Introduction

Papaya (Carica papaya L.) is an edible tropical fruit crop which has several medicinal and nutritional benefits. It is diploid (2n=9), dicotyledonous plant with a small genome size of 372 Mbp (Arumuganathan and Earle, 1991; Damasceno et al., 2009; Araujo et al., 2010). It is believed to be originated from Central and South America. Papaya belongs to the order Brassicales and family Caricaceae. It is closely related to the genus Vasconcellea. It shared a common ancestor with the member of order Brassicales e.g. Arabidopsis thaliana (Ming et al., 2008). Papaya is short lived, semi-woody, herbaceous and perennial plant that can be grown upto 10 m in height and produces fruits in nine to ten months from germinating period. It exhibits palmately-lobed leaves and clustered at the top of plant (Morton, 1987; OECD, 2005).

Papaya fruit is found to be most nutritious and ranked first among 35 commonly used fruits according to the percentage of US recommended daily allowances for antioxidant vitamins (A, C and E), thiamine, folate, riboflavin, niacin, potassium, iron, calcium and fibre (Chandrika et al., 2003; Ming et al., 2008). It is low in calories, fat and sodium and contains no starch. The fresh fruit is mainly consumed but it is also used in drinks, jams and as a dried and crystallized fruit candy. The entire plant produces a proteolytic enzyme, papain (EC: 3.4.22.2) which is commonly used in food processing to tenderize meat, clarify beer and juice (chillproofing), produce chewing gum, and coagulate milk. Papain can also be used in wide range of medical applications such as to help in digestion, reduce swelling, in fever and in treatment of ulcers (Aravind et al., 2013). In addition, it is also utilized for making soap, shampoo, lotions, skin care products and toothpastes in pharmaceutical/chemical industries (Morton, 1987). It is used as important fruit model crop owing to their numerous seed production and small genome size.

Papaya is trioecious species with three sex types: male, female and hermaphrodite. Among these sex types, hermaphrodite plants are preferred for commercial cultivation in tropical regions due to their pyriform shaped fruits (Magdalitan and Mercado, 2003), while female plants are grown mainly for papain production (Parasnis et al., 1999, 2000). Male plants are not useful for economic purposes as they do not produced fruits and hence they should be removed from the field which increases production cost (Bedoya et al., 2007). However, sex type cannot be identified phenotypically at early seedling stages until the plant flowers (i.e. 3-4 months after germination). This process increased the cost, labor and waste time. In order to save this it is necessary for farmers that the sex type of this crop is identified before transplanting.

1 Sex Phenotypes and Floral Morphology

1.1 Types of flowers

Papaya is polygamous species and possesses three sex forms namely; female, male and hermaphrodite (Yu et al., 2008a). Flowers are slightly fragrant, fleshy and waxy, yellow to cream in color. Papaya flowers are basically characterized into three types: staminate flower, pistillate flower and hermaphrodite flower. They borne on cymose inflorescences and appears in the axils of the leaves. The inflorescence type varies according to the sex of the plants. Flowering occurs generally within 9-12 months after germination. Male trees are characterized with long inflorescence, bearing dozen of flowers having yellow in color, arise in clustered form and possess ten stamens without ovary (Figure 1 a, b). Female trees are characterized with short inflorescence having few flowers, are white or cream in color with large rounded superior ovary without stamens (Figure 1 c, d). Hermaphroditic trees are having short inflorescence, bearing bisexual flowers and functional ovary along with stamens (Figure 1 e, f). Hermaphrodite plants are further classified into four types: elongate, intermedia or carpelloid, pentandria and barren or sterile types. Elongate type is having ten stamens in two clusters, smooth and elongated functional ovary (Figure 1 g). Intermedia or carpelloid type possesses irregularly ridged ovary and two to ten mostly distorted stamens (Figure 1 h). Pentandria form is characterized with five stamens attached with the base of rounded ovary (Figure 1 i). Barren or sterile type is having ten functional stamens but the ovary aborts (Figure 1 j) (Ming et al., 2007; Silva et al., 2007).

1.2 Segregation ratio

The three sex types of papaya are inherited in unexpected ratios because male dominant alleles linked with a lethal factor (Table 1). These unexpected ratios have been becoming the topic of extensive studies. After the self-pollination of hermaphrodite plants, their seeds always segregate into ratio of 2:1 of hermaphrodites and females. If female trees were fertilized by pollen from a male tree, then seeds of female plants segregate at the male to female ratio of 1:1. A similar ratio of 1:1 hermaphrodite to female obtained when female fertilized by pollen from a hermaphrodite tree. When the males self-pollinated occasionally (in optimal growing conditions some male flowers do not undergo their carpel abortion and form fruits), then male trees occur at a ratio of 2 male: 1 female. When male pollen fertilizes the female organ of hermaphrodite trees, then a ratio of 1 male: 1 hermaphrodite: 1 female is obtained. Therefore, no male trees are produced, when hermaphrodites are self pollinated or when hermaphrodites are used as a pollen source to fertilize female trees.

1.3 Effect of genetics and environment on sex expression

Many reports have shown that genetic and environmental factors (including temperature, moisture etc.) influence the sex expression in hermaphrodite and male plants of papaya (Awada, 1958; Allan et al., 1987; Silva et al., 2007a), while the female flowers are most stable (Ming et al., 2007). Following genetic and environmental factors influence the sex expression in papaya:

(1) Genetic influence: It is reported that genetic influence of genotype has great effect on sex expression. Silva et al., (2007a) reported in segregating back cross 1 (BC1) papaya population where elongate type of hermaphrodite flower changes into carpelloid (6-9 stamens) and pentandric (5 stamens) owing to their genetic inheritance of the genotype. Ramos et al., (2011) similarly reported that the variation of hermaphrodite elongate type to carpelloid or pentandric form is mainly due to genetic component.

(2) Temperature: During winter, cool temperature changes hermaphrodite (elongate form) to a carpelloid or the pentandria form. Fusion of stamens to the ovary wall leads to the decrease in number of stamens. During summer, high temperature produces sterile/barren type flowers which ultimately lead to no fruit formation (Awada, 1958). Allan et al. (1987) reported that male trees also showed reversion of sterile staminate to elongate type hermaphrodite flowers under night temperatures of 12^◦C and short daylengths.

(3) Moisture and Nitrogen: In addition, both moisture and nitrogen also affect the sex expression of papaya. Carpelloid and pentandric are developed in the presence of high nitrogen level and excess soil moisture (Awada, 1957). Hermaphrodite trees produces barren type flowers in drought condition (Chan, 2009).

2 Importance of Sex Determination Study in Papaya

The study of sex type identification is valuable in papaya because sex of the papaya plant cannot be predicted morphologically at early seedling stages. Among the three sex types hermaphrodite plants are grown for its pyriform-shaped fruits that are preferred for consumption and female plants are important for commercial papain production, while male plants are non-desirous (Urasaki et al., 2002). However, female trees require presence of small number (6-10%) of male trees in the field for fruit production (Eustice et al., 2008). Papaya seeds produce seedlings of unidentified sex, therefore farmers have to remove the male plants from the field and leave the female or hermaphrodite plants on the basis of floral morphology which can be performed only after three to four months from germination (Ma et al., 2004). If the prediction of sex of papaya could be done at early seedling stage, then an expected male and female plants ratios (5% males: 95% females) would be maintained by removing excess male trees. This would save the resources such as space required for planting, fertilizers, water and labor etc. otherwise spent on these undesirable male trees. Prediction of papaya sex at seedling stage using morphological traits have been attempted by many researchers but success began to achieved with advancements in genomics, molecular tools and techniques. In this paper we have reviewed the genetics of sex determination and highlighted on several methods including morphological, cytological or isozyme and molecular markers based techniques for sex determination in papaya.

3 Molecular Genetics of Sex Chromosomes in Papaya

In papaya, females possess homogametic X chromosome (XX), while male and hermaphrodite have heterogametic XY and XY^h chromosomes, respectively. The sex inheritance in papaya depends on three alleles including a recessive “m” allele for females and a dominant “M” allele for males and “M^h” allele for hermaphrodites. Female (homozygous recessive; mm), male (heterozygous dominant; Mm) and hermaphrodite (heterozygous dominant; M^hm) are the three viable genotypes (Figure 2). The dominant homozygous combinations (M^hM^h, M^hM, and MM) would be lethal and therefore non-viable (Hofmeyr, 1967). It was assumed that dominant alleles M and M^h represent genetically inactive regions of “sex chromosomes” which slightly vary in their length and functional genes are lost in these regions. Therefore, these homozygous dominant genotype would be lethal, while Mm and M^hm would be remain viable because an “m” sex chromosome is present in each genotype.

Recently, Ming et al., (2007) proposed that two genes (stamen suppressing gene in female flower and carpel suppressor gene in male flower) play an important role for determination of sex forms. Stamen-suppressing gene as the name indicates causes abortion of stamen before or at the stage of initiation of stamen primordia in female flower. On the other hand, carpel suppressor or male fertility gene aborts the carpel at a later developmental stage in male flower.

3.1 Suppression of recombination around sex determination locus

High-density genetic linkage mapping of the papaya genome was done for the purpose of cloning of sex determination genes, which revealed severe recombination suppression around the sex determination locus (Ma et al., 2004). This mapping result validated the Storey’s (1953) hypothesis stating that the region containing sex determination genes behaves as a unique factor that does not undergo crossing over. A total of 1501 markers including 1498 Amplified fragment length polymorphism (AFLP), morphological sex type, fruit flesh color and the papaya ringspot virus coat protein markers were mapped onto 12 linkage groups (LGs) of papaya. Out of these 1501 mapped markers, 225 were found to be co-segregated with sex types. This linkage mapping has also demonstrated that the genomic region around the sex determination locus possess high polymorphism.

3.2 Physical mapping of papaya genome

A high-density genetic map of papaya was constructed utilizing bacterial artificial chromosome (BAC) end sequences derived microsatellite markers (Chen et al., 2007). This genetic map demonstrated that 12 LGs of papaya were covered a total of 707 markers, containing706 microsatellite loci and one morphological marker (fruit flesh color). These 12 LGs were made up of three minor and nine major LGs. The nine major LGs are equivalent to nine chromosomes of papaya. LG1, one of the members of nine LGs is largest and present the sex chromosome of papaya. The position of recombination suppression was observed around male specific Y chromosome portion (MSY) on LG1as well as at the centromere portion of other LGs. Segregation distortion was observed on two LGs of papaya, i) distortion on LG1 around the MSY was due to abortion of the homozygous YY genotype during post-zygotic selection at 25-50 days after pollination and, ii) distortion on LG6 was due to an unknown reason.

In another study, the physical map was linked with genetic map of papaya.  For integration purpose, a sequence-tagged high density genetic map and BAC end sequences were utilized. This study revealed that the location of recombination suppression is found across the genome as well as around the MSY on LG1.The size of recombination suppressed portion i.e. MSY on LG1 was predicted about 8-9 Mb on the basis of integrated genetic and physical mapping. The rate of recombination slowly rises as the distance from MSY portion increases and rapidly rise at location about 10 Mb away from MSY to seven-fold of the genome coverage, then decreased again. These rise and fall in rate of recombination in 10 Mb away from MSY and suppression of recombination in 8-9 Mb of MSY portion suggests that recombination rate in these portions gradually developed during the early stages of sex-chromosome evolution (Yu et al., 2009).

More recently, physical maps for MSY region (Gschwend et al., 2011), and the hermaphrodite-specific Y^h chromosome portion (HSY) and its X counterpart (Na et al., 2012) were constructed using BAC libraries. These physical mapping results are significant to study the early events occurring during evolution of sex chromosome, identify genes responsible for sex-determination and helpful in sequencing the sex specific regions of papaya.

3.3 Origin of sex chromosomes

The MSY portion possesses several types of chromosomal rearrangements such as insertions, deletions, inversions and duplications. Expansion was identified on two regions of the MSY which suggested that at the molecular level, homomorphic sex chromosomes (cytologically similar) are heteromorphic. The divergence between X and Y^h were estimated to be between  0.5 and 2.2 million years based on the gene found on HSY and X BACs, which indicates the recent origin of the papaya sex chromosomes (Yu et al., 2008b).

Yu et al. (2008a) studied the characteristics of dioecious X and Y BACs and a comparison was done in between the sequences of both dioecious and gynodioecious X, Y and Y^h BACs chromosomes. Several chromosomal rearrangements including insertions, duplications, deletions, and inversions were detected between the X and Y-specific BACs and expansion was predicted on the Y BAC due to suppression of recombination in this region. Both Y and Y^h-specific BACs shared high degree of sequence identity in DNA. X-specific BACs were found to be almost identical in both dioecious and gynodioecious. Y and Y^h chromosomes were diverged approximately 73,000 years ago which suggested that a common ancestral Y chromosome is responsible for the evolution of both Y and Y^h chromosomes.

3.4 Features of male-specific portion of the Y chromosome

A pachytene chromosome-based cytogenetic mapping of MSY was done to study the features of MSY. The MSY region constitute 13% portion of the Y chromosome. A high level of methylation and centromere is present within MSY of the Y chromosome. This methylation and centromere play a valuable role in silencing the gene and suppression of recombination in evolution of Y chromosome (Zhang et al., 2008).

3.5 Evolution of sex chromosomes

Expansion of both Y chromosome (Yu et al., 2008a) and X chromosome (Gschwend et al., 2012) occurs which is the common feature during early stages of evolution of sex chromosomes. The recently originated papaya X chromosome was compared to its homologous autosome in close relative monoecious Vasconcellea monoica for revealing the evolutionary history of the X chromosome. The V. monoica genome size (626 Mb) was found 41% larger than the papaya genome (372 Mb) which suggested the expansion in X chromosome of papaya. The reason behind the expansion of papaya X chromosome is due to the higher accumulation of repetitive sequences as compared to the autosomal sequence.

3.6 Gene responsible for sex determination

Urasaki et al., (2012) performed a digital transcriptome analysis to identify sex determining gene in papaya. This analysis was done by utilizing floral samples from male, female and hermaphrodite papaya plants. 312 unique tags were located to sex chromosomes (Y^h and X; most of them mapped on X chromosome), and 30 were mapped on both Y^h and X chromosomes. In addition, Y and Y^h chromosome-specific gene i.e. MAD-box gene was identified. It regulates the expression of other distant genes such as increased the expression of genes in the female or reduced the expression in the male which plays role in sex-determination.

3.7 Sequencing of sex chromosomes

The sequencing of the sex chromosomes HSY and its X counterpart were done. The main purpose of sequencing was to study the events occurring at the time of early stages of HSY and X chromosome evolution. This study revealed that HSY differs from X regions in respect of size of chromosomes, physical size of inversions and gene content etc. A high amount of retrotransposons are present in HSY portion, which makes HSY portion larger in size than X. HSY and X possess two main inversions namely, inversion 1 and 2. A similarity was found in the physical size of inversion 1 portion of HSY and X chromosome regions, as they possess similar amount of repetitive sequence (80.7% in HSY and 76.5% in X chromosome, respectively). Since recombination suppression accumulates high amount of repetitive sequences in HSY region (80.2% in HSY against 60.5% in the X) therefore the size of inversion 2 in HSY chromosome was more than twice that of X chromosome. A total of 16 transcripts-encoding sequences (nine HSY-specific genes and seven pseudogenes) were identified in HSY which is lesser in number as compared to X containing 28 transcript-encoding sequences (24 X-specific genes and four pseudogenes). In addition, the sequencing of sex determining regions HSY and corresponding X regions yields 8.1 Mb and 3.5 Mb pseudomolecules, respectively. The emergence of sex chromosomes were reported about 7.0 million years ago (Wang et al., 2012).

More recently, Vanburen et al. (2015) did the sequencing and resequencing of MSY and HSY portions using BAC-by-BAC approach. This study reported that high similarity in gene content was predicted in both MSY and HSY regions and differs from each other by only 0.4% sequences. Three different populations of Y chromosomes (MSY1, MSY2, and MSY3) were identified from wild papaya males. Findings revealed that the MSY1 and MSY2 haplotype groups were found from the two opposite coasts of Costa Rica. Third population of Y chromosome i.e. MSY3 as well as all HSY haplotype groups were identified from the north Pacific region of Costa Rica.

3.8 Sex-specific repeats

The sex chromosomes of papaya contain sex specific repeats. Twenty HSY-specific and one X-specific repeats were identified. The portion of HSY in which expansion occurs, contains HSY-specific repeats (from 2.0 to 4.0 and 5.0 to 7.5 Mb). Expansion in HSY occurs mostly due to accumulation of these sex-specific repeats. Both X and HSY possess highest amount of repeat Ty3-gypsy retrotransposons. X region contains 67.2% repetitive element, which is lower than HSY and Y chromosome. The importance of HSY and X-specific repeats may be in developing the molecular markers specific for sex identification (Na et al., 2014).

3.9 Role of sRNAs in sex determination

Aryal et al. (2014) analyzed small non-coding RNAs (sRNA) in the libraries prepared from female, male and hermaphrodite flowers of papaya. sRNA plays an significant role in gene silencing and DNA methylation, suggesting its involvement in sex differentiation in plants. lllumina libraries were made from the floral male, female, and hermaphrodite papaya plants for the study of sRNA reads. A total of 29 micro RNAs (miRNAs) were detected using these sRNA reads. In one library, a total of 65 miRNAs (34 new and 31 conserved miRNAs) were identified. From these miRNAs, only 14 miRNAswere differentially expressed among male, female and hermaphrodite flowers. Six miRNAs (miR160, miR167a, miR167b, miR169 and miR393) were expressed higher in papaya male flowers that regulate the genes in auxin signaling pathway which indicates that auxin plays a main role in carpel development. Two miRNAs (miR159 and miR166) were expressed higher in female flowers, which played an important role in regulating the genes responsible for floral meristem identity and the embryo patterning. Four miRNAs (miR156a, miR156b, miR168b and miR_novel_39) were expressed higher in hermaphrodite and male flowers. Two miRNAs (miR171 and miR394) were expressed higher in female and male flowers. The results indicate potential role of these sRNAs in papaya sex determination.

4 Papaya Genome Sequencing

A draft genome sequence of papaya was generated from a SunUP female plant using the whole-genome shotgun approach with Sanger method. It was assembled into 271Mb contigs and unassembled portion may contain repetitive sequences. Papaya genome contains 24,476 genes with average gene length 2,373 bp. Total 35.5% GC content is present in the genome and average intron length is 479 bp (Ming et al., 2008). Papaya genome sequences could be is a valuable source to study the mechanism of sex determination at molecular level.

5 Methods for Identification of Sex Types

Papaya sex identification at early plant development stage has been a problem since long back which led to the researchers to develop many marker techniques such as, (1) morphology based, (2) biochemical markers, (3) polymerase chain reaction (PCR)-based markers and (4) sequencing-based markers (Figure 3). Morphological methods differentiate the sex types on the basis of traits such as leaf or root morphology, rate of growth and seed coat color etc. (Reddy et al., 2012; Demandante et al., 2014). Several other cytological methods (Datta, 1971) and Isozyme (biochemical) markers have been used for sex type identification (Sriprasertsak et al., 1988). Molecular markers are divided into two types (i) PCR-based molecular markers and (ii) sequence-based molecular markers. PCR based molecular markers includes: (a) Random amplified polymorphic DNA (RAPD), (b) AFLP, (c) Inter simple sequence repeat (ISSR), and sequence-based molecular markers includes (a) simple sequence repeats (SSR), and (b) single nucleotide polymorphism (SNP).

5.1 Morphological identification

Morphological and cytological studies were the first markers to be used for early diagnosis of gender in papaya. Some morphological traits such as color of seed coat and shape or morphology of root have been linked with the sex type of papaya. Females are presumed to possess a light color seed coat and branched root shape, while males have dark seed coat and straight root shape. However, these studies have not been verified scientifically for sex type identification (Magdalita and Mercado, 2003). A morphological study was done to determine the maleness or femaleness in papaya based on the leaf morphology and rate of growth. It revealed that the male exhibit more number of three lobed leaves with slow growth rate whereas female seedlings show more vigor and faster growth and are abundant in five lobed leaves (Reddy et al., 2012). Demandante et al., (2014) identified the sex types of papaya based on morphology of leaf shape using Elliptic Fourier Analysis (EFA). The distribution of leaf shape was determined by making a scatter plot using the principle components of papaya male, female and hermaphrodite plants leaves. This analysis showed a little orientation from central axis of principal components in male while, in female and hermaphrodite leaf shape was found to be distant away from the central axis of first two principal components. Another test, Kruskal-Wall was failed to show much change in the leaf shape distribution of papaya different sex types.

5.2 Cytological identification

Several cytological studies have also been done to identify either any chromatin body or presence of a heteromorphic pair of chromosome in papaya which could help in identifying the different sex forms. However, these studies failed due to none of the above have been identified (Datta, 1971).

5.3 Isozymes markers

Isozymes are isoforms of single enzyme that differs in amino acid sequences yet catalyzed the same chemical reaction (Markert and Moller, 1959). Alteration in amino acid occurs due to mutation in DNA which changes the net electric charge of the protein. Electrophoresis techniques can be used to detect such differences in ionic charge and size of the protein and resolved using enzyme specific stains which results in a small number of specific bands. Isozyme markers are co-dominant in nature. Cationic peroxidase isozyme was used for gender determination in papaya; males could be differentiated from females on the basis of banding pattern. However, females failed to differentiate from hermaphrodites (Sriprasertsak et al., 1988). Isozyme has some limitations for sex type identification such as post transcriptional modification, affected by environmental conditions; their expression varies from tissue to tissue. The failure of morphological traits study, cytological evidences (Parasnis et al., 1999, 2000; Magdalitan et al., 2003; Gangopadhyay et al., 2007) and isozyme markers to identify or differentiating the different sex forms of papaya at the early juvenile seedling stage has encouraged for the development and utilization of PCR-based molecular markers.

5.4 PCR-based gender-linked markers

Mullis and Faloona (1987) discovered polymerase chain reaction (PCR) technology that leads to the development of many novel fingerprinting techniques. In PCR-based methods, only one primer or a primer pairs are used for DNA amplification reaction. These PCR based markers which are also termed second generation markers like RAPD, SCARs, ISSRs, AFLPs, SSRs and third generation marker like SNPs are advantageous over the first-generation markers [hybridization-based markers; e.g. restriction fragment length polymorphism (RFLP)], as they require much less DNA (10-100 ng) of relatively lower quality, avoid DNA blotting and use of radioactivity, amenable to automation and are much more user-friendly. RAPD (Welsh and McClelland, 1990;William et al., 1990),AFLP (Vos et al.,1995), ISSR (Zietkiewicz et al., 1994), SSR (Akkaya et al., 1992) and SNP (Jordan and Humphries, 1994) are most commonly used PCR based DNA marker techniques that have been used to develop gender/sex-linked markers in papaya and in various other dioecious taxa.

5.4.1 Random amplified DNA polymorphism (RAPD)

Welsh and McClelland, (1990) and William et al., (1990) independently developed a new PCR based marker technique called arbitrarily primed polymerase chain reaction (AP-PCR) or random amplified polymorphic DNA (RAPD) technique, respectively. This technique utilizes short synthetic oligonucleotides (usually 10 bases long) primer of random sequence. It is simple, cheap and no prior sequence information of template DNA is required. It is dominant marker (scored as either ‘present’ or ‘absent’); showing high levels of polymorphism and required a small quantity of DNA (Jiang, 2013). RAPD is most popular marker system for sex determination in papaya.

Several RAPD based sex-specific markers have been generated in C. papaya (Table 2). Magdalita and Mercado (2003) used two 20mer primer pairs to predict the sex type in three papaya varieties (‘Cavite’, ‘Cariflora’ and ‘Sinta’ hybrid). Females produced a single band of 0.8 kb; hermaphrodites produced two distinct bands of 1.3 kb and 0.8 kb, while males had no band. The frequency of males, hermaphrodites and females identified as such both by field observation and PCR, showed 100% accuracy in the prediction. Bedoya and Nuenz (2007) developed sex-linked RAPD marker in Colombian dioecious genotypes of C. papaya. Results demonstrated in this study that a RAPD marker OPY7 (900 bp) was found in male plants but not in females or hermaphrodites. In another study, OPC09 (1.7 kb) and OPE03 (0.4 kb) markers were identified in male and hermaphrodite plants whereas OPE19 (2.18 kb) in female plants (Niroshini et al., 2008). Many other male specific RAPD markers were developed such as OPF2 (800 bp) and OPY7 (369 bp) (Parasnis et al., 2000; Shivkumar et al., 2014).

Despite these, RAPD have some limitations such as high sensitivity to variations in PCR amplification conditions resulting low reproducibility and reveals only homology (Mishra et al., 2014).

5.4.2 Sequence characterized amplified region (SCAR)

Due to high sensitivity of RAPD to variations in PCR amplification conditions, these markers are converted into more reliable and stable marker termed specific sequence characterized amplified region (SCAR; Paran and Michelmore, 1993). SCAR markers are sequence specific, highly reproducible and simple to use. These are developed by cloning the amplified bands of RAPD, then sequencing their ends. The sequence information is used to design forward and reverse SCAR primers (22-24 nucleotides long) and amplified using PCR followed by bands visualized on agarose gel. Polymorphisms either detected as length polymorphisms (co-dominant) or as presence or absence of amplified band (dominant; Singh and Singh, 2015).

Several male-hermaphrodite specific RAPD markers were developed into SCAR markers (Deputy et al., 2002; Lemos et al., 2002). Urasaki et al., (2002) identified a 450 bp fragment, named PSDM (Papaya Sex Determination Marker) in all male but not in the female plants. From this RAPD marker a SCAR was developed that amplified fragments from the genomes of male and hermaphrodite plants, but not the female ones. A SCAR marker was also developed from RAPD marker OPY7 (900 bp) to differentiate plants of hermaphrodite and male from female plants in Colombian papaya genotype (Bedoya and Nuenz, 2007). Results indicated that sequences utilized for the development of SCAR marker is present on Y chromosome. Chaturvedi et al. (2014) have validated the SCAR marker W11 among different cultivars of dioecious and gynodioecious papaya genotypes (Table 2).

5.4.3 Inter Simple Sequence Repeat (ISSR)

ISSR (Zietkiewicz et al., 1994) is PCR based DNA fingerprinting technique utilizes single primer that contains microsatellite sequences, usually 15-30 nucleotide long and amplifies regions between adjacent, inversely oriented SSR-microsatellites (Gupta et al., 1996). There are two types of primer, non-anchored primer consists of microsatellite sequences and anchored primer containing microsatellite sequences in addition usually, two nucleotides long) arbitrary sequence either at the 3′ or 5′end of the primer. ISSR marker offers several advantages such as no prior sequence data is required for primer synthesis, dominant marker and low quantity of DNA (5-50ng/reaction) are needed (Singh and Singh, 2015).

ISSR markers have been employed for sex identification in various dioecious plants (Table 2). A 500 bp band was observed in solo group (SS72/12) particularly in hermaphrodite plants of papaya and further investigations were done to validate these results. A marker around 500 bp was found co-segregating with sex in three genotypes viz., Solo group (SS72/12), hybrid (Tainung H), and Formosa group (Tailândia) (Da Costa et al., 2011). Gangopdhayay et al. (2007) utilized three microsatellite probes (CAG)₅, (GACA)₄ and (CAA)₅ for sex-identification in papaya. Out of three primers, only primer (GACA)₄ generated one female-specific band which was detected in all female and hermaphrodite plants. Parasnis et al. (1999) utilized (GATA)₄ microsatellite probe which generated a 5 kb male-specific band. Papaya Y chromosome is morphologically identical to the X chromosome. To study this, probes, such as (GAA)₆ and (GATA)₄ were utilized. But, the differences were observed in the plants of male and female at molecular level. This indicates that the way of divergence occurs between the genetic material of papaya chromosomes X and Y is sex-specific. Due to some disadvantages of ISSR such as low reproducibility and limited number of bands generated and labour involved during analysis process makes this marker system less interesting among researchers for sex-identification in plants.

5.4.4 Amplified fragment length polymorphism (AFLP)

A novel PCR based technique was developed by Vos et al. (1995) which involves PCR amplification of restriction fragment of sample DNA. This technique is used for generating fingerprints of DNA of any origin or complexity. It is highly efficient marker offers many advantages such as high reproducibility, high level of polymorphism, high genomic abundance, detects multiple loci and no prior knowledge of sequence information is required. Polymorphism is analyzed on the basis of presence and absence of restriction fragments (Mishra et al., 2014).

No AFLP marker is still available for sex determination in C. papaya, but it has been utilized in several other plant species (Table 2). Wang et al. (2011) utilized 64 pairs of AFLP primer combinations to develop sex-specific AFLP markers in Eucommia ulmoides Oliv. One male-specific marker (350 bp) was generated from E-ACA/M-CTT primer combination. Further, a 247 bp SCAR marker was developed by utilizing this 350 bp male-specific AFLP marker. Male-specific AFLP markers have also been identified in other plants species including hemp (Flachowsky et al., 2001), Broussonetia papyrifera (Lianjun et al., 2012), whereas two male-specific markers of 525 bp and 325 bp and a female-specific marker of 270 bp have been identified in Simmondsia chinensis (Agarwal et al., 2011). The AFLP method is rarely used for early sex diagnosis of seedlings among plants due to some drawbacks such as high cost, more time consuming and laborious analysis method.

5.5 Sequence-based molecular markers

5.5.1 Simple Sequence Repeat (SSR)

SSRs are consisting of one to six (bp) tandem repeats (mono-, di-, tri-, tetra and penta-, hexanucleotides), and are found throughout all genomes including prokaryotes (Li et al., 2004; Thiel et al., 2003). They are also termed as simple sequence length polymorphisms (SSLPs; Tautz, 1989), microsatellite (Litt and latty, 1989), short tandem repeats (STRs; Edwards et al., 1991).They are found in both coding and non-coding regions (Toth et al., 2000). They are more valuable molecular marker than other PCR-based markers like RAPD, ISSR and AFLP due to their sequence-specificity, multiallelic nature, co-dominant inheritance, abundance in the genome, high rate of transferability, high level of polymorphism and reproducibility (Powell et al., 1996; Zane et al., 2002; Theil et al., 2003). In addition, it does not required high quality of DNA and performs well with low quantity of template DNA (10-100ng/reaction). The polymorphic nature of SSR was observed by Litt and Luty (1989). The length polymorphism of SSR is generated due to variation in repeats number (Ellegren, 2004).The variations in these repeats occur due to slippage of strand which creates mispairing (Levinson and Gutman, 1987) and repetitive errors generated at the period of replication of DNA (Schlotterer and Tautz, 1992; Kattiet al., 2001), or unequal crossing-over between sister chromatids during meiosis (Innan et al., 1997). The principle of polymorphism detection involves the designing of primers from flanking sequences near the portion of microsatellite repeat motif. Amplification is performed using PCR and running agarose or denaturing polyacrylamide gel for visualization of variations in alleles. There are two types of SSRs on the basis of their location: (1) SSRs that are distributed throughout the genome are called genomic-SSRs, (2) SSRs that are found only within genes (i.e. inside exons, exon-intron junctions or introns) are called as genic-SSRs or Expressed Sequence Tags-SSRs (EST-SSRs).

With the advancement of functional genomics a large numbers of ESTs and other DNA sequences of various organisms are available in various data banks. Availability of these large amounts of freely accessible data led to the development of EST-based SSR markers through data mining. Development of EST-SSRs or genic-SSRs in silico has become a fast, efficient, and relatively inexpensive method compared with the development of genomic-SSRs (Gupta et al., 2003; Senan et al., 2014). Genic-SSRs act as functional markers owing to their origin from expressed portion of genome. They possess several advantages such as ease of use, less time consuming, cheapest to develop, occurrence in expressed portion, sequence-specificity and high rate of transferability i.e. the ability to effectively transfer SSR markers across species and genera so they provide the better estimate of polymorphism (Gupta et al., 2003). Genic-SSRs can also be used for comparative genomics study. EST-SSRs developed for one species can be utilized for the related plant species for which small amount of data on ESTs and SSRs is available in public databases by identifying rate of transferability in these species. It is believed that EST-SSRs in the genetic maps revealed about the distribution of genes along the genetic map. They can also be used for comparative mapping study (comparing the gene order of identical genes) in related plant species owing to their origin from conserved region of the genome (Varshney et al., 2005).

One report is available for sex determination using microsatellite system in papaya. Chiu et al. (2015) analyzed the sex characters in all hermaphrodite cultivar (Taichun Sunrise; TS) and typical hermaphrodite cultivar (Taiwan Seed Station No.7; T7) of papaya and their F1 progeny using SSR markers. They performed SSR analysis of three mating combinations, TS x TS, and TS x T7 and T7 x T7 and their F1 plants to check the segregation pattern of papaya sex. The first two mating combinations i.e., TS x TS, and TS x T7 yielded 2:1 ratio of hermaphrodites vs dioecious females offspring which was supported by chi-square test but T7 x T7, resulted in all F1 plants hermaphrodite with no females. On the basis of the results obtained they concluded that a lethal recessive gene could be linked to T7 cultivar and this lethal allele causes the fatality in female offspring.

SSR markers have also been developed in several other plants such as in hemp (Rode et al., 2005), hop (Jakse et al., 2008) and date palm (Elmeer et al., 2012; Maryam et al., 2016). Rode et al., (2005) identified first time sex-linked SSR markers in hemp. Ten SSRs were found to be polymorphic in population 00/50. Out of these, three SSR markers CS301, CS308 and CS501 were identified as sex-linked markers. In CS308 SSR marker, three different marker alleles were detected, 185 bp and 192 bp in the male parent and 183 bp and 185 bp in the female parent, respectively. Jakse et al., (2008) utilized microsatellite marker HlAGA7 which produced an allele of 165 bp size in all males, it indicated a tight linkage between male characters in hop. Two groups (Elmeer et al., 2012; Maryam et al., 2016) utilized microsatellite markers to differentiate between male and female in date palm. First group reported that the primer mPdCIR048 produced one locus with the size of 160/190 bp reoccurred in 4 male samples but not detected in any of the female samples. Similarly second group observed that SSR Primer mpdCIR48 produced a specific locus (250/250) in all male samples only. Primer DP-168 produced a locus of 300/310 bp reoccurred in 5 date palm male samples, which indicated that these are potential markers to identify sex at early seedling stages in date palm. SSR marker utilization in other plant species highlighted that these marker could be developed to identify sex types in papaya (Table 2).

5.5.2 Single nucleotide polymorphism (SNP)

SNP is new generation marker based on the principle of the single nucleotide change (A, T, C or G) in DNA sequences of different individuals of species of genome. They are commonly present in animals and plants. The frequency range of SNP is one SNP every 100-300 bp in plants. Distribution of SNP in coding and non-coding region of genomes is hetergenous. They are generated by either transition: purine to purine or pyrimidine to pyrimidine exchanges (A or G to C or T and vice-versa) or transversion: purine to pyrimidine or pyrimidine to purine exchanges (A or T to C or T and vice-versa). They possess several advantages such as co-dominant and biallelic nature, often linked to gene, highly polymorphic and showing high reproducibility which makes them highly efficient marker system over RAPD, ISSR and AFLP (Jiang, 2013). These markers are evolutionarily stable due to low mutation rate. Polymorphism in SNPs arises due to insertion and deletion with respect to single base in the genome. They cannot be resolved by conventional methods like agarose, and polyacrylamide gel electrophoresis. Their detection includes sequenced genomes and next-generation sequencing technologies (Martin et al., 2010), capillary electrophoresis (Drabovich et al., 2006) and mass spectrometry (Griffin et al., 2000) etc. They are important in detection of functional polymorphism if present in coding region because change in amino acid sequence resulting altered phenotype (Singh and Singh, 2015).

No SNP study has been done yet in papaya for sex identification, but recently sex-linked SNP marker was identified in Pistacia vera (Kafkas et al., 2015) using restriction site-associated DNA (RAD) sequencing (Table 2). Thirty eight putative sex-linked SNP markers were produced from 28 reads by RAD sequencing and further validation of these sex-linked markers were done by SNaPshot analysis. This study demonstrated that eight SNP loci could effectively differentiate sex types. Further, high-resolution melting (HRM) analysis along with real-time PCR was done by utilizing these eight SNP loci. Out of these eight SNP loci, only four SNP loci (SNP-PIS-133396, SNP-PIS-136404, SNP-PIS-167992, and SNP-PIS-174431) were successfully separating sex in all 166Pistacia plants. Similarly, SNP could be utilized for identification of gender in C. papaya due to their abundance in the genome and highly polymorphic nature.

Conclusion

The purpose of present article is to reflect the genetics of sex determination of economically and medicinally important papaya fruit crop and to know the current approaches employed for identification of sex at juvenile stage in papaya. Genetics study highlighted the characteristics of papaya sex chromosome including expansion of X and Y chromosome, events occurring during sex chromosome evolution and sex determination genes which provides the better understanding of sex determination mechanism in papaya. Several studies based on morphological, cytological characters and biochemical markers have been developed for sex type identification in papaya but none of them is found to be reliable yet. Hence, molecular methods provides important tool for good and easy identification of sex type at any stage of growth and development. This review provides a comprehensive view on the wide range of sex linked molecular markers identified in papaya. Although sex linked RAPD, ISSR and AFLP markers have been identified but these are not being utilized commercially for sex identification in papaya due some limitations. Hence at present markers relevant to sex-determination in papaya are still limited. Therefore, more precise, reliable, cost-effective, highly reproducible and relatively faster molecular markers are needed for sex type identification at juvenile stage. SSRs and SNPs are providing a good opportunity to develop sex-linked markers for sex identification in papaya as both have several advantages such as highly abundant, less time consuming, rapid and cost effective genotyping.

Acknowledgements

The financial assistance in the form of Fast Track Research Project on papaya sex determination sanctioned by Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India, New Delhi and Junior Research Fellowship (to Priyanka) by Department of Biotechnology (DBT), Government of India, New Delhi is gratefully acknowledged.

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