Genetics, Vol. 166, 419-436, January 2004, Copyright © 2004

High-Density Linkage Mapping Revealed Suppression of Recombination at the Sex Determination Locus in Papaya

Hao Maa,b, Paul H. Moorec, Zhiyong Liub, Minna S. Kimd, Qingyi Yua,b, Maureen M. M. Fitchc, Terry Sekiokad, Andrew H. Patersone, and Ray Mingb
a Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii 96822,
b Hawaii Agriculture Research Center, Aiea, Hawaii 96701,
c U.S. Department of Agriculture-ARS, Pacific Basin Agricultural Research Center, Aiea, Hawaii 96701,
d Department of Tropical Plant and Soil Sciences, University of Hawaii, Honolulu, Hawaii 96822
e Applied Genetic Technology Center, University of Georgia, Athens, Georgia 30602

Corresponding author: Ray Ming, 99-193 Aiea Heights Dr., Suite 300, Aiea, HI 96701., rming{at}harc-hspa.com (E-mail)

Communicating editor: J. A. BIRCHLER


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

A high-density genetic map of papaya (Carica papaya L.) was constructed using 54 F2 plants derived from cultivars Kapoho and SunUp with 1501 markers, including 1498 amplified fragment length polymorphism (AFLP) markers, the papaya ringspot virus coat protein marker, morphological sex type, and fruit flesh color. These markers were mapped into 12 linkage groups at a LOD score of 5.0 and recombination frequency of 0.25. The 12 major linkage groups covered a total length of 3294.2 cM, with an average distance of 2.2 cM between adjacent markers. This map revealed severe suppression of recombination around the sex determination locus with a total of 225 markers cosegregating with sex types. The cytosine bases were highly methylated in this region on the basis of the distribution of methylation-sensitive and -insensitive markers. This high-density genetic map is essential for cloning of specific genes of interest such as the sex determination gene and for the integration of genetic and physical maps of papaya.

PAPAYA (Carica papaya) is a member of the family Caricaceae that is the flowering plant family closest to the family Brassicaceae (which includes Arabidopsis, whose genome is completely sequenced) (BREMER et al. 1998 Down; ARABIDOPSIS GENOME INITIATIVE 2000). Papaya is a widely cultivated fruit crop in tropical and subtropical regions worldwide. It is believed to be native to tropical America where it has undergone a long period of selection (STOREY 1976 Down). Papaya is polygamous with three basic sex types: female, male, and hermaphrodite. Hermaphrodite trees produce a pyriform-shaped fruit that is preferred in the market. However, seeds from hermaphrodite trees always segregate into hermaphrodites and females at the ratio of 2:1 and the sex types of the plants can be determined only by inspection of the flowers. Therefore, it is a general practice for farmers to plant three to five seedlings in one hill, allowing them to grow for 4 to 6 months until the sex types are identified, and then to remove the undesired plants to develop the orchards with only hermaphrodite plants.

On the basis of segregation ratios from crosses among three sex types, STOREY 1938 Down and HOFMEYR 1938 Down proposed that sex determination in papaya is controlled by a single gene with three alleles: M, Mh, and m. Male individuals (Mm) and hermaphrodite individuals (Mh m) are heterozygous, whereas female individuals (mm) are homozygous recessive. The dominant combinations of MM, MhMh, and MMh are lethal, resulting in a 2:1 segregation of dominant markers on the linkage group (LG) where the sex determination gene is located. Later, STOREY 1953 Down revised the hypothesis to state that sex is determined not by one gene, but rather by a complex of genes that are confined to a small region on the sex chromosome within which crossing over is precluded. The different segments in this region are so closely linked together that they behave as unit factors. HOFMEYR 1967 Down proposed that the symbols M1 (M) and M2 (Mh) represent inactivated regions of slightly different lengths from which vital genes are missing. On the basis of interspecific hybridization in Caricaceae, HOROVITZ and JIMENEZ 1967 Down proposed that the sex determination is of XX-XY type. The genotype of the male is XY, female XX, and hermaphrodite XY2, where Y2 is the modified Y chromosome. The Y chromosome has a lethal region and the Y2 chromosome preserves this lethal region. A more recent modification of the model proposed to explain the papaya sex expression is that the three alleles encode different trans-acting factors to direct the expression of the different flower forms (SONDUR et al. 1996 Down).

Papaya is diploid with nine pairs of chromosomes and a small genome size of 372 Mbp (STOREY 1941 Down; ARUMUGANATHAN and EARLE 1991 Down). Over the past 10 years, three major advancements have enhanced papaya genetic and genomic research:

  1. Transgenic improvement. Papaya transformed with the papaya ringspot virus coat protein (PRSVCO) gene was successfully developed and released to save Hawaii's papaya industry from collapse because of susceptibility to papaya ringspot virus disease (FITCH et al. 1992 Down; GONSALVES 1998 Down).

  2. Sex-linked DNA markers. Four sequence-characterized amplified region (SCAR) markers tightly linked to sex forms were developed (PARASNIS et al. 2000 Down; DEPUTY et al. 2002 Down; URASAKI et al. 2002 Down) as ways to sex the plant prior to flowering since known sex-linked vegetative characters are too far from the sex-determining locus to be of practical use. These DNA sex markers are now used in selection of desired sex types at the seedling stage for more efficient papaya production (DEPUTY et al. 2002 Down).

  3. The genetic linkage map. The first genetic map was reported more than 60 years ago and consisted of only three morphological markers: sex form, flower color, and stem color (HOFMEYR 1939 Down). In 1996, SONDUR et al. 1996 Down developed a second map based on 62 randomly amplified polymorphic DNA (RAPD) markers and mapped the sex determination gene on linkage group 1.

Considerable efforts have gone into the genetic mapping of many plant species. High-density genetic mapping not only is the first step toward isolating and cloning the genes of interest via chromosome walking or chromosome landing but also is an important tool for genomic dissection, marker-assisted selection, comparative analysis of the plant genomes, and integration of genetic, physical, and cytomolecular maps (MARTIN et al. 1993 Down; TANKSLEY et al. 1995 Down; KLEIN et al. 2000 Down; PATERSON et al. 2000 Down; DRAYE et al. 2001 Down). Genetic linkage maps have been reported for many plant species over the last 15 years. However, high-resolution genetic maps, which are essential for genomic studies, have been limited to the model plant Arabidopsis (PETERS et al. 2001 Down) and to major crop species, such as maize (DAVIS et al. 1999 Down), rice (HARUSHIMA et al. 1998 Down; WU et al. 2002 Down), tomato, potato (TANKSLEY et al. 1992 Down; HAANSTRA et al. 1999 Down), wheat (BOYKO et al. 2002 Down), soybean (KEIM et al. 1997 Down), and rapeseed (LOMBARD and DELOURME 2001 Down).

Among the crop species, the genetic mapping of papaya lagged behind that of many other plant species, due partly to the low level of polymorphism among existing germplasm (SHARON et al. 1992 Down; STILES et al. 1993 Down; KIM et al. 2002 Down). The objective of this study was to develop a high-density genetic map of papaya and to characterize its sex locus. This map will serve as the first and essential step for conducting extensive genomic research on this crop.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Plant materials:
The F2 mapping population was grown at the Kunia substation on Oahu, Hawaii, along with the parents Kapoho, SunUp, and a set of F1 plants. Plant tissues from young leaves of 34 hermaphrodite and 20 female plants were collected for DNA isolation.

AFLP analysis:
DNA extraction protocol used to extract the papaya DNA was described previously (STEIGER et al. 2002 Down). Amplified fragment length polymorphism (AFLP) analyses were performed on a Li-Cor IR2 automated DNA sequencer (Li-Cor, Lincoln, NE) with selective amplification prepared from fluorescent-labeled EcoRI or PstI primers and MseI primers as described by KIM et al. 2002 Down. Selective amplifications were carried out using various combinations of two- to four-nucleotide extensions to EcoRI (E-) or PstI (P-) primers with MseI (M-) primers (E-2/M-2, E-2/M-3, E-2/M-4, E-3/M-3, E-3/M-2, P-3/M-2, P-3/M-3, and P-3/M-4; Table 1).


 
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  		Table 1. List of AFLP primers used in linkage mapping in papaya

Map construction:
Goodness of fit was tested by chi-square analysis for the expected segregation of dominant (3:1), sex-linked (2:1), and codominant (1:2:1) markers in the F2 population. The linkage map was constructed using the program MAPMAKER 3.0 (LANDER et al. 1987 Down) employing a minimum LOD score of 5.0 and a maximum recombination rate ({theta}) of 0.25. First, the codominant markers were used for defining linkage groups. After this, the codominant markers in a total of nine linkage groups were combined with the middle-sized (80–350 bp) markers, fitting either 3:1 or 2:1 ratios, and assigned to linkage groups. After ordering the markers in each group, the anchor markers were selected at regular intervals (one marker/15–20 cM). The anchor markers were mixed with all the remaining markers for a final grouping and ordering by using "first order," "try," and "ripple" functions of the program. Linkage maps were generated using the Kosambi mapping function.

Poisson distribution test:
Marker distribution on our map was determined by counting the number of AFLP markers in a sliding 10-cM interval over the total length of each linkage group. The AFLP marker distribution was analyzed using a Poisson distribution function P(x) = e-µµx/x!, where µ is the average number of markers in a 10-cM interval over the entire map and x is the actual marker count in each interval.

Marker distribution and DNA methylation analysis:
At any site where the number of markers in the interval was greater than, or equal to, the minimum upper limit number of markers of the chi-square-tested expectation, that site was designated as a cluster. The distribution of PstI/MseI markers was compared to that of the EcoRI/MseI markers. If methylation occurred randomly throughout the genome, the frequency of PstI/MseI markers in each linkage group or on the sex locus would be expected to be equal to the frequency of EcoRI/MseI markers observed in the same group or region. Significant deviation from an equal distribution supports the hypothesis of an alternate methylation status.


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

DNA polymorphism:
The genomic DNA of parental cultivars Kapoho and SunUp, with F1 and 54 F2 plants, was amplified with 987 AFLP primer combinations (Table 1). Of the 987 primers, 781 generated 58,173 bands. A portion of a TIF image for AFLP products amplified by the primer pair E-GCT/M-AG is shown in Fig 1. The number of nucleotides in EcoRI/MseI and PstI/MseI primer extensions were significantly correlated to the number of bands (r = -0.8703***) but not to the number of markers (r = -0.3468). A two-nucleotide extension of MseI-** primer with a two-nucleotide extension of EcoRI-A* generated the most bands, with an average of 78 bands/primer pair (Table 2). A two-nucleotide extension of MseI-** primer with a three-nucleotide extension of EcoRI-A** generated the most polymorphic markers, with an average of 2.5 markers/primer pair.



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  		Figure 1. A portion of a TIF image showing AFLP products amplified by the primer pair E-GCT/M-AG. The wells are denoted at the top of the figure. Polymorphic markers are identified as fragments that are present in one of the original parents and some of the F2 samples but absent from others (arrows). The SunUp dominant marker between 200 and 204 bp is cosegregating with sex.


 
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  		Table 2. AFLP fragments generated with different primer combinations

Of the 987 primer pairs used in this study, 106 did not generate any polymorphic markers; the remaining 881 primer pairs generated 1812 AFLP markers. Theoretically, the AFLP markers generated by the shorter nucleotide extension primer pairs should include the markers generated by longer nucleotide extension primer pairs as well as additional new markers. Forty-five of the EcoRI/PstI and MseI primer pairs having two to four extra nucleotide extensions were found to generate redundant markers. After pairing the data set to remove the redundant markers, a total of 1767 polymorphic markers, ranging from 25 to 700 bp, remained for linkage analysis

Among all 1767 markers, 644 were derived from the female parent, Kapoho, 983 were from the hermaphrodite (pollen donor) parent, SunUp, and 140 were inherited in a codominant fashion. Each primer pair generated an average of 1.8 polymorphic markers. The EcoRI/MseI primer pairs generated more polymorphic markers than the PstI/MseI primer pairs did, with an average of 2.0 and 1.2 markers, respectively.

Segregation analysis:
The {chi}2 test showed that 71.4% of the AFLP markers fit the expected 3:1 and 1:2:1 Mendelian segregation ratios. When the papaya sex-linked segregation (2:1) markers were added, those fitting the expected ratios increased to 89.4%. The remaining 187 markers were skewed significantly from the expected ratios.

Genetic linkage map:
A total of 1501 markers, including 1498 AFLP markers, the papaya ringspot virus coat protein marker, morphological sex type, and fruit flesh color, were mapped into 12 linkage groups at a LOD score of 5.0 and a recombination frequency of 0.25. The 12 major linkage groups covered a total length of 3294.2 cM, with an average distance of 2.2 cM between adjacent markers. The remaining 269 AFLP markers were assigned into smaller groups or remained unlinked. Our linkage group 1 corresponded to the LG1 of the RAPD map where the sex locus was located (Fig 2; SONDUR et al. 1996 Down). Because we have no common markers with the previous RAPD map, we designated LGs 2–12 in descending order according to the length of each group generated from our AFLP data (Fig 2).


















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  		Figure 2. AFLP genetic map of papaya linkage groups 1–12. The sex determination locus was mapped on LG1 with 225 cosegregating markers. Transgenic PRSVCO and Fcolor were mapped on LG7. The alphanumeric code describes the AFLP marker as scored. The first letter of the marker represents the size category of the fragments ("S," <80 bp; "M," 80 ~ 350 bp; and "L," >350 bp); the four numerals following represent EcoRI-/PstI- and MseI-primer combination code (see Table 1), and the first two numerals are the code for the EcoRI- or PstI-primer, while the last two numerals are the code for the MseI-primer; the sixth element of the code is an alphabetical order of the polymorphic loci generated by each primer pair; then the seventh element indicates from which parent the marker was derived (K, Kapoho; S, SunUp; C, codominant); the last element of the code is information about the {chi}2 test (0, not fit expected segregation; 2, fit 2:1; 3, fit 3:1; 5, fit 2:1 and 3:1; C, fit 1:2:1). Cosegregating markers are in boxes.

Striking features of LG1 include 225 markers that cosegregated with the sex locus and all 344 dominant markers on LG1 that were derived from the pollen donor parent SunUp.

The PRSVCO and fruit flesh color (Fcolor) were mapped to LG7 where each was flanked by two markers that were 3.7/4.7 cM and 3.4/3.7 cM distant from them, respectively (Fig 2).

Distribution of AFLP markers over the papaya genome:
The distribution of the AFLP markers over the 12 linkage groups varied greatly. LG1 had the highest marker density (0.85 cM/interval), while LG10 had the lowest (3.9 cM/interval). The number of markers per linkage group ranged from 27 to 400; the length of the linkage groups ranged from 86.2 to 695.3 cM. Under the assumption of random marker distribution, the number of 10-cM intervals containing a given number of markers would follow a Poisson distribution. Our observed and expected number of frame markers/10-cM interval for all the linkage groups deviated significantly from Poisson expectations ({chi}2 = 717.42, d.f. = 8, P < 0.0001; Fig 3). The deviation was greatest in those intervals containing 1, 7, or >7 markers.



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  		Figure 3. The distribution of AFLP markers compared to that expected by Poisson distribution.

A {chi}2 test confirmed that the mapped AFLP markers were not evenly distributed throughout the linkage groups. Clustering was prevalent for all markers, frame markers (markers on each linkage group excluding cosegregating markers), and EcoRI-only frame markers in each group analyzed (Table 3). Although PstI frame markers distributed evenly when the {chi}2 test included all the linkage groups, in LG1 (obs = 5; exp = 13) and LG6 (obs = 21; exp = 11), they still deviated significantly from the expected number.


 
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  		Table 3. {chi}2 tests for the distribution of AFLP markers among the linkage groups

On the basis of the total number of frame markers and the length of the linkage groups, 6 markers/interval would be the minimum upper limit for the number of markers that is statistically different from the average of 2.2 markers/10 cM (P = 0.05). When the frame markers were counted, 14 clusters were identified on 8 linkage groups. When the cosegregating markers were included in the analysis, 59 clusters were identified on the 12 linkage groups. The cluster covering the sex locus and containing 232 markers is the largest among the 59 clusters.

DNA methylation analysis:
If the EcoRI and PstI restriction sites were distributed randomly throughout the genome and the genome were not methylated, then the PstI markers should occur in each linkage group at the same frequency as the EcoRI markers. This was not the case. We calculated a significant deviation from equal distribution of the PstI markers in all the linkage groups (Table 3). This deviation was caused mainly by LG1 (obs = 28, exp = 50) and LG2 (obs = 45, exp = 58). These results suggest a higher level of DNA methylation for LG1 and LG2 compared to the level of methylation in the other 10 linkage groups.

We assessed the potential level of DNA methylation of the sex locus by determining the frequency of sex cosegregating markers derived from EcoRI/MseI vs. those from the PstI/MseI primer pairs. A total of 16.3% of the 1285 mapped markers or 14.2% of all the 1479 polymorphic markers produced by EcoRI/MseI primers cosegregated with sex; only 7.3% of the 204 mapped markers or 5.2% of all 288 PstI/MseI markers cosegregated with sex. Thus, there were significantly fewer (P < 0.01) PstI/MseI markers than EcoRI/MseI markers associated with the sex locus.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

Characteristics of the sex determination locus:
A total of 225 markers, or a total of 66% of all 342 markers on LG1, cosegregated with the sex determination locus. Our results showed that recombination was severely suppressed in the region surrounding the sex determination locus and validated the hypothesis that crossing over is repressed within the region containing the sex determination gene of papaya so that it behaves as a unit factor (STOREY 1953 Down). DNA sequence divergence often causes regional suppression of recombination (LUKACSOVICH and WALDMAN 1999 Down). The large number of cosegregating markers at the sex determination locus suggested extensive sequence divergence resulting in a high polymorphism rate. This might be the consequence of evolution to preserve the function of the sex determination gene that led to dioecy. Suppression of recombination was reported around another gene with evolutionary significance that controls apospory in buffelgrass (OZIAS-AKINS et al. 1998 Down; JESSUP et al. 2002 Down).

All 342 dominant markers on LG1 were derived from the pollen donor parent SunUp, and all but 2 of these markers fit a 2:1 segregating ratio. This situation arises from the lethal effect of the homozygous dominant genotype at the sex determination locus. For a dominant marker linked to the recessive sex allele m from the female parent Kapaho to be mapped, its counterpart recessive marker (absent band) is linked to the dominant sex allele Mh from the hermaphrodite parent SunUp since only one hermaphrodite F1 plant was used to generate the mapping population. When the seeds of homozygous dominant genotypes (MhMh) were aborted, the class of homozygous recessive markers as the counterparts to the Kapaho-derived dominant markers were aborted as well. The segregating ratio of sex-linked dominant markers from Kapoho become 3:0 and thus cannot be mapped.

Excluding the SunUp dominant markers on LG1, the dominant markers derived from each parent are almost equal: 644 from Kapoho and 639 from SunUp, suggesting a similar level of genetic variation on each parental cultivar as reported previously (KIM et al. 2002 Down).

It is known that DNA markers are clustered in the centromeric region due to the suppression of recombination in the heterochromatic regions surrounding centromeres (ALONSO-BLANCO et al. 1998 Down; COPENHAVER et al. 1999 Down; HAANSTRA et al. 1999 Down). However, the scale of suppression of recombination that we found at the sex determination locus of papaya has not been previously reported in any centromeric clusters found in high-density maps of any other plant species (DAVIS et al. 1994 Down; ROUND et al. 1997 Down; BOYKO et al. 2002 Down; MENZ et al. 2002 Down). Suppression of recombination in the papaya sex region is less likely the effect of the centromere and more likely the consequence of sex evolution in this plant species. This hypothesis is supported by the fact that another four clusters with at least seven cosegregating markers are mapped on this linkage group. Because several marker clusters, not just one, were mapped on LGs 2–8, it is difficult to predict the position of centromeres.

DNA methylation:
In LG1, the sex locus had an extremely low frequency of PstI markers. The lower level of sex cosegregating markers generated by the methylation-sensitive enzyme than by the nonsensitive enzyme indicates that the cytosine bases are highly methylated around the sex locus. A positive correlation has been reported for other plant species among marker clusters, centromeric regions, high levels of cytosine methylation, and heterochromatic regions (VUYLSTEKE et al. 1999 Down; YOUNG et al. 1999 Down). Our analysis of DNA methylation is in accord with the marker cluster results. Since most expressed genes are associated with hypomethylated regions (YOUNG et al. 1999 Down), our results suggest that relatively few active genes are located around the sex locus of papaya. These results strongly support the hypothesis that Hofmeyr proposed more than 30 years ago (HOFMEYR 1967 Down).

High-density genetic map:
Although our high-density linkage map was constructed from 1767 AFLP markers, 2 morphological markers, and 1 transgene marker, gaps still remain. A total of 1501 markers were mapped into 12 linkage groups corresponding to the nine pairs of chromosomes. Theoretically, the 3 "extra" linkage groups should eventually cojoin the other 9 groups to eliminate those gaps that are so large they currently necessitate the reported 12 linkage groups. The differences in length among the linkage groups might be due to recombination rate and nucleotide composition difference of each chromosome, or they may be due to chromosomal translocations during meiosis and/or methods used in our map construction.

If chromosomal translocation had occurred, at least half of the seeds derived from F1 might not be viable (KLUG and CUMMINGS 1994 Down). A seed germination test that we conducted (data not shown) failed to support the hypothesis that a translocation event was the basis for the large linkage groups. The largest linkage group, LG2, likely represents more than one chromosome since cytological observations indicate its chromosomes are physically similar in size (STOREY 1941 Down). However, we were not able to break this linkage group using our current mapping population and the type of DNA markers used. This may be resolved in the future by integration of the genetic and physical maps of papaya and by chromosome in situ hybridization of selected bacterial artificial chromosome clones on the physical map.

Crop species such as maize, rice, and sorghum have a bigger genome size than papaya (ARUMUGANATHAN and EARLE 1991 Down), but the lengths of their high-density genetic maps are <2000 cM (HARUSHIMA et al. 1998 Down; VUYLSTEKE et al. 1999 Down; MENZ et al. 2002 Down). Although papaya has a relatively smaller genomic size, the length of its genetic map is 3294.2 cM. This disparity could be due partially to a high genome homology between the two papaya parental cultivars that were derived from the same gene pool (KIM et al. 2002 Down). A higher genome homology results in a higher recombination rate, which expands the genetic map. For example, in potatoes, the genetic map generated from an intraspecific cross is 165% the size of the map from an interspecific population (BONIERBALE et al. 1988 Down; GEBHARDT et al. 1991 Down). Another reason for the large size of the papaya map is the nature of dominant markers that were used for its construction. Since dominant markers represent both homozygous and heterozygous dominant genotypes, the resolution of dominant markers is 50% lower than that of codominant markers for mapping in an F2 population of a diploid species. The direct effect of mapping dominant markers expands the map size by 50%.

The inserted PRSVCO was mapped on LG7 on this high-density linkage map, verifying that there was a single insertion when this gene was transformed (FITCH et al. 1992 Down). Currently, a high-density map of the inserted gene is required for registering genetically modified food products. This map can be used for registration of transgenic papaya in foreign countries to open new markets.

Papaya breeders have long been interested in developing DNA markers linked to fruit flesh color for selection. Dominant yellow color is associated with firmness, while recessive red color is associated with good flavor but shorter shelf life. Experiments are underway to develop SCAR markers linked to flesh color from the flanking AFLP markers on LG7.

Efficiency of the length of nucleotide extension:
The number of amplified products generated by the AFLP technique is theoretically related to the size of the genome and the length of the nucleotide primer extensions (VOS et al. 1995 Down). Papaya has a relatively small genome with 372 Mb (ARUMUGANATHAN and EARLE 1991 Down). The combination of EcoRI-A** primer with MseI primer that contained two-, three-, and four-nucleotide extensions gave 73, 53, and 31 bands, respectively (Table 2). As reported for barley, flax, and onion (VAN TREUREN 2001 Down), shortening the nucleotide extension increased the number of bands in steps by 1.7- and 2.4-fold. This was considerably less than the 4-fold increase expected on a theoretical basis. However, the varied length of extended nucleotides of primers from four to seven in our study resulted in a statistically significant negative correlation with the number of amplified products.

Likewise, the number of polymorphic markers was not strictly related to the length of nucleotide extensions of the primer pairs. The short length of selective nucleotides, such as two-nucleotide extensions of EcoRI-A* and EcoRI-G* with MseI-**, produced less informative profiles than did a three-nucleotide extension of the same EcoRI primer with MseI-**. The reasons for the less informative profile appear to be saturation and homoplasy, which were produced during selective amplification. Saturation and homoplasy have been reported in a range of crop species (VAN TREUREN 2001 Down). Other plant species may have optimal nucleotide extensions for an effective AFLP amplification on the basis of fragment saturation and homoplasy (HAN et al. 1999 Down). In papaya the five primer extensions of EcoRI-A**/MseI-** and EcoRI-G**/MseI-** were most efficient for AFLP marker generation (Table 2). The PstI primers generated fewer markers than the EcoRI primers did, possibly because the PstI restriction nuclease is a methylation-sensitive enzyme and large portions of plant genomes are methylated (INAMDAR et al. 1991 Down; WARNER 1996 Down; YOUNG et al. 1999 Down), thus dramatically reducing the number of restriction sites.

Polymorphisms and segregation:
Although the primer pairs of EcoRI-A**, EcoRI-G**, and PstI-A** with MseI-** resulted in an average of 73, 59, and 50 bands, respectively (Table 2), the number of bands is fewer than that produced in other plant species such as Alstroemeria spp. (HAN et al. 1999 Down), lettuce (VAN TREUREN 2001 Down), sugarcane (HOARAU et al. 2001 Down), and tomato (HAANSTRA et al. 1999 Down). Also each primer pair generated an average of only 1.8 AFLP markers in papaya, which is very low compared to polymorphism observed in other plant species such as Populus deltoids (WU et al. 2000 Down), tomato (HAANSTRA et al. 1999 Down), rice (MACKILL et al. 1996 Down), and Arabidopsis (PETERS et al. 2001 Down). The low rate of polymorphic AFLP markers in papaya is the result of the low level of polymorphism between the two original parents, since Kapoho and SunUp are both Hawaiian Solo types derived from the same gene pool. This is at first surprising because the two parents are morphologically quite different. This result is consistent with other reports that papaya has a narrow genetic base (SHARON et al. 1992 Down; STILES et al. 1993 Down; SONDUR et al. 1996 Down; KIM et al. 2002 Down).

The effect of AT/GC content in selective nucleotide sequence:
The effect of AT/GC content in selective nucleotide sequences on the number of AFLP bands has been reported in soybean (KEIM et al. 1997 Down), Alstroemeria (HAN et al. 1999 Down), and Pinyon pine (TRAVIS et al. 1998 Down). Those studies used EcoRI/MseI primer pairs and showed that a high level of GC content will produce a low number of bands for soybean and Alstroemeria, but a high number of bands for pinyon pine. Our data were generated from both EcoRI/MseI and PstI/MseI primer pairs and indicated that the number of AFLP bands and markers was not statistically associated with either the AT or the GC content of the nucleotide extensions in the primers. However, AT-rich EcoRI and PstI selective nucleotide primers produced significantly more sex cosegregating markers than did the GC-rich primers. In Alstroemeria aurea, DE JEU et al. 1997 Down found that heterochromatic regions were related to AT-rich sequences. A more detailed description of the genomic structure of the papaya sex locus will require studies involving in situ hybridization and sequence analyses.

Marker clusters:
Because greatly suppressed recombination would produce an apparent tight clustering of markers while high levels of recombination would produce widely dispersed markers, the clusters found in our linkage map can be interpreted as reflecting possible chromosome structures. A similar pattern has been reported for other plant species, including tomato (TANKSLEY et al. 1992 Down), Arabidopsis (ALONSO-BLANCO et al. 1998 Down), soybean (YOUNG et al. 1999 Down), and maize (VUYLSTEKE et al. 1999 Down). In those cases, marker clusters were frequently associated closely with the chromosome centromere. If we limit our present analysis to only the frame markers, papaya has only a few marker clusters that are present on only eight linkage groups. A possible reason for this paucity of clusters in papaya compared to other plant species, with the notable exception of Arabidopsis, could be the small size of the Arabidopsis and papaya genomes with a greatly reduced "junk" DNA component in the heterochromatin regions. Notably, if the cosegregating markers were added to the analysis, the sex locus is the biggest cluster. This indicates that the sex locus is particularly differentiated from the other cluster regions in our map.


*  ACKNOWLEDGMENTS

We thank Susan Ancheta for technical assistance, Leslie Poland for growing and maintaining papaya plants at the Hawaii Agriculture Research Center Kunia substation, and Melinda Moore, Robert Osgood, Ming-Li Wang, and Stephanie Whalen for reviewing the manuscript. This work was supported by a U.S. Department of Agriculture-Agricultural Research Service Cooperative Agreement (no. CA 58-5320-8-134) with the Hawaii Agriculture Research Center and a USDA-ARS agreement for Minor Crops Research through the University of Hawaii.

Manuscript received June 18, 2003; Accepted for publication September 24, 2003.


*  LITERATURE CITED
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

ALONSO-BLANCO, C., A. J. M. PEETERS, M. KOORNNEEF, C. LISTER, and C. DEAN et al., 1998  Development of an AFLP based linkage map of Ler Col and Cvi Arabidopsis thaliana ecotypes and construction of a Ler/Cvi recombinant inbred line population. Plant J. 14:259-271.[CrossRef][Medline]

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