Genetic characterization of guava (Psidium guajava L.) germplasm in the United States using microsatellite markers V. Sitther, D. Zhang, D. L. Harris, A. K. Yadav, F. T. Zee, L. W. Meinhardt & S. A. Dhekney Genetic Resources and Crop Evolution An International Journal ISSN 0925-9864 Genet Resour Crop Evol DOI 10.1007/s10722-014-0078-5
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Author's personal copy Genet Resour Crop Evol DOI 10.1007/s10722-014-0078-5
RESEARCH ARTICLE
Genetic characterization of guava (Psidium guajava L.) germplasm in the United States using microsatellite markers V. Sitther • D. Zhang • D. L. Harris • A. K. Yadav • F. T. Zee • L. W. Meinhardt S. A. Dhekney
•
Received: 14 August 2013 / Accepted: 7 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Genetic diversity of 35 Psidium guajava L. accessions and three related species (P. guineense Sw., P. sartorianum (O. Berg) Nied. and P. friedrichsthalianum (O. Berg) Nied.) maintained at the U.S. Department of Agriculture (USDA), National Plants Germplasm System, Hilo, HI, was characterized using 20 simple sequence repeat (SSR) markers. Diversity analysis detected a total of 178 alleles ranging from 4 to 16 alleles per locus. The observed mean heterozygosity (0.2) and inbreeding coefficient (0.8) indicated a high level of inbreeding among the accessions tested. Multi-locus DNA fingerprints based on the 20 SSR loci unambiguously differentiated all accessions and revealed the absence of duplicated samples. Ordination and cluster analyses suggested that the genetic
relationships between majorities of the accessions could be explained by geographic origin, mainly including tropical America, Southeast Asia and Hawaii. A Bayesian cluster analysis partitioned the accessions into two groups and the partition was largely compatible with the result of ordination analyses. Distance-based cluster analyses further indicated that accessions from same geographical region or breeding programs grouped together in spite of the inter-regional exchange of germplasm. Accessions from Southeast Asia were dominantly white fleshed, whereas accessions from tropical America and Hawaii exhibited diverse flesh colors. The results also indicated that accessions from the same region were likely derived from a small number of common
V. Sitther (&) Department of Biology, Morgan State University, 1700 E. Cold Spring Lane, Baltimore, MD 21251, USA e-mail:
[email protected]
F. T. Zee Tropical Plant Genetic Resources and Disease Research, Hilo, HI 96720, USA
D. Zhang L. W. Meinhardt Sustainable Perennial Crops Laboratory, USDA-ARS, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA
S. A. Dhekney University of Wyoming, Sheridan Research and Extension Center, Sheridan, WY 82801, USA
D. L. Harris Saint Martin’s University, 5000 Abbey Way SE, Lacey, WA 98503-7500, USA A. K. Yadav Agricultural Research Station, Fort Valley State University, 1005 State University Drive, Fort Valley, GA 31030-4313, USA
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ancestors or progenitors. All 20 SSRs were transferable to P. guineense, P. sartorianum and P. friedrichsthalianum, indicating a close relationship between the cultigens and wild relatives. Application of SSR markers in the USDA/Agricultural Research Service germplasm collection provides new insight into the diversity of the guava germplasm, which will be valuable in future breeding endeavors and the conservation of guava genetic resources. Keywords Cultivar identification Fingerprinting Genetic variation Homozygosity Inbreeding Molecular markers Psidium guajava Simple sequence repeat
Introduction Psidium guajava L. is an important fruit crop in tropical regions of the United States, where it is cultivated for production of fresh fruit, jam, jelly and juice. It is an excellent source of health beneficial compounds including high amounts of vitamins C, dietary fiber, b-carotene, and it is known for its ability to reduce LDL cholesterol and triglycerides (Preece 1981; Setiawan et al. 2001; Singh et al. 1992). An increased demand for guava consumption resulted in increased production from 1.3 million lbs (2010) to 1.9 million lbs in 2011 in the state of Hawaii alone (http://www.nass.usda.gov/Statistics_by_State/Hawaii/ Publications/Fruits_and_Nuts/guavaFF.pdf). The origin of guava, although uncertain, is believed to be in Central and South America (Nakasone and Paull 1998). The genus Psidium (2n = 22) includes about 150 species and is widely distributed in tropical regions of the world. While P. guajava is the most commonly cultivated and widely studied species, other Psidium species including P. cattleianum Sabine, P. friedrichsthalianum (O. Berg) Nied., P. guineense Sw., P. montanum Sw. and P. sartorianum (O. Berg) Nied. are fairly well distributed. Adequate genetic variation has been observed in P. guajava for the development of commercial cultivars with economic importance (Pathak and Ojha 1993). Selected guava cultivars are commercially grown worldwide including South Asia, Central and South America, subtropical regions of the continental U.S., Hawaii and Australia. Guava is a self-pollinated crop with 35–40 % outcrossing (Nakasone and Paull 1998;
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Singh and Sehgal 1968). It is common to select progeny with desirable characteristics from a seedling population; this often results in the development of commercial cultivars that may not be homozygous to the parents from which they are derived. The economic importance of guava has driven significant efforts to accelerate the exploitation of Psidium resources for development of new cultivars. However, technological advances through high-input agriculture in the last 50 years were based on performance of a limited number of selected varieties. The narrow genetic diversity of many commercial crops has increased the vulnerability of agriculture to disease, climatic and insect problems. More importantly, large- scale monoculture increases the rapid displacement of landraces by few selected commercial cultivars. The goal for any germplasm collection is to capture and conserve the genetic diversity through the preservation of the entire germplasm in a species, which includes cultivated varieties, primitive cultivars, landraces and wild and weedy relatives. Although a large number of clonally propagated accessions are maintained in germplasm collections worldwide, their use for crop improvement is limited by inadequate information on genetic diversity, population structure and appropriate phenotypic assessment. The identification, characterization and evaluation of collections will lead to the utilization of the germplasm for development of improved cultivars. In the United States, the University of Hawaii, College of Tropical Agriculture and Human Resources is involved in guava improvement and has significantly impacted the development of new cultivars. From 1948 to 1969, 21 guava cultivars from seven countries were introduced and evaluated (Morton 1987; Nakasone and Paull 1998). ‘Beaumont’, the first guava cultivar released to the island’s processing industry, was selected from an open-pollinated seedling population in 1954. The variety ‘Ka Hua Kula’, selected from 1,200 open-pollinated ‘Beaumont’ seedlings and released in 1978, has fruit qualities similar to ‘Beaumont’ but with a stronger pink color and higher acid content. A growing interest in the exploration of guava genetic resources and obstacles in procuring international plant germplasm make it paramount to characterize accessions currently available with the U.S. Department of Agriculture/Agricultural Research Service (USDA/ARS), National
Author's personal copy Genet Resour Crop Evol
Plant Germplasm System (NPGS), which maintains an ex situ collection in Hilo, HI. A thorough evaluation of available germplasm can provide valuable information to breeders for selection of parents for development of improved cultivars. Various DNA markers have been used to differentiate guava cultivars at the molecular level (FeriaRomero et al. 2009; Rodriguez et al. 2007). Of these, simple sequence repeat (SSR) markers are widely preferred as they are co-dominant, highly polymorphic, exhibit transferability among closely related genera and can be scored unambiguously (Bucheli et al. 2001; Tautz 1989). SSRs for P. guajava have been developed using genomic libraries of the species for the simple sequence repeats (GA)n and (GT)n (Risterucci et al. 2005). These microsatellite loci have proved effective for the genetic analyses of guava accessions from various countries (Kanupriya et al. 2011; Valde´s-Infante et al. 2007). The present study aimed at dissecting the genetic structure of the largest guava germplasm collection in the United States, which maintains highly diverse accessions, wild species and selections of improved cultivars. Although the origin of the accessions is unknown, the collection represents cultivars with diverse horticultural characteristics. The objectives of the study were to characterize the genetic variation among guava accessions, determine the genetic structure of the cultivar collection and evaluate the transferability of SSR markers developed for P. guajava to other wild species of the genus.
Materials and methods Plant material Psidium accessions characterized in this study, their source and flesh color are listed in Table 1. Leaf samples were obtained from the USDA/ARS, Pacific Basin Tropical Plant Germplasm Resource Center (PBARC) in Hilo, HI. In addition to 35 P. guajava accessions, one each of the wild species including P. friedrichsthalianum, P. guineense, P. sartorianum were included to test the cross-transferability of the SSR markers. Actively growing shoot and leaf samples from all accessions and wild species were harvested and immediately frozen to -80 °C prior to being shipped overnight on dry ice. Immediately upon
arrival, the tender shoots were separated from stems, ground to a fine powder in liquid nitrogen and stored at -80 °C. DNA isolation and quantification Total genomic DNA was extracted from 500 mg of leaf tissue using a Qiagen DNeasy Plant Maxi Kit (Qiagen, Valencia, CA, USA). Prior to DNA extraction, samples were placed in a Qiagen tissue lyser (Qiagen, Valencia, CA, USA) for 2 min at 30 Hz to disrupt leaf material. Quality of DNA was checked on a 1 % (w/v) agarose gel, and its concentration was determined using a spectrophotometer (NanoDrop, Wilmington, DE, USA). A portion of the DNA was diluted in molecular grade water to 10 ng lL-1 concentration and stored at -20 °C. PCR amplification and microsatellite identification A set of forty PCR primers amplifying twenty microsatellite loci cloned and sequenced by Risterucci et al. (2005) were used to characterize guava accessions (Table 2). These markers have been previously demonstrated to be useful in distinguishing guava genotypes (Kanupriya et al. 2011; Valde´s-Infante et al. 2007). PCR amplifications were performed using WellRED fluorescent dye-labeled primers (Beckman Coulter, Inc., Fullerton, CA, USA). Reactions were carried out in 25 lL volume containing 10 ng genomic DNA, 0.4 lM dNTPs, 0.1 lM fluorescent-labeled forward and reverse primers, 3.0 mM MgCl2, and 0.1 U of 29 Taq DNA polymerase (GoTaq DNA Polymerase, Promega Corporation, Madison, USA) mixed in reaction buffer (pH 8.5). DNA amplification was performed using a Bio-Rad iCycler ver. 1.259 system (Bio-Rad, Hercules, CA, USA). Conditions for PCR reactions were: one cycle at 94 °C for 5 min, 30 cycles at 94 °C for 45 s, 55 °C for 1 min, 72 °C for 1 min and a final cycle of 72 °C for 8 min. The amplified loci were separated by capillary electrophoresis with a 400 bp size standard and analyzed on a CEQTM8000 eight-channel capillary genetic analysis system (Beckman Coulter, Fullerton, CA, USA). SSR fragments were generated using the CEQ 8000 Fragment Analysis software version 7.0.55 according to manufacturers’ recommendations (Beckman Coulter, Inc.) and fragment sizes were automatically
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Author's personal copy Genet Resour Crop Evol Table 1 List of the 38 guava accessions, code, source, and flesh color as recorded in the USDA/ARS, PBARC in Hilo, HI Code
Species
Accession
Source
Flesh color
HPSI 3
Psidium guajava
‘Indonesian Seedless’
Florida
White
HPSI 5
Psidium guajava
‘Hong Kong White’
Hawaii
White
HPSI 6 HPSI 7
Psidium guajava Psidium guajava
‘Patillo’ ‘Pink Acid’
Florida Florida
Pale pink Pink
HPSI 8
Psidium guajava
‘Thailand Seedless’
Thailand
White
HPSI 15
Psidium guajava
‘Hong Kong Pink’
Hawaii
Pink
HPSI 27
Psidium guajava
‘J.B. White’
Singapore
White
HPSI 34
Psidium guajava
‘Fan Retief’
South Africa
Pink
HPSI 38
Psidium guajava
‘Poamoho Pink’
Hawaii
Pale pink
HPSI 41
Psidium guajava
‘Thai Maroon’
Malaysia
Red
HPSI 42
Psidium guajava
‘Diminuitive’
Hawaii
Pale yellowish
HPSI 44
Psidium guajava
‘Bon Dov’
Israel
White
HPSI 48
Psidium guajava
‘Indian Red’
Florida
Pale pink
HPSI 52
Psidium guajava
‘Klom Ampom’
Thailand
White
HPSI 53
Psidium guajava
‘Klom Sa Lee’
Thailand
White
HPSI 54
Psidium guajava
‘Bangkok Apple’
Thailand
White
HPSI 55
Psidium guajava
‘Khao Sawaive’
Thailand
White
HPSI 57
Psidium guajava
‘Holmberg’
Hawaii
Pink
HPSI 60 HPSI 67
Psidium guajava Psidium guajava
‘Klom Toonklao’ ‘Rica’
Thailand Brazil
White Pink
HPSI 68
Psidium guajava
‘Gema De Doro’
Brazil
Yellow
HPSI 61
Psidium guajava
‘Pearl guava’
Taiwan
White
HPSI 12
Psidium guajava
‘Golden’
Taiwan
White
HPSI 50
Psidium guajava
‘Alahabad Safeda’
Australia
Pale pink
HPSI 56
Psidium guajava
‘Red Indian’
Florida
Pink
HPSI 26
Psidium guajava
‘Gushiken Sweet’
Hawaii
White
HPSI 13
Psidium guajava
‘Pear’
Taiwan
Pink
HPSI 14
Psidium guajava
‘Ruby 9 Supreme’
Florida
White
HPSI 62
Psidium guajava
‘Less Seed’
Taiwan
Pink
HPSI 49
Psidium guajava
‘Lucknow’
Australia
Pale pink
HPSI 47
Psidium guajava
‘Uma’
San Diego, CA
White
HPSI 16
Psidium guajava
‘Puerto Rico’
Puerto Rico
Pink
HPSI 37
Psidium guajava
‘Beaumont’
Hawaii
Pink
HPSI 35 HPSI 20
Psidium guajava Psidium guajava
‘Ka Hua Kula’ ‘Waiakea’
Hawaii Hawaii
Pink Dark pink
HPSI N-97-46
Psidium guineense
N-97-46
Australia
Pale orange
HPSI N-95-20
Psidium friedrichsthalianum
‘Costa Rican’
El Salvador
White
HPSI N-97-49
Psidium sartorianum
N-97-49
Australia
Greenish
calculated by the CEQTM8000 Genetic Analysis System. Allele calling was performed using the CEQ 8000 binning wizard software (CEQTM8000 software version 7.0.55, Beckman Coulter, Inc.) and edited based on the bin list using a SAS program (SAS 1999).
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Data analysis Summary statistics for each marker locus including allelic richness, heterozygosity, gene diversity, number of alleles and inbreeding coefficient were computed
Author's personal copy Genet Resour Crop Evol Table 2 Characteristics of 20 simple sequence repeat markers (Risterucci et al. 2005) used in the analysis of genetic diversity of the U.S. guava germplasm collection EMBL Accession no.
Repeat motif
Primer sequences (50 –30 ) Forward
Reverse
Allele size ranges
mPgCIR01
AJ639775
(GA)17
TAGTGCTTTGGTTGCTT
GCAGGTGGATATAAGGTC
237
mPgCIR03
AJ639754
(GA)40
TTGTGGCTTGATTTCC
TCGTTTAGAGGACATTTCT
158
mPgCIR04
AJ639755
(GA)25
TTCAGGGTCTATGGCTAC
CAACAAGATACAGCGAACT
148
mPgCIR05
AJ639756
(GA)31
GCCTTTGAACCACATC
TCAATACGAGAGGCAATA
252
mPgCIR07
AJ639757
(CA)13AA(GAA)3
ATGGAGGTAGGTTGATG
CGTAGTAATCGAAGAAATG
149
mPgCIR08 mPgCIR09
AJ639758 AJ639759
(GA)12 (GA)19
ACTTTCGGTCTCAACAAG GCGTGTCGTATTGTTTC
AGGCTTCCTACAAAAGTG ATTTTCTTCTGCCTTGTC
214 173
mPgCIR10
AJ639760
(CT)12
GTTGGCTCTTATTTTGGT
GCCCCATATCTAGGAAG
261
mPgCIR11
AJ639761
(CT)17
TGAAAGACAACAAACGAG
TTACACCCACCTAAATAAGA
298
mPgCIR13
AJ639762
(AC)12(AT)4G(GA)2
CCTTTTTCCCGACCATTACA
TCGCACTGAGATTTTGTGCT
245
mPgCIR15
AJ639764
(GA)8GG(GA)9
TCTAATCCCCTGAGTTTC
CCGATCATCTCTTTCTTT
147
mPgCIR16
AJ639765
(TC)25
AATACCAGCAACACCAA
CATCCGTCTCTAAACCTC
292
mPgCIR17
AJ639766
(CT)23
CCTTTCGTCATATTCACTT
CATTGGATGGTTGACAT
231
mPgCIR19
AJ639768
(CT)16
AAAATCCTGAAGACGAAC
TATCAGAGGCTTGCATTA
274
mPgCIR20
AJ639769
(CT)14(CA)17
TATACCACACGCTGAAAC
TTCCCCATAAACATCTCT
266
mPgCIR21
AJ639770
(AG)15GG(AG)7
TGCCCTTCTAAGTATAACAG
AGCTACAAACCTTCCTAAA
154
mPgCIR22
AJ639771
(GT)9(GA)14
CATAAGGACATTTGAGGAA
AATAAGAAAGCGAGCAGA
235
mPgCIR23
AJ639772
(TA)4(GT)7
GTCTATACCTAATGCTCTGG
CCCAGGAAAATCTATCAC
185
mPgCIR25
AJ639773
(GA)24
GACAATCCAATCTCACTTT
TGTGTCAAGCATACCTTC
124
mPgCIR26
AJ639774
(GT)2(GA)17
CTACCAAGGAGATAGCAAG
GAAATGGAGACTTTGGAG
185
Primer
using GenAlEx 6.5 (Peakall and Smouse 2006, 2012). Inbreeding coefficient followed the definition of Wright (1965). Multilocus matching was performed to identify duplicates in the data set, and the same program was used for genotype matching. Accessions with different names that were completely matched at the 20 loci were considered as duplicates. Pair-wise genetic distances as defined by Peakall et al. (1995) were computed using the DISTANCE procedure implemented in GenAlEx 6.5. The same program was also used to perform a principle coordinate analysis (PCoA). A cluster analysis using the Neighbor Joining method was used to further examine the genetic relationship among accessions. Kinship coefficient was chosen as genetic distance measurement to ensure a direct estimate of shared ancestry among the individual accessions (n = 38). The computation was executed using microsatellite analyzer (Dieringer and Schlo¨tterer 2002) with 100 bootstrapping. A dendrogram of the consensus tree was generated from the resulting distance matrix using the neighbor-
joining algorithm (Saitou and Nei 1987) available in PHYLIP (Felsenstein 1989) and visualized using the TreeView Version 1.6.6 (Page 1996). A model-based clustering algorithm implemented in the STRUCTURE software program (Pritchard et al. 2000) was applied to the SSR data in order to verify the kinship coefficients from distance-based cluster analyses. The model-based Bayesian algorithm attempted to identify genetically distinct subpopulations based on allele frequencies. The admixture model was applied and the number of clusters (K value), indicating the number of subpopulations the program attempted to find, was set from 1 to 10, and analyses was carried out without assuming any prior information about the genetic groups. Ten independent runs were assessed for each fixed number of clusters (K), each consisting of 1 9 106 iterations after a burn-in of 2 9 106 iterations. The DK value was used to detect the most probable number of clusters (Evanno et al. 2005) and the computation was performed using the STRUCTURE HARVESTER
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program (Earl and vonHoldt 2012). Of the 10 independent runs, the one with the highest Ln Pr (X|K) value (log probability or log likelihood) was chosen and represented as bar plots.
Table 3 Summary statistics for 38 guava accessions assessed with 20 simple sequence repeats (Risterucci et al. 2005) Ne
Ho
He
a
6.0
3.4
0.0
0.7
1.0
mPgCIR03
4.0
1.3
0.0
0.2
1.0
mPgCIR11
11.0
6.0
0.0
0.8
1.0
mPgCIR16
7.0
4.9
0.2
0.8
0.8
mPgCIR17
11.0
5.1
0.3
0.8
0.7
mPgCIR21
11.0
4.2
0.1
0.8
0.9
mPgCIR04
12.0
5.8
0.5
0.8
0.4
mPgCIR05
8.0
3.1
0.1
0.7
0.8
mPgCIR08
11.0
6.2
0.2
0.8
0.8
mPgCIR09
16.0
8.7
0.3
0.9
0.6
mPgCIR10
7.0
3.9
0.1
0.7
0.8
mPgCIR13
9.0
2.0
0.3
0.5
0.5
mPgCIR15
10.0
5.1
0.2
0.8
0.7
mPgCIR19
8.0
2.9
0.0
0.7
1.0
mPgCIR20 mPgCIR22
8.0 7.0
4.2 2.6
0.4 0.1
0.8 0.6
0.5 0.8
mPgCIR23
8.0
3.1
0.2
0.7
0.7
mPgCIR25
9.0
3.8
0.0
0.7
1.0
mPgCIR26
9.0
5.6
0.1
0.8
0.9
mPgCIR07
6.0
2.6
0.1
0.6
0.8
Mean
8.9
4.2
0.2
0.7
0.8
Locus mPgCIR01
Cross-species transferability All 20 loci were evaluated for amplification on three wild guava species namely P. friedrichsthalianum, P. guineense and P. sartorianum. Sample collection, DNA isolation, PCR amplification and detection of SSR profiles were performed according to the procedure described above for P. guajava.
Results All 20 SSRs tested generated amplicons and detected polymorphisms among the guava accessions evaluated. The number of alleles ranged from 4 to 16 across the 20 loci, with an average of 8.9 (Table 3). The highest number of polymorphic alleles was detected at locus mPgCIR09 with a total of 16 alleles. Expected heterozygosity values (He) ranged from 0.24 to 0.9, with an average of 0.7, while observed heterozygosity (Ho) values ranged from 0.00 to 0.5 with an average of 0.2. The inbreeding coefficient among accessions tested ranged from 0.4 to 1.0, with an average of 0.8. Four of the twenty loci (mPgCIR01, mPgCIR03, mPgCIR11 and mPgCIR25) generated complete homozygous genotypes among the 38 accessions tested. Individual genotype matching (pair-wise comparisons) based on multi-locus SSR profiles did not detect matching pairs, demonstrating that each accession analyzed was a distinct genotype. Results of principle coordinate analysis performed to provide spatial representation of the relative genetic distances among individuals revealed three distinct groups, with the wild species interspersed among the P. guajava accessions (Fig. 1). The plane of the first three PCoA axes accounted for 72.1 % of the total variation (first axis = 37.8 %, second = 19.2 % and third = 15.1 %). Two wild species, P. friedrichsthalianum and P. sartorianum along with five other P. guajava accessions clustered in the first group. The second group comprised of 13 accessions, the majority of which were clones collected from Southeast Asia. Cultivars and breeding lines that were developed at the
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Na
IC
Na = number of alleles, Ne = number of effective alleles, Ho = observed heterozygosity, He = expected heterozygosity, IC = inbreeding coefficient a
Definition of inbreeding coefficient according to Wright (1965)
University of Hawaii’s guava improvement program and those selected by the Institute of Food and Agricultural Sciences, University of Florida (Crane and Balerdi 2012) were included in the third group. P. guineense closely clustered with the P. guajava accessions in this group. Neighbor joining dendrogram based on kinship coefficient placed the 38 guava accessions into six clusters (Fig. 2), which was partially supported by the results of principle coordinate analysis. The first cluster comprised of almost identical accessions as those revealed in the PCoA. This cluster comprised of eight P. guajava accessions with varied parentage and included the three wild species, P. friedrichsthalianum, P. guineense, and P. sartorianum. In this cluster, accessions ‘Beaumont’ and ‘Ka Hua Kula’ grouped closely and accurately reflected their parent–offspring relationship. Cluster II comprised of three accessions, all of which were obtained
Author's personal copy Genet Resour Crop Evol Principal Coordinates Poamoho Pink Holmberg Indonesian Seedless
Coord. 2
Fig. 1 Principle coordinate analysis of 38 guava accessions from USDA/ ARS, Pacific Basin Tropical Plant Germplasm Resource Center, Hilo, Hawaii. Clustered groups are represented by different colors. The plane of the first three PCoA axes account for a total of 72.1 % of the total variation (first axis = 37.8 %, second = 19.2 % and third = 15.1 %). (Color figure online)
Alahabad safeda Hong Kong White Waiakea Patillo Lucknow N-97-46 Hong Kong Pink Rica Gushiken Sweet Less Seed Pink Acid Bon Dov Diminuitive Ruby x Supreme Indian Red Thai Maroon Red Indian Uma Pear Beaumont Klom Sa Lee
Kahuakula
Klom Toonklao Pearl guava Khao Sawaive
Costa Rican
N-97-49
Gema De Doro
J.B. White Klom Ampom Golden
Fan Retief
Thailand Seedless
Puerto Rico
Bangkok Apple
Coord. 1 Group I
from Hawaii. Most accessions in cluster III were processing type guavas with pink-fleshed acidic fruits. The acidic processing guavas, ‘Pink acid’ and ‘Patillo’ grouped very closely in this cluster. The pink-fleshed accessions, ‘Poamoho pink’ and ‘Holmberg’ that were developed at the Poamoho station, University of Hawaii, grouped closely in cluster V along with five other accessions. ‘Pear’ that has crispy firm flesh and ‘Alahabad Safeda’with soft flesh clustered closely with ‘Lucknow’ in this cluster. The largest number of accessions were grouped in cluster VI comprising 12 of the 38 accessions. This cluster included the white crispy dessert guava ‘Klom Toonklao’, ‘Klom Ampom’, ‘Thailand Seedless’ and ‘Bangkok Apple’ from Thailand; ‘Golden’ and ‘Pearl Guava’ from Taiwan, and ‘J. B. White’ from Singapore, which are selections developed from Thai dessert guava germplasm. Although ‘Hong Kong White’ and ‘Hong Kong Pink’ were collected from the University of Hawaii germplasm site, they grouped in different clusters. Population stratification of the accessions based on DK value computed by STRUCTURE HARVESTER revealed two clusters as the most probable number of K (Evanno et al. 2005) (Figs. 3 and 4), and the partition was partially compatible with principle coordinate
Group II
Group III
analysis (Fig. 1) as well as the dendrogram based on kinship coefficient (Fig. 2). All accessions that grouped together in the first cluster of PCoA and dendrogram were included in cluster I of the structure analysis. In addition, accessions in Group I of Bayesian clustering included most of the accessions in clusters II, III and IV of the kinship based dendrogram, whereas Group II of the Bayesian cluster included most accessions in clusters IV, V and VI. Accessions ‘Hong Kong White’, ‘Hong Kong Pink’ ‘Costa Rica’, ‘Indian Red’, ‘Pink Acid’, ‘Ruby 9 Supreme’ and ‘Red Indian’ and ‘Patillo’ were hybrids between the two different groups. Most of these hybrid types fell in clusters II, III and IV in the NJ dendrogram (Fig. 2). The Bayesian clustering assigned cultivar groups supporting the classification based on flesh color to a certain extent. There was a noticeable difference in flesh color among the two groups (Fig. 3). While most white-fleshed accessions from Thailand clustered together in Group I (red), pink-fleshed accessions were interspersed in Group II (red). Of a total of 15 white-fleshed cultivars tested in the study, only two were assigned in Bayesian Group 1. All SSRs markers tested were capable of amplifying DNA in the different Psidium species tested. While
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Author's personal copy Genet Resour Crop Evol Fig. 2 Neighbor-joining dendrogram depicting the relationship among the 38 guava accessions from USDA/ARS, Pacific Basin Tropical Plant Germplasm Resource Center in Hilo, Hawaii. Identification of accessions correspond to samples listed in Table 1
Accession
Source
'Gema De Doro'
Yellow Brazil 'Costa Rican' El Salvador White N-97-46 Australia Pale Orange N-97-49 Australia Greenish 'Ka Hua Kula' Hawaii Pink 'Beaumont' Hawaii Pink 'Fan Retief' South Africa Pink 'Puerto Rico' Puerto Rico Pink Pale Yellowish 'Diminuitive' Hawaii 'Gushiken Sweet' Hawaii White 'Waiakea' Hawaii Dark pink 'Less Seed' Taiwan Pink Pale Pink 'Patillo' Florida Pink 'Pink Acid' Florida Pink 'Hong Kong Pink' Hawaii ‘Ruby’ x ‘Supreme’ Florida White 'Red Indian' Florida Pink 'Indian Red' Pale Pink Florida White 'Indonesian Seedless'Florida 'Lucknow' 'Pear' 'Alahabad Safeda' 'Hong Kong White' 'Rica' 'Holmberg' 'Poamoho Pink' 'Uma' 'Bon Dov' 'Thai Maroon' 'Khao Sawaive' 'Klom Sa Lee' 'Golden' 'Klom Toonklao' 'Pearl guava' 'J.B. White' 'Klom Ampom'
Australia Taiwan Australia Hawaii Brazil Hawaii Hawaii California Israel Malaysia Thailand Thailand Taiwan Thailand Taiwan Singapore Thailand 'Bangkok Apple' Thailand 'Thailand Seedless' Thailand
19 out of the 20 SSRs tested in the study produced amplification profiles in the expected range allele range, several cultivated and the wild species showed out of range alleles at loci mPgCIR04. In general, cross-species amplification was observed in all three Psidium species. Of the 20 SSRs tested, three primer pairs did not produce amplifications in the exact allele range in P. guineense, P. sartorianum and P. friedrichsthalianum, respectively, as described by Risterucci et al. (2005).
Discussion Assessment of genetic variability in guava is a first step towards rational conservation and efficient use of
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Flesh Color
I
II
III
IV
Pale Pink Pink Pale Pink White Pink Pink Pale Pink
V
White White Red White White White White White White White White White
VI
genetic resources for crop improvement. Accurate identification of accessions in a germplasm collection is an important challenge faced in crop improvement programs. Germplasm obtained from a reliable source such as the original breeder, an institutional collection or those from identified sources by a knowledgeable collector are more reliable than those obtained from open markets. For germplasm identification, co-dominant molecular markers such as microsatellites are powerful tools as they are objective and offer reproducible means of identification, independent of environmental influences. The use of SSR markers has provided valuable information on the genetic diversity among guava accessions in India and Cuba (Kanupriya et al. 2011; Valde´s-Infante et al. 2007). In the present study polymorphic SSR markers developed
Author's personal copy Genet Resour Crop Evol
Fig. 4 Plot of Delta K (filled circles, solid line) calculated as the mean of the second-order rate of change in likelihood of K divided by the standard deviation of the likelihood of K, m(|L00 (K)|)/s [L(K)]
Fig. 3 Inferred clusters based on Bayesian analysis among 38 guava accessions obtained from USDA/ARS, Pacific Basin Tropical Plant Germplasm Resource Center in Hilo, HI. Each vertical line represents an individual multilocus genotype. Each color represents the most likely ancestry of the cluster from which the genotype or partial genotype was derived. Delta K is the potential number of genetic clusters that may exist in the overall sample of individuals. Individuals with multiple colors have admixed genotypes from multiple clusters. (Color figure online)
by Risterucci et al. (2005) enabled identification of cultivated and wild type accessions and establishment of multilocus profiles of guava germplasm in the U.S. SSRs used in the study were consistent in their ability to amplify specific loci to discriminate alleles. In spite of the varying heterozygosity and number of
alleles, the combination of the 20 SSR markers used in the present study clearly differentiated all cultivated and wild type Psidium accessions at the USDA/ARS. High levels of expected heterozygosity (He) were observed among the guava accessions (0.7), while observed heterozygosity (Ho) was much lower with an average of 0.2. This large difference between He and Ho indicates the tendency of high inbreeding or ‘‘founder effect’’ during domestication. The high inbreeding coefficient (0.8) corroborates previous observations that guava has an estimated outcrossing rate of 35–40 % (Nakasone and Paull 1998). The pattern of allelic diversity observed in our study was similar to that reported by Kanupriya et al. (2011) where 23 SSRs were used to characterize pink-fleshed, white-fleshed, and segregating selections of open pollinated cultivars in India. The average number of alleles detected in the present study (8.9) was similar to their findings where 6.4 alleles per locus were detected. The levels of observed heterozygosity (Ho = 0.2) in the present study is lower than that reported by Valde´sInfante et al. (2007) in which open pollinated seedlings from a Cuban breeding program showed an average observed heterozygosity of 0.38. This suggests that cross-incompatibility may play a significant role in hindering the effectiveness of creating true hybrids and recombining favorable alleles (from parental clones) in the guava breeding program.
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Author's personal copy Genet Resour Crop Evol
Testing and identification of cross-incompatibility groups, followed by controlled hybridization between cross-compatible parents may be needed for the development of improved guava cultivars. However, since we have evaluated a small set of SSR loci, there is possibility that the low heterozygosity and large inbreeding coefficient was partially due to the sampling bias of SSR markers. Testing of additional guava-specific SSR markers on diverse guava germplasm could verify this observation. It was interesting to note that the processing type pink-fleshed acidic guavas, ‘Pink acid’ and ‘Patillo’ grouped very closely in the NJ dendrogram (Fig. 2). In addition, accessions ‘Poamoho Pink’ and ‘Holmberg’ developed at the research station in Hawaii grouped closely (Cluster V), and most accessions that grouped in cluster VI were selections developed from Thai dessert guava germplasm. The clustering of these closely-related accessions indicates the efficacy of the SSR markers used in the study. The grouping patterns revealed by PCoA (Fig. 1), NJ dendrogram (Fig. 2) and Bayesian clustering analysis (Figs. 3 and 4) were largely compatible. In general, accessions collected from the same geographical region or breeding program tended to group together, indicating that despite the widespread distribution of guava in the tropical world and more than 100 years cultivation in the Asia and Pacific region, the exchange of germplasm among regions has been limited. Additionally, it appears that only a small number of guava germplasm have been used in breeding programs. The pattern of clustering could also be explained by consumer preferences and breeding for specific traits. For example, six out of seven accessions from Thailand were consistently grouped in one cluster in the PCoA, NJ tree and Bayesian clustering analyses, and all of them were white fleshed. This apparently is due to a regional preference of varieties with white flesh and crunchy texture. The 20 microsatellite markers showed transferability among closely related genera. One or two alleles per locus were produced in the wild guava species (2n = 44). The interspersed distribution of wild type guavas along with the cultivated species as observed in the PCoA, NJ dendrogram and Bayesian clustering analysis indicate the high levels of genetic similarity among them. It is not surprising to notice few non-
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specific and out of range amplifications in the wild species and this has been reported previously (Risterucci et al. 2005). During the isolation and characterization of guava SSRs used in this study, non-specific amplifications were observed in 6 of the 23 loci tested. It was interesting to note that mPgCIR21 produced out of range amplifications in several cultivated and wild accessions as well. Among the wild guava accessions tested, 19 of the 20 SSRs produced PCR products with the exception of mPgCIR04 in P. friedrichsthalianum. Repeated DNA extraction and amplifications did not yield PCR products indicating the possibility of mutation at the annealing site causing null alleles. Cross-species transferability of the SSR markers indicates the widespread use of this molecular tool. Chromosomal segments conserved in several related species enable these markers to be valuable tools in comparative genomic studies and subsequently in breeding programs. Such attributes are highly beneficial as they minimize the efforts required for developing additional primers. The present study provides an insight on the molecular characterization of guava germplasm using co-dominant markers. The information is highly useful to curators in guiding conservation management practices of ex situ collection for guava germplasm. However, it needs to be pointed out that guava samples tested in this study represent germplasm available in the United States only; other wild species and landraces remain unrepresented and genetic relatedness has not been fully comprehended. A broader assessment with world-wide representation would provide a deeper insight on the distribution of genetic relatedness in this crop. Information generated in the present study paves the way for selection of appropriate parents for guava improvement. The high level of homozygosity suggests an immediate need for improving genetic diversity of available germplasm through controlled cross-pollination. Such heterozygosity created in the population could provide an additional source of genetic variation for development of commercially desirable cultivars. Additional research targeting the use of wild species will be pursued to increase fitness among parental accessions in the breeding program, which will avoid the expression of deleterious recessive genes caused by inbreeding.
Author's personal copy Genet Resour Crop Evol Acknowledgments Financial support of the USDA-CSREES through an award Agreement 2004-38814-15128 to The Fort Valley State University is greatly appreciated.
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