Acta Botanica 2-2014.indd ACTA BOT. CROAT. 73 (2), 2014 291 Acta Bot. Croat. 73 (2), 291–298, 2014 CODEN: ABCRA 25 ISSN 0365-0588 eISSN 1847-8476 PCR detection assay for sex determination in papaya using SCAR marker KANUPRIYA CHATURVEDI1, PADMAKAR BOMMISETTY1, ARPITA PATTANAIK1, VASUGI CHINNAIYAN2, DINESH M. RAMACHANDRA2, ASWATH CHENNAREDDY1* 1 Division of Biotechnology, Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Hesaraghatta, Bangalore 560 089, India. 2 Division of Fruit Crops, Indian Institute of Horticultural Research, Hesaraghatta, Bangalore, India Abstract – Papaya (Carica papaya L., 2n = 18), a polygamous angiosperm, is a major fruit crop in tropical and subtropical regions. It is trioecious with three sex forms: male, female, and hermaphrodite, where sex determination is controlled by the XY chromosome pair with two slightly different Y chromosomes i.e. Y for male and Yh for hermaphrodite. Sex type determination in papaya, which cannot be determined either by embryo shape or morphology at the juvenile developmental stage, is an essential pre-requisite for crop im- provement processes as it helps in identifi cation of fruitful plants. Hence, molecular profi l- ing could be used as an alternative that provides a quick and reliable identifi cation of sex types in plantlets at initial stages only. In the present study we have validated the sex- linked sequence characterized amplifi ed region (SCAR) marker W11 using PCR detection assay among different cultivars of papaya i.e. dioecious with either female or male and gynodioecious with either female or hermaphrodites and also performed a double-blind test for validating the seedlings of 84 F1 plants, which resulted in their sex determination. The assay clearly gives 800 bp band in male plants in dioecious types and hermaphrodite in gynodioecious plants. Keywords: Carica papaya, dioecious, double-blind test, gynodieocious, papaya, SCAR marker, sex determination Introduction Carica papaya L., a native of Latin America, is a diploid (2n = 18) belonging to the family Caricaceae. Papaya is one of the most important fruit crops of India with a production of 4.71 million tons grown over an area of 0.1 million ha (NATIONAL HORTICULTURE BOARD 2012). It is widely grown in many other countries also (Tab.1). Papaya is widely cultivated * Corresponding author, e-mail: aswathiihr@gmail.com Copyright® 2014 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. CHATURVEDI K., BOMMISETTY P., PATTANAIK A., CHINNAIYAN V., RAMACHANDRA D. M., CHENNAREDDY A. 292 ACTA BOT. CROAT. 73 (2), 2014 for its edible fruits and milky latex that yields papain, a proteolytic enzyme, and carpain alkaloids that are of great economic importance. As the yields and proteolytic activity of the crude papain obtained from the female fruits are greater than that obtained from hermaphro- dites (MADRIGAL et al. 1980), dioecious papaya cultivars are preferred for the extraction of papain. The papaya is polygamous plant species with three main sex types, namely pistillate or female, staminate or male, and hermaphrodite (STOREY 1941). The results of earlier reports (HOFMEYR 1938, STOREY 1938, 1941, 1953) on the genetic analysis of sex determination in papaya suggest that sex determination is controlled by three alleles. The genetics of sex is very intriguing in the papaya. Among several hypotheses, the genetic balance hypothesis explaining sex determination in papaya (where factors determining femaleness are present in the sex chromosome and that of maleness in the sum total of autosomes) has been widely accepted (HOFMEYR 1938). It is further assumed that M1 and M2 respectively represent the inert segments of sex chromosomes. The M1 is slightly larger than the M2 segment. It is presumed that genes responsible for life are eliminated in these inactive regions and cause the lethality of the genotypes M1M1, M1M2, and M2M2. However, the genotypes M1m and M2m will be viable due to the presence of an active sex chromosome. Recent reports have proven that sex determination in papaya is controlled by a recently evolved XY chromosome pair, with two slightly different Y chromosomes controlling the development of males (Y) and hermaphrodites (Yh) (GSCHWEND et al. 2011, NA et al. 2012, WANG et al. 2012). Traditionally, the propagation of papaya plants is through seeds, which would give rise to a population of generally 1:3 for male to female. However, the sex of these plants cannot be deduced from the external morphology of embryonic or juvenile forms. The sex of the seedlings can be detected only after the plants attain reproductive maturity, i.e. after 5–8 months. However, if the sex of the dioecious plants is identifi ed at the seedling stage, prior to their transplantation to the fi eld, then a desired ratio of male to fe- male plants (5% males/95% females) can be achieved, thereby saving on the input cost. In Tab. 1. Global papaya production in million tons (source: NATIONAL HORTICULTURE BOARD 2012). Countries 2002 2003 2004 2005 2006 2007 2008 2009 2010 India 2.15 1.69 2.54 2.14 2.48 2.91 3.63 3.91 4.71 Brazil 1.60 1.71 1.61 1.57 1.90 1.81 1.89 1.79 1.87 Indonesia 0.61 0.63 0.73 0.55 0.64 0.62 0.72 0.77 0.70 Nigeria 0.76 0.80 0.86 0.76 0.76 0.77 0.69 0.76 0.70 Mexico 0.88 0.96 0.79 0.71 0.80 0.92 0.64 0.71 0.62 Ethiopia 0.23 0.23 0.26 0.30 0.26 0.23 0.25 0.26 0.23 Democratic Republic of the Congo 0.21 0.21 0.21 0.22 0.22 0.22 0.22 0.22 0.23 Colombia 0.09 0.09 0.10 0.14 0.16 0.22 0.21 0.19 0.26 Thailand 0.35 0.31 0.28 0.03 0.13 0.20 0.20 0.21 0.21 Guatemala 0.05 0.07 0.08 0.10 0.11 0.18 0.19 0.20 0.20 Other 1.41 1.46 1.39 1.49 1.43 1.37 1.41 1.44 1.49 TOTAL 8.32 8.17 8.85 8.01 8.90 9.45 10.050 10.460 11.220 SEX DETERMINATION ASSAY IN PAPAYA ACTA BOT. CROAT. 73 (2), 2014 293 the case of gynodieocious types, the hermaphrodite plants give pyriform fruits and the fe- male round fruits. However, from the marketing point of view, pyriform fruits are desirable. As the segregation in this case is also 1:1 for female to hermaphrodite, identifi cation at the early stage is desirable. Molecular markers have been studied earlier for sex determination in papaya (SONDUR et al. 1996, SOMSRI et al. 1998, PARASNIS et al. 1999, 2000, NIROSHINI et al. 2000, 2008, URA- SAKI et al. 2002a, b, DEPUTY et al. 2002, LEMOS et al. 2002). Recently a transcriptomics study has been conducted for sex determination in papaya (URASAKI et al. 2012). However, none of these studies validated the use of sequence characterized amplifi ed region (SCAR) mark- ers across a large population of dioecious and gynodieocious cultivars and a population with an intergeneric cross. In this paper the SCAR marker W11 (DEPUTY et al. 2002) was initially tested among di- oecious and gynodieoecious cultivars and was later used to determine the sex of 84 seed- lings of an intergeneric cross of Carica papaya × Vasconcella caulifl ora containing a mix- ture of male, female and hermaphrodite plants. Materials and methods A total of 98 papaya plants of which a known set of 14 cultivars comprising 6 dioecious and 8 gynodieocious plants (Tab. 2) and unknown set of 84 F1 seedlings of Carica papaya × Vasconcella caulifl ora maintained at the Indian Institute of Horticultural Research in Ban- galore were taken for the study. In the unknown set all the seedlings were marked to confi rm SCAR marker results when the seedlings produced their fi rst fl ower. Healthy and mature leaf tissue (2 g) was ground in liquid nitrogen until it formed a very fi ne powder, and ge- nomic DNA was extracted by using modifi ed CTAB (hexadecyltrimethylammonium bro- mide) method (DOYLE and DOYLE, 1990). Purifi ed DNA was quantifi ed using GeneQuant UV-spectrophotometer (GE Health Care Bio-sciences Ltd, U.K.) and diluted accordingly for further analysis. Tab. 2. List of dioecious and gynodioecious papaya (Carica papaya L.) varieties used for validation. Type Sample number Name of the variety and sex type Dioecious 1 2 3 4 5 6 Line 21 Male Line 21 Female Shilong Male Shilong Female Nigeria Male Nigeria Female Gynodioecious 7 8 9 10 11 12 13 14 Arka Prabhat Female Arka Prabhat Hermaphrodite Surya Hermaphrodite Surya Female Thailand Hermaphrodite Thailand Female Dwarf Lilly Female Dwarf Lilly Hermaphrodite CHATURVEDI K., BOMMISETTY P., PATTANAIK A., CHINNAIYAN V., RAMACHANDRA D. M., CHENNAREDDY A. 294 ACTA BOT. CROAT. 73 (2), 2014 PCR amplifi cation profi le was carried out in 25 mL volume containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM each dNTP, 0.5 mM each primer, 50 ng ge- nomic DNA and 0.6 units of TaqDNA polymerase (Bangalore Genei, Bangalore). Reactions were carried out in an Eppendorf mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany) using the following temperature profi le: an initial step of 5 min at 95 °C, 30 cy- cles of 1 min at 95 °C, 1 min at 58.5 °C and 1 min at 72 °C, and a fi nal step of 7 min at 72 °C. Amplifi cation products were screened by electrophoresis using 1.5% agarose gel. Results Diverse dioecious and gynodioecious papaya cultivars were initially screened for link- age of W11 to sex type. The reaction profi le of this SCAR marker assay was optimized by modifying the earlier mentioned protocol (DEPUTY et al. 2002) through a change of the con- centrations of PCR ingredients, such as a decrease in MgCl2 concentration, an increase in the concentration of dNTPs, primers, DNA and Taq polymerase. Amplifi ed fragment of 800 bp were obtained in hermaphrodite and male papaya plants only, but not in female plants. This amplifi cation pattern was confi rmed by initial screening and validated in true male, female and hermaphrodite plants of both dioecious and gynodioecious cultivars of Carica papaya and Visconcella caulifl ora (Fig. 1 a, b), which were used as controls for later valida- tion assay. Fig. 1. Amplifi cation pattern of W11 marker among male, female and hermaphrodite specimens of dioecious and gynodioe cious papaya plants.W11 scar marker giving an amplicon of 800 bp size only in male and hermaphrodite, but not in female plants; M – 1 kb ladder; a) from 1 to14: male, female and hermaphrodite papaya cultivars (refer to table 2 for details); b) 1 – ‘Surya’ (female), 2 – ‘Shillong’ (male), 3 – ‘Surya’ (hermaphrodite), 4 – V. caulifl ora (fe- male), 5 – V. caulifl ora (male). We used 84 F1 two month old seedling plants of an intergeneric cross of Carica papaya × Visconcella caulifl ora, information about the sex of which was unknown, for validation purpose. Genomic DNA was extracted from tender and healthy leaves of 10 day old leaves. These seedlings were maintained until adult stage and used for cross-verifi cation in the fi eld. Of 84 papaya seedlings, a screened amplifi ed 800 bp fragment was obtained in only three SEX DETERMINATION ASSAY IN PAPAYA ACTA BOT. CROAT. 73 (2), 2014 295 plants (results not shown) indicating that they might be either hermaphrodite or male, while those lacking 800 bp fragment were considered to be females. The analysis was repeated three times, confi rming the reproducibility of results. Internal controls were used during each step of the analysis. After fl owering we observed that the plants that showed the band and were tagged were all hermaphrodites and rest were females. Discussion Prior information about the sex type in papaya is necessary before an elite breeding program is planned. From studies on controlled pollinations over years (SINGH 1990), it was established that in the progenies of a cross whose parents are known (pure line), the propor- tion of males and bisexual or female follows a defi nite ratio. In the dioecious types, if fl ow- ers of female plants are pollinated with the fl ower of male plants the progenies obtained will have approximately 50% males and 50% females. In the gynodioecious types where either bisexual plants are selfed or the female is cross pollinated by bisexual fl owers, the progenies obtained will be 50% females and 50% hermaphrodites. The gynodioecious types are better in fruit production as both female and hermaphrodite plants produce fruits, while in dioecious types only female plants produces fruits. Previous studies have provided preliminary data indicating that random amplifi ed poly- morphic DNA (RAPD) markers might be useful for detecting sex expression in papaya (SONDUR et al. 1996, SOMSRI et al. 1998, NIROSHINI et al. 2000, 2008, PARASNIS et al. 2000, LEMOS et al. 2002, URASAKI et al. 2002a, b), but these reports utilized a relatively small number of plants. However, DEPUTY et al. (2002) reported SCAR markers that are specifi c for male and hermaphrodite plants in a large number of plants. DEPUTY et al. (2002) cloned three RAPD-PCR products showing linkage to the gene that determines fl ower sex, Sex1, in papaya. Two of these RAPD products, T12 and T1, had been previously mapped to 7 cM fl anking the Sex1 gene. The third product W11, which we validated, was chosen because W11 is closer to Sex1 than either T12 or T1. SCAR W11 and SCAR T12 were mapped in a papaya ‘Sun-Up’ and ‘Kapoho’ cross. Both of these cultivars are of the Hawaiian type. All Hawaiian types have previously been shown to be quite similar at the DNA level (STILES et al.1993). SCAR W11 and SCAR T12 showed linkage in all 182 plants, indicating these markers are within 0.3 cM of Sex1. There were no crossovers between SCAR T12 and Sex1 in the 182 plants, indicating a linkage signifi cantly closer than the 7.0 cM previously reported by SONDUR et al. (1996). SCAR W11 produced products almost exclusively in males and hermaphrodites but not in females; however, it is not clear at this time whether the difference is the result of a lim- ited number of base changes in the SCAR binding sites or more substantial alterations such as deletions of the binding sites or even the entire regions. In the present study the W11 SCAR marker validated in the cultivars of Carica papaya and Visconcella caulifl ora, am- plifi ed a discriminating band in hermaphrodites and male papaya plants, but not in females, (Figs. 1 a, b). The reason may be that the specifi c chromosomal region for sex type determi- nation in papaya shares an identical segment of the Y chromosome of males and hermaph- rodite plants and is absent in females. This result is consistent with the expected result and as reported by DEPUTY et al. (2002). LIU et al. (2004) identifi ed the male specifi c (MSY) re- gion in hermaphrodite and male papaya plants and found that some sequences of this region CHATURVEDI K., BOMMISETTY P., PATTANAIK A., CHINNAIYAN V., RAMACHANDRA D. M., CHENNAREDDY A. 296 ACTA BOT. CROAT. 73 (2), 2014 are common in both male and hermaphrodite plants. In our studies the fragments of 800 bp shared identical sequences between male and hermaphrodite type papaya, and this may support the above study (data not shown). Detection of sex linked RAPD markers as well as the SCAR markers have been at- tempted in several dioecious species. Thirty two male-specifi c RAPD bands were identifi ed in hop (Humulus lupulus L.) by the screening of 900 random primers (POLLEY et al. 1997). Others found one RAPD fragment of 400 bp size, closely linked with the male sex type of hemp (Cannabis sativa L.) (MANDOLINO et al. 1999). The pointed gourd (Trichosanthes dio- ica Roxb.) has also been studied and found to have a RAPD marker associated with fe- males that is absent in all male plants (SINGH et al. 2002). Similarly, the presence of a fe- male-specifi c band in nutmeg (Myristica fragrans Houtt.) has also been reported by the screening of 60 operon primers (SHIBU et al. 2000). Others have detected two RAPD mark- ers linked to M locus (maleness) in asparagus (Asparagus offi cinalis L.) and successfully converted one of these bands to a SCAR marker (JIANG and SINK 1997). The results illustrate the possibility of developing a molecular marker based method to identify sex at seedling stage in other agriculturally important dioecious plants including nutmeg (M. fragrans Houtt.), hemp (C. sativa L.), pistachio (Pistacia vera L.), kiwifruit (Actinidia chinensis P.), asparagus (A. offi cinalis L.). Farming of these crops could greatly benefi t from the development of methods for sex detection at an early stage as a suffi ciently larger number of productive hermaphrodite or female (depending on the market preference) plants could be cultivated by minimizing the number of unproductive male trees. In our study the assay kit has been successfully used to identify the hermaphrodite plants in the segregating population (F1) of Carica papaya × Vasconcellea caulifl ora, which was developed for papaya ring spot virus (PRSV) resistance. The results of this molecular based detection of sex type in papaya were in congruent with the morphological observa- tions among 84 F1 papaya plants. The three plants that showed an amplifi cation of the 800 bp fragment turned out to be hermaphrodites while the remaining 81 plants were females. Thus the present assay clearly identifi ed the true sex type in the seedling stage of the papa- ya plants. This assay is more like a double blind test where the plants were marked before fl owering as per our test and validated once the fl owering occurred. It saved time and also increased the confi dence level of the marker. This can go a long way in reducing the period taken for developing resistant lines for PRSV. In the intergeneric hybridization, the female parent used was gynodioecious Carica papaya and the male was the dioecious type Vasconcellea caulifl ora. The sex of the segre- gants if identifi ed in the F1 or in F2 generation after screening would help in reducing the population size for further sib mating, which would be easily manageable. References DEPUTY, J. C., MING, R., MA, H., LIU, Z., FITCH, M. M. M., WANG, M., MANSHARDT, R., STILES, J. I., 2002: Molecular markers for sex determination in papaya (Carica papaya L.). Theoretical and Applied Genetics 106, 107–111. DOYLE, J. J., DOYLE, J. L., 1990: Isolation of plant DNA from fresh tissue. Focus 12, 13–15. 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