar, Merck, Germany) added by 7 for RAPD markers was 95°C/3 min (initial denaturation), gr/L oatmeal were used for DNA isolation. The Quality and 95°C/30 s, annealing temperature/50 s, 72°C/2 min (40×) and

19 2nd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2012) June 30-July 1, 2012 Bali

72°C/10 min (final extension). Also, PCR program for MSP has several applications. Technical application like markers was 94°C/3 min, 94°C/30 s, 50°C for (GTG)5 and morphological traits prediction using molecular data is one of 44°C for M13/50 s, 72°C/2 min (35×) and 72°C/10 min. PCR the samples. Also, if we evaluate a germplasm from a species amplified products of RAPD and ISSR primers were subject regarding important traits for special condition using to horizontal gel electrophoresis using 1.2% agarose gel in 1X molecular markers, we will be able to use these valuable TBE buffer at 95 V for 30 mins using HU-150 and HU-70 findings to generalize for other germplasm for mentioned (Padideh Pars Co., Iran) standard gel electrophoresis unit. As condition. It will be possible to recognize special desire size marker we used a DNA ladder (GeneRuler 1kb DNA, genome in laboratory condition [16]. Fermentas, SM0313 100bp, Germany) and the ethidium bromide stained gels were documented using gel document (ULIdoc Inc. UK). IV. ACKNOWLEDGMENT I am grateful to Dr. N. Mohebbali-pour for his assistance in D. Data Analysis statistics analysis. Also, I need to appreciate Mr. Majid Mokhtari and Mehdi Mianaji as technical support in Molecular and phenotypic (both quantitative and Biotecnology Laboratory of Islamic Azad University- qualitative) data were separately entered in Microsoft Excel Miyaneh Branch. Also thanks to Mr. Siamak Salahi and Mrs. 2010, and transferred to SPSS 16. Stepwise multiple Rana Dastjerdi whom I received some fungal isolates from. regressions were done using SPSS ver. 16, SPSS Inc., USA15. Molecular data and morphological data were assumed as independent and dependent data, respectively and analyzed REFERENCES using step wise multiple regression technique. [1] Behdad, E. "Plant protection encyclopedia of Iran: pests, diseases and weeds”, Yad-boud Publisher, Isfahan, 1991. III. RESULTS AND DISCUSSION [2] Belisario, A. "Anthracnose,"In Compendium of Nut Crop Diseases. In A. RAPD markers Temperate Zones, B. L. Teviotdale, T. J. Michailides and J. W. Pscheidt, Eds. USA: APS Press, pp. 77-78, 2002. There were some electrophoretic band in all used RAPD [3] Belisario, A. and Hubbes, M. "Genetic variability in Gnomonia primers which were had significant relationship with leptostyla,"Acta Horticulture, 442, pp. 385-387, 1992. quantitative and qualitative phenotypics’. On the whole, 11 [4] Belisario, A., Scotton, M., Santori, A. and Onofri. S. "Variability in the Italian population of Gnomonia leptostyla, homothallism and resistance of and 4 informative RAPD markers had significant correlation Juglans species to anthracnose, "Forest Pathology, 38, pp. 129–145, with quantitative and qualitative traits, respectively. By the 2008. way, RAPD12 and RAPD211 bands had the most correlation [5] Bosso, N. A., Fall, A., Corr, N., Agyemang, K. "Genetic improvement with quantitative and qualitative traits, respectively. programme of trypanotolerant cattle, sheep and goats in the gambia: an overview of the main features and achievements". S. Münstermann, ITC. Therefore, qualitative traits had less correlation with RAPD (eds.) Livestock Research for development. In: Proc. of the markers. RAPD211 had two positive (informative) markers PROCORDEL National Conference, The Gambia, 2003. with qualitative traits. The most significantly correlated traits [6] Draper, N. and Smith, H. "Applied Regression Analysis, "2nd Edition, 2 New York, John Wiley & Sons, Inc. 1981 were CD and LML (5 and 6 bands). The R coefficient was [7] Jamshidi, S. and Salahi, S. "Distribution and etiology of walnut 2 obtained in LML (38%) and CC (35%). R of DAM and ML anthracnose in Northwest of Iran, "Journal of Modern Science of had the weakest correlation (0.5%). There was three RAPD19 Sustainable Agricultural, 6(21), 1-14, 2011. [8] Jamshidi, S. and Zare, R. "Molecular phylogeny of Ophiognomonia bands with significant correlation with DI as a pathological leptostyla isolates collected from Iran based on ITS nrDNA sequencing," trait (Table II), promising an specific primer gaining in the Proc. of International Conference of Biotechnology and Food Science. future using this finding and replacing it instead of Indonesia, pp. 130-132, 2011. [9] Jamshidi, S., Zare, R., Zamanizadeh H. R. and Rezaee, S. "Study on sexual pathogenicity tests to find resistant walnut genotypes. reproduction and some morphological and pathological traits of Ophiognomonia leptostyla in Iran," Rostaniha, 10(2), 161-177 (2010). [10] Liu, D., C. Sue, R. Baird and Pedersen, J. "Rapid Mini-Preparation of Fungal DNA for PCR," Journal of Clinical Microbiology, 38(1), 471-471 B. ISSR Markers 2000. [11] Miyanaji, M., Abdollahi, H. and Jamshidi, S. "Genetic variation of ISSRs had less correlation with quantitative traits Ophiognomonia leptostyla (Fr.) Sogonov, in ITS and IGS regions by morphological comparing RAPDs, more with qualitatives’. CAPS marker in Northwest of Iran," Journal of New Agricultural Science, Nine out of 24 ISSR markers were informative. The most 6(19), 79-89, 2010. correlation (3 bands) was between M13 with PE. Seventh [12] Naz, N. A. and Ahmad, M. "Genetic and phenotypic correlations for some sexual maturity traits in nili ravi buffalo heifers," Pakistan band of (GTG)5 primer had significant correlation with three Veternary Journal, 26(3), 141-143 (2006). traits including LAL, ALM and IGP as three quantitative [13] Salahi, S., Javan Nikkhah, M. and Jamshidi, S. "Study on population structure of Gnomonia leptostyla, causal agent of walnut anthracnose in traits. On the whole (GTG)5 and M13 were more correlated East Azarbaijan province, Iran," New Agricultural Science Journal, 3(6) with qualitative and qualitative traits, respectively. There was 53-68, 2009. a significant correlation between DAL as pathological trait [14] Salahi, S. and Jamshidi, S. "Evaluation of different varieties of walnut to and ISSRs (M13-16) (Table III). Gnomonia leptostyla, causal agent of walnut anthracnose," Journal of New Agricultural Science, 5(16): 55-61, 2010. Study of correlation between molecular and morphological

20 2nd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2012) June 30-July 1, 2012 Bali

[15] SAS Institute Inc. "SAS/STAT User's Guide, Ver. 6," 4th Edition, Vol. 2, biochemical and molecular AFLP markes in Silynum marianum L.," Cary, NC: SAS Institute Inc. 1989. Iranian Medicinal Plants Researches Journal, 24 (3), 278-292, 2008. [16] Shokr-pour, M., Mohammadi, S. A., Moghaddam, M., Ziaei, S. A. and [17] Sogonov, M. V. Castlebury, L. A. Rossman, A. Y. Mejia L. C. and Javan-shir, A. "Analysis of correlation between morphologic and White, J.F. Leaf-inhabiting genera of the Gnomoniaceae, Diaporthales, Studies in Mycology, 62, 1–79, 2008.

TABLE II REGRESSION COEFFICIENTS IN MULTIPLE REGRESSION BETWEEN PHENOTYPIC AND RAPD BANDS Regression coefficient

Marker-band Quantitative traits Qualitative traits CD DAM ADM LAL LML MAL MML DI IGP CC MDM ME Constant 15.600 198.925 22.108 22.488 24.744 110.629 6.942 1.602 1.455 2.810 1.801 0.25 RAPD1-2 - 32.070 - 4.382 RAPD1-5 - 0.365 RAPD1-7 -3.727 RAPD12-9 - 2.585 RAPD12-10 2.850 RAPD12-11 - 1.223 RAPD12-12 1.162 - 3.955 RAPD12-15 - 57.146 - 9.873 RAPD12-17 0.850 RAPD19-20 1.155 - 44.692 - 0.896 RAPD19-23 - 4.630 - 59.129 - 0.859 RAPD211-29 30.408 4.129 - 1.013 0.264 RAPD211-30 0.513 RAPD211-31 - 0.397 Adjusted R2 0.355 0.054 0.072 0.247 0.384 0.166 0.055 0.112 0.078 0.045 0.181 0.062 Informative 6 1 1 3 5 2 1 2 1 1 2 1 markers no.

TABLE III REGRESSION COEFFICIENTS IN MULTIPLE REGRESSION BETWEEN PHENOTYPIC AND ISSR BANDS

Regression coefficient Marker-band Quantitative traits Qualitative traits

LAL LML MAL DAL IGP CC ME PE Constant 156.993 17.533 6.633 16.944 2.094 2.867 0.500 - 0.499

(GTG) -2 - 0.232 5 5.238 (GTG)5-3 (GTG)5-7 22.053 0.433 - 0.416 M13-12 0.449 M13-14 0.217 M13-16 - 1.315 M13-23 - 0.444 - 0.533 M-24 0.783 2 Adjusted R 0.059 0.05 0.076 0.069 0.084 0.136 0.044 0.176

Informative markers no. 1 1 1 1 2 1 1 3