Identification and Parentage Analysis of Citrus Cultivars Developed in Japan by CAPS Markers

Home , Japanese citrus, Setoka

This article is an Advance Online Publication of the authors’ corrected proof. Note that minor changes may be made before final version publication.

The Horticulture Journal Preview e Japanese Society for doi: 10.2503/hortj.OKD-026 JSHS Horticultural Science http://www.jshs.jp/

Keisuke Nonaka1*, Hiroshi Fujii1, Masayuki Kita2, Takehiko Shimada1, Tomoko Endo1, Terutaka Yoshioka1 and Mitsuo Omura3

1NARO Institute of Fruit Tree and Tea Science, Shizuoka 424-0292, Japan 2NARO Western Region Agricultural Research Center, Zentsuji 765-8508, Japan 3Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

To protect the rights of breeders of the major citrus cultivars developed under breeding programs by the national institute of Japan, we developed a method of cultivar identification based on cleaved amplified polymorphic sequence (CAPS) markers, and used it to evaluate their identity and parentage. We selected 19 CAPS markers that had a single-locus origin and moderate polymorphism, and used them to construct genotyping data for 59 citrus cultivars (including American accessions), local varieties, and selections. Of the 19 CAPS markers, 8 were sufficient to discriminate among all accessions except ‘Mato’ buntan (Citrus grandis Osbeck) and ‘Hirado’ buntan (Citrus grandis Osbeck). Among the 33 Japanese cultivars, the parentage of 30 agreed with that reported, but ‘Setoka’, ‘Southern Red’, and ‘Reikou’ had discrepancies at one or more loci. Using 15 to 18 CAPS markers to validate the putative parentage revealed that the seed parent of ‘Setoka’ was ‘KyEn No. 4’, not ‘Tsunonozomi’, and the pollen parent of ‘Southern Red’ was ‘Osceola’, not ponkan (C. reticulate Blanco). The seed parent of ‘Reikou’ remains unknown.

Key Words: breeder rights, genotype, ‘Reikou’, ‘Setoka’, ‘Southern Red’.

simultaneously consider how to protect the rights of Introduction farmers and plant breeders from unwarranted damage Citrus is one of the most important genera of fruit concerning the outflow of a new cultivar overseas and trees that are adapted to temperate and subtropical areas the inflow of such products back to the host country. of Japan. The citrus cross-breeding program in Japan Therefore, it has become necessary to develop cultivar began in 1937 at the National Horticulture Research identification techniques (Kunihisa et al., 2003; Station of the Ministry of Agriculture and Forestry, now Ninomiya et al., 2015). The rights of plant breeders are the Okitsu Citrus Research Station of the National of increasing interest worldwide. The International Agriculture and Food Research Organization Institute Union for the Protection of New Varieties of Plants of Fruit Tree Science (NIFTS). The program has con- (UPOV; http://www.upov.int/index_en.html, June 14, tinued since 1964 at the NIFTS Okitsu and Kuchinotsu 2016) has provided and promoted effective systems of Citrus Research Stations. It is focused mainly on im- plant variety protection. For example, UPOV’s working proving fruit quality, with the goals of a high sugar con- group on Biochemical and Molecular Techniques, and tent, excellent flavor, seedlessness, ease of peeling, and DNA-profiling in particular (BMT), has encouraged the presence of a thin locular membrane, which are charac- use of DNA profiling techniques to protect the rights of teristics specific to mandarins. The program released 41 plant breeders (Shoda et al., 2012). new citrus cultivars by means of controlled cross- To help identify citrus cultivars developed in Japan breeding between 1963 and 2014. by using DNA-profiling techniques, several research When we develop a new plant cultivar, we must groups have reported the development of DNA markers (Matsuyama et al., 1992; Omura et al., 2004; Ueda et al., 2003). However, information on these markers, Received; June 14, 2016. Accepted; August 17, 2016. First Published Online in J-STAGE on October 4, 2016. their polymorphisms and their application to important * Corresponding author (E-mail: [email protected]). commercial cultivars has not been made fully available

© 2016 The Japanese Society for Horticultural Science (JSHS), All rights reserved. 2 K. Nonaka, H. Fujii, M. Kita, T. Shimada, T. Endo, T. Yoshioka and M. Omura to the public. Recently, Ninomiya et al. (2015) reported et al., 1999d; Moriya et al., 2011; Ninomiya et al., that 33 citrus cultivars or accessions, including 7 local 2015; Sawamura et al., 2004, 2008; Yamamoto et al., varieties and 11 cultivars collected from abroad, could 2003). However, citrus cultivars developed in the be identified from polymorphisms at 11 cleaved ampli- NIFTS breeding program have generally not been in- fied polymorphic sequence (CAPS) markers. However, vestigated to confirm their parent–offspring relation- this information was not sufficient because the acces- ships, even though they have frequently been used as sions they examined included only 8 of 41 cultivars de- parents in other breeding programs. veloped by NIFTS that are commercially produced and Here, we aimed at establishing a method for identifi- frequently used as parents in citrus breeding programs cation of the citrus cultivars developed by the citrus in Japan. breeding program in Japan by using a subset of the In Citrus, a variety of DNA marker types have been CAPS markers developed by Shimada et al. (2014). We developed. These include random amplified polymor- also aimed at confirming the parent–offspring relation- phic DNA (RAPD), restriction-fragment-length poly- ships of these cultivars by adding related cultivars and morphism (RFLP), amplified-fragment-length selections in the analysis to increase the reliability of polymorphism (AFLP), CAPS, and simple sequence re- the identification. Using the results of this analysis, we peat (SSR) markers (Cai et al., 1994; Chen et al., 2008; investigated candidate parents of the cultivars that had Jarrell et al., 1992; Ollitrault et al., 2010; Omura et al., discrepancies in the parent–offspring relationships. 2003; Ruiz and Asins, 2003; Shimada et al., 2014). Materials and Methods CAPS markers utilize amplified DNA fragments digested with a restriction endonuclease to reveal Plant material and DNA preparation restriction-site polymorphisms (Konieczny and We selected 59 citrus cultivars, local varieties and se- Ausubel, 1993). CAPS markers have several advan- lections that were developed or used as parents in the tages for the identification of cultivars and for parent- NIFTS citrus breeding program (Table 1), for use in the age analysis. They are more applicable to a wide range CAPS analysis. ‘Okitsu-wase’, ‘Trovita’, and ‘Yoshida’ of cultivars and selections than is the case for RAPD were chosen for the analysis as representative mutants and RFLP molecular-marker systems. In addition, they of satsuma mandarin (Citrus unshiu Marc.), sweet are easy to assay, require only a few nanograms of orange (C. sinensis (L.) Osbeck), and ponkan DNA for the PCR amplification, and are mainly in- (C. reticulata Blanco), respectively. All plant materials herited in a codominant manner. CAPS markers are ro- were obtained from the NIFTS collections at bust because an amplified product is always obtained, Kuchinotsu Citrus Research Station (Nagasaki, Japan) whereas RAPD and AFLP markers have inherently null and Okitsu Citrus Research Station (Shizuoka, Japan). alleles (Iwata et al., 2001). Shimada et al. (2014) devel- Total DNA was isolated from fully expanded fresh oped 708 CAPS markers from sequenced-tagged-site leaves by using the Qiagen DNeasy Plant Mini Kit (STS) primers with designs based on cDNA (i.e., ex- (Qiagen, Hilden, Germany), following the manufactur- pressed sequence tags; ESTs) and used them to con- er’s instructions. struct a linkage map for citrus. Pedigree information is also important for traditional CAPS analysis fruit tree breeding to estimate the values of various pa- The CAPS genotypes were identified by using 37 of rameters for genetic analysis or to predict the target trait the citrus CAPS markers developed by Shimada et al. characteristics of the offspring (Sato et al., 2006; (2014) that showed high versatility in the cultivars and Yamada et al., 1994, 1995b, 1997). As high-throughput selections used in this study (Table 2). Each CAPS am- single nucleotide polymorphism (SNP) genotyping plification was conducted in 12.5 μL of 10 mM technologies have developed rapidly, a genotyping Tris·HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, method for SNPs based on the GoldenGate assay 0.16 mM each dNTP, 10 pM each forward and reverse (Illumina, San Diego, California, USA) has been devel- primers, 10 ng of genomic DNA, 1.25 units of oped for citrus (Fujii et al., 2013b). Unfortunately, mis- AmpliTaq Gold DNA polymerase (Roche, Branchburg, genotyping often occurs in high-throughput genotyping NJ, USA), and the manufacturer’s PCR buffer for the (Close et al., 2009). Fujii et al. (2013b) showed that polymerase. After 10 min of denaturation at 94.5°C, parentage information could be used to detect mis- amplification was performed using 35 cycles of 1 min genotyping. Therefore, it is also important to confirm denaturation at 94°C, 1 min annealing at 52 to 64°C parent–offspring relationships for cultivars by using tra- (Table 2), and a 2 min extension at 70°C, followed by a ditional molecular markers and to provide the correct 10-min final extension at 70°C. The PCR products were pedigree information for use in fruit tree breeding pro- checked using a 200-bp ladder marker using 1.5% aga- grams. In perennial fruit tree species, discrepancies in rose gel electrophoresis. parent–offspring relationships of some cultivars have The PCR products were digested with restriction en- been found and corrected by using molecular markers zymes (Takara Bio Inc., Shiga, Japan) (Table 2) under (Kimura et al., 2003; Kitahara et al., 2005; Matsumoto the following conditions. We mixed 4 μL of the PCR Table 1. Citrus cultivars, local varieties, and other selections usedHort. in the J. present Preview study. Cultivars 1 to 33 were developed by the citrus breeding 3pro- gram at the national institute of Japan and were used for parentage analysis. Table 1. Citrus cultivars, local varieties, and other selections used in the present study. Cultivars 1 to 33 were developed by the citrus breeding program at the national institute of Japan and were used for parentage analysis.

Described parentage (seed parent × pollen Code Sample name Type and Origin Reference Results for parentage analysis in the present study parent) or speciesz 1 ‘Akemi’ ‘Kiyomi’ × ‘Seminole’ Cultivar, bred by NIx in Japan Yoshida et al. (2000b) No discrepancy 2 ‘Amaka’ ‘Kiyomi’ × ‘Encore’ Cultivar, bred by NI in Japan Matsumoto et al. (2001) No discrepancy 3 ‘Amakusa’ ‘KyOw No. 14’ × ‘Page’ Cultivar, bred by NI in Japan Matsumoto et al. (1999b) No discrepancy 4 ‘Ariake’ ‘Seike’ navely × Clementine Cultivar, bred by NI in Japan Yamada et al. (1995a) No discrepancy 5 ‘Asumi’ ‘Okitsu 46 gou’ × ‘Harumi’ Cultivar, bred by NI in Japan Kita et al. (2012) No discrepancy 6 ‘Benibae’ ‘HF No. 9’ × ‘Encore’ Cultivar, bred by NI in Japan Takahara et al. (2006) No discrepancy 7 ‘Benimadoka’ ‘Mato’ buntan × ‘Hirado’ buntan Cultivar, bred by NI in Japan Yamada et al. (1993) No discrepancy 8 ‘Harehime’ ‘E-647’ × ‘Miyagawa Wase’y Cultivar, bred by NI in Japan Yoshida et al. (2005d) No discrepancy 9 ‘Hareyaka’ ‘Encore’ × ‘Nakano 3 gou’ ponkany Cultivar, bred by NI in Japan Matsumoto et al. (1999d) No discrepancy 10 ‘Harumi’ ‘Kiyomi’ × Ponkan ‘F-2432’y Cultivar, bred by NI in Japan Yoshida et al. (2000a) No discrepancy 11 ‘Hayaka’ ‘Imamura’ unshuy × ‘Nakano 3 gou’ ponkany Cultivar, bred by NI in Japan Okudai et al. (1991b) No discrepancy 12 ‘Kankitsu Chukanbohon Nou 5 Gou’ ‘Lee’ × Mukaku kishu Cultivar, bred by NI in Japan Yoshida et al. (2005a) No discrepancy 13 ‘Kankitsu Chukanbohon Nou 6 Gou’ King × Mukaku kishu Cultivar, bred by NI in Japan Yoshida et al. (2005b) No discrepancy 14 ‘Kiyomi’ ‘Miyagawa Wase’ × ‘Trovita’ orangey Cultivar, bred by NI in Japan Nishiura et al. (1983a) No discrepancy 15 ‘May Pummelo’ Hassaku × ‘Hirado’ buntan Cultivar, bred by NI in Japan Ueno et al. (1985) No discrepancy 16 ‘Mihaya’ ‘Tsunonozomi’ × ‘No. 1408’ Cultivar, bred by NI in Japan Nonaka et al. (2012) No discrepancy 17 ‘Miho-core’ ‘Miho Wase’y × ‘Encore’ Cultivar, bred by NI in Japan Matsumoto et al. (1999c) No discrepancy 18 ‘Nankou’ ‘Miho Wase’y × Clementine Cultivar, bred by NI in Japan Okudai et al. (1991a) No discrepancy 19 ‘Nishinokaori’ ‘Kiyomi’ × ‘Trovita’ orange Cultivar, bred by NI in Japan Matsumoto et al. (2003a) No discrepancy 20 ‘Reikou’ ‘KyEn No. 5’ × ‘Murcott’ Cultivar, bred by NI in Japan Yoshioka et al. (2009) Unidentified 21 ‘Seinannohikari’ ‘EnOw No. 21’ × ‘Youkou’ Cultivar, bred by NI in Japan Yoshioka et al. (2015) No discrepancy 22 ‘Setoka’ ‘Tsunonozomi’ × ‘Murcott’ Cultivar, bred by NI in Japan Matsumoto et al. (2003b) The predicted parentage is ‘KyEn No. 4’ × ‘Murcott’ 23 ‘Shiranuhi’ ‘Kiyomi’ × ‘Nakano 3 gou’ ponkany Cultivar, bred by NI in Japan Matsumoto (2001) No discrepancy 24 ‘Southern Red’ ‘Kara’ × Ponkany Cultivar, bred by NI in Japan Kobayashi (1995b) The predicted parentage is ‘Kara’ × ‘Osceola’ 25 ‘Southern Yellow’ ‘Tanikawa’ buntan × Mukaku kishu Cultivar, bred by NI in Japan Kobayashi (1995a) No discrepancy 26 ‘Summer Fresh’ Hassaku × Natsudaidai Cultivar, bred by NI in Japan Shichijo et al. (1983) No discrepancy 27 ‘Sweet Spring’ ‘Ueda’ unshuy × Hassaku Cultivar, bred by NI in Japan Nishiura et al. (1983b) No discrepancy 28 ‘Tamami’ ‘Kiyomi’ × ‘Wilking’ Cultivar, bred by NI in Japan Yoshida et al. (2005c) No discrepancy 29 ‘Tsunokagayaki’ ‘KyOw No. 14’ × ‘Encore’ Cultivar, bred by NI in Japan Imai et al. (2008) No discrepancy 30 ‘Tsunokaori’ ‘Kiyomi’ × ‘Okitsu Wase’y Cultivar, bred by NI in Japan Matsumoto et al. (1991) No discrepancy 31 ‘Tsunonozomi’ ‘Kiyomi’ × ‘Encore’ Cultivar, bred by NI in Japan Yoshioka et al. (2011) No discrepancy 32 ‘Yellow Pummelo’ Hassaku × ‘Hirado’ buntan Cultivar, bred by NI in Japan Ueno et al. (1985) No discrepancy 33 ‘Youkou’ ‘Kiyomi’ × ‘Nakano 3 gou’ ponkany Cultivar, bred by NI in Japan Matsumoto et al. (1999a) No discrepancy 34 Clementine Citrus clementina hort. Ex Tanaka Local variety, Algeria Hodgson (1967) 35 ‘E-647’ ‘Kiyomi’ × ‘Osceola’ Selection, bred by NI in Japan 36 ‘Encore’ King × ‘Willowleaf’ Cultivar, USA Hodgson (1967) 37 ‘EnOw No. 21’ ‘Encore’ × ‘Okitsu Wase’ Selection, bred by NI in Japan 38 Hassaku C. hassaku hort. Ex Tanaka Local variety, Japan Hodgson (1967) 39 ‘HF9’ ‘Hayashi’ unshu × ‘Fukuhara’ orangey Selection, bred by NI in Japan 40 ‘Hirado’ buntan C. grandis Osbeck Local variety, Japan Hodgson (1967) 41 ‘Kara’ ‘Owari’ unshu × King Cultivar, USA Hodgson (1967) 42 ‘Kawano’ Natsudaidai C. natsudaidai Hayata Local variety, Japan 43 King C. nobilis Lour. Cultivar, USA Hodgson (1967) 44 ‘KyEn No. 5’ ‘Kiyomi’ × ‘Encore’ Selection, bred by NI in Japan 45 ‘KyOw No. 14’ ‘Kiyomi’ × ‘Okitsu Wase’ Selection, bred by NI in Japan 46 ‘Lee’ Clementine × ‘Orlando’ Cultivar, USA Hodgson (1967) 47 ‘Mato’ buntan C. grandis Osbeck Local variety, China Hodgson (1967) 48 Mukaku kishu C. kinokuni hort. ex Tanaka Local variety, China Hodgson (1967) 49 ‘Murcott’ Unknown Cultivar, USA Hodgson (1967) 50 ‘No. 1408’ ‘EnOw No. 21’ × ‘No. 2681’ Selection, bred by NI in Japan 51 ‘Okitsu 46 gou’ ‘Sweet Spring’ × ‘Trovita’ orange Selection, bred by NI in Japan 52 ‘Okitsu Wase’ C. unshiu Marc. Cultivar, bred by NI in Japan Iwasaki et al. (1966) 53 ‘Page’ ‘Minneola’ × Clementine Cultivar, USA Hodgson (1967) 54 ‘Seminole’ ‘Duncan’ grapefruit × ‘Dancy’ Cultivar, USA Hodgson (1967) 55 ‘Tanikawa’ buntan C. grandis Osbeck Cultivar, Japan Hodgson (1967) 56 ‘Trovita’ orange C. sinensis (L.) Osbeck Cultivar, USA Hodgson (1967) 57 ‘Wilking’ King × ‘Willowleaf’ Cultivar, USA Hodgson (1967) 58 ‘Yoshida’ ponkan C. reticulata Blanco Local variety, Taiwan 59 ‘Osceola’ Clementine × ‘Orlando’ Cultivar, USA Hodgson (1967) z Citrus species are based on Tanaka (1954). y ‘Okitsu Wase’, ‘Trovita’, and ‘Yoshida’ were used in the analysis as representatives of mutants of satsuma mandarin, sweet orange, and ponkan, respectively. x NI is abriviation of the National Institute. Table 2. Characteristics of the CAPS markers used in this study. The first 19 CAPS markers were used in the cultivar identification and parentage analysis for all 33 NIFTS cultivars; the next 18 markers were used to clarify any parentage discrepancies revealed by the first 19 markers. 4 K. Nonaka, H. Fujii, M. Kita, T. Shimada, T. Endo, T. Yoshioka and M. Omura type Segregation aa:ab aa:ab:bb aa:ab aa:ab ab:aa aa:ab:bb ab:aa aa:ab aa:ab ab:aa aa:ab:bb aa:ab aa:ab:bb ab:aa aa:ab:bb ab:aa aa:ab aa:ab ab:aa ab:aa aa:ab:bb aa:ab:bb ab:aa ab:aa ab:aa aa:ab:bb aa:ab:bb ab:aa ab:aa aa:ab:bb ab:aa ab:aa aa:ab:bb aa:ab aa:ab:bb aa:ab:bb ab:aa (bb:bc) w y 1 2 1 1 1 2 1 1 1 1 2 1 2 1 2 1 1 1 1 1 2 2 1 1 1 2 2 1 1 2 1 1 2 1 2 2 d.f. 1 (1) x 8.4* 5.7* -value 2.1ns 2.6ns 1.2ns 0.6ns 6.6ns 1.7ns 5.1ns 0.3ns 0.6ns 0.1ns 5.8ns 0.0ns 3.5ns 5.2ns 0.9ns 2.0ns 0.6ns 1.9ns 0.6ns 3.3ns 8.4ns 3.3ns 1.8ns 0.0ns 1.4ns 6.5ns 2.7ns 1.1ns 7.2ns 2.3ns 2.2ns 7.5ns 7.8ns 0.1ns 2 χ 0.1ns (0.0ns) Type of segregation Type aa aa aa aa aa aa aa aa aa aa aa aa aa aa ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab parent Pollen aa (bc) parents aa aa aa aa aa aa aa aa aa ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab Genotype of Seed parent ab (bb) z 8921411 6411254 3485637 2701440 7583093 5254755 8685984 7049430 2509737 7928192 6643448 22844111 25511133 11614367 11830875 11715646 31250356 19608059 43863744 24612654 23595492 37192231 23782088 35363026 22737521 18070262 21960709 40630241 16716984 22193554 24905716 17074755 15058632 23459052 16912088 38571337 16233365 End position Start position 3483334 2697793 31244440 19605473 43862440 11611994 8914744 24606501 23593516 37189653 7579900 23776796 35354776 22840947 22730839 5253281 6409311 8683898 18068297 7924189 21953129 40628244 16712914 11826472 7047024 22191056 24898769 17057658 11707953 2501520 15056834 25506446 6632695 16909492 38566192 23456630 16232756 Scaffold scaffolf_9 scaffold_3 scaffold_8 scaffold_4 scaffold_3 scaffold_6 scaffold_3 scaffold_6 scaffold_6 scaffold_5 scaffold_9 scaffold_1 scaffold_2 scaffold_1 scaffold_8 scaffold_3 scaffold_3 scaffold_2 scaffold_1 scaffold_6 scaffold_8 scaffold_5 scaffold_6 scaffold_6 scaffold_3 scaffold_8 scaffold_1 scaffold_4 scaffold_6 scaffold_7 scaffold_4 scaffold_4 scaffold_8 scaffold_6 scaffold_5 scaffold_8 scaffold_4 EST annotation of Clementine Ver1.0 annotation of Clementine EST z cM When the annealing temperature reached 54°C, amplification was continued at 54°C for 30 cycles. 25.049 73.105 79.515 55.503 54.726 96.565 43.295 37.102 41.367 46.823 46.197 27.489 17.966 29.144 20.703 29.035 42.609 84.912 58.42 29.152 30.721 39.296 44.663 93.624 45.092 25.22 25.752 43.879 95.613 69.928 45.051 86.113 47.41 88.433 42.789 13.499 43.542 (AGI map) Position in the integration map group AGI-05 AGI-08 AGI-03 AGI-07 AGI-05 AGI-08 AGI-05 AGI-04 AGI-04 AGI-09 AGI-03 AGI-01 AGI-06 AGI-01 AGI-08 AGI-05 AGI-05 AGI-09 AGI-01 AGI-07 AGI-08 AGI-09 AGI-07 AGI-08 AGI-05 AGI-08 AGI-01 AGI-07 AGI-08 AGI-02 AGI-07 AGI-07 AGI-09 AGI-08 AGI-09 AGI-08 AGI-07 Linkage u 600 700 800 1000 (500) common both allele by 2°C for every 2 cycles. fragment of 750 350 250 200 400 650 200 250 350 600 450 400 250 300 550 b allele 700 850 900 750 500 450 600 400 300 600 900 550 800 300 300 800 900 300 300 600 700 350 600 450 400 800 750 600 850 700 450 450 600 1000 1000 1200 1000 Polymorphic fragment size (bp) 800 800 800 900 900 500 650 600 500 900 550 400 800 950 500 650 700 850 600 800 1100 1100 1100 1100 1100 1600 1000 1200 1400 1200 2200 1400 1000 1400 1200 1250 1200 a allele 800 800 900 500 600 600 550 400 900 950 500 700 800 600 800 (bp) PCR 1100 1100 1100 1100 1400 1600 1000 1200 1400 1800 1400 1800 2200 1000 1200 1400 1000 1600 1400 1200 1250 1200 product size test). 2 v 66 62 64 58 56 56 58 56 60 60 60 58 56 58 56 62 56 54 60 58 58 64 54 Td Td Td Td Td Td Td Td Td Td Td Td Td Td (°C) 0.05 (χ

Annealing < Temperature Temperature

The annealing temperature starting at 64°C was reduced P I enzyme Nde II Hind III Dra I Hinf I Dra I Hinf I Msp I Msp I STS Msp I EcoR Hae III Msp I Msp I Pvu II Msp I Nde II Sty I Hinc II Hinf I Dra I Rsa I Sty I Hinf I Rsa I Hae III Rsa I Hha I Hha I Hinf I Hinc II Dra I Pvu II Pvu II Msp I Msp I Hae III Restriction Characteristics of the CAPS markers used in this study. The first 19 CAPS markers were used in the cultivar identification and parentage analysis for all 33 NIFTS cultivars; the next 18 markers were markers 18 next the cultivars; NIFTS 33 all for analysis parentage and identification cultivar the in used were markers CAPS 19 first study.The this in used markers CAPS the of Characteristics used to clarify any parentage discrepancies revealed by the first 19 markers. down PCR. Touch name Marker Cp2223 Tf0250 Tf0300 Tf0348 Tf0350 Tf0364 Tf0386 Wy0003 Cp0336 Al0413 Al0636 Bf0028 Bf0036 Bf0145 Bf0158 Cp1624 Al0326 Bf0029 from Shimada et al. (2014). d.f., degrees of freedom. III and the values in parentheses of values in parentheses Tf0250/ Hind III and the analysis of and ‘Kankitsu Chukanbohon Nou 5 Gou’. Segregation Tf0250/ Hind III are ‘Okitsu 46 gou’ except parents al. (2014). Seed and pollen et from Shimada and ‘Setoka’. of the cross between ‘E-647’ Fb0159/ Hinc II were analyzed on the basis of offspring showed the fragment size of c allele. Gn0043 If0208 Mf0097 Tf0062 Tf0079 Tf0150 Tf0168 Tf0235 Tf0271 Tf0326 Bf0136 Cp0975 Fb0159 Tf0165 Gn0071 If0211 Ov0305 Tf0069 Gn0040

ns, not significant; *, Td.

z y x w v u Table 2. Hort. J. Preview 5 product with 1.0 μL of the buffer and 2 to 3 units of the electrophoresis results. Table 3 shows the observed het- restriction enzyme, and then adjusted the volume to a erozygosity (Ho) and polymorphism information con- total of 10 μL with sterilized water. After digestion at tent (PIC) of each CAPS marker, which could be used

37°C for more than 2 h, the segmentation patterns were to evaluate their genetic diversities. Ho was calculated analyzed by means of 1.5% agarose gel electrophoresis as the percentage of individuals with heterozygous geno- with ethidium bromide staining. types that were actually observed in the 59 accessions, Of the 37 CAPS markers, 19 were used for the culti- and PIC was calculated according to the method of var identification and parentage analysis (Table 3), and Botstein et al. (1980). Ho for the CAPS markers ranged the other 18 for parentage analysis for cultivars that from 0.246 to 0.820, and averaged 0.461. PIC ranged showed discrepancies in the previously reported parent– from 0.207 to 0.375, and averaged 0.325. These mark- offspring relationship. ers were assigned to the reference genetic map of the Clementine (C. clemetina hort. ex Tanaka) scaffolds Identification of citrus cultivars and parentage analysis v. 1.0 developed by Ollitrault et al. (2012), and at least We used MinimalMarker software (Fujii et al., one marker was located in each of the nine scaffolds 2013a) to select minimal CAPS marker subsets for (Table 2). Segregation of the genotypes of the 19 mark- identification of tested cultivars on the basis of the anal- ers in the F1 progeny is shown in Table 2. All markers ysis results with the 19 markers. We then used the geno- except Bf0028/HaeIII showed Mendelian segregation types defined by the selected CAPS markers to identify which fitted the expected ratio deduced from the parent 45 cultivars, 7 local varieties, and 7 selections. genotypes (χ2 test, P > 0.05), indicating that each CAPS We used MARCO software (Fujii et al., 2010) to marker was derived from one locus. The Bf0028/HaeIII identify the parent–offspring relationship on the basis marker showed a significant χ2 result (P < 0.05), but of the inheritance of one allele at each CAPS locus was nonetheless judged to be a suitable marker because being transmitted from the parents to the offspring. allele inheritance based on it was evaluated using more MARCO could arbitrarily set a cultivar as the “parent” than 10 combinations of parents and their offspring, and or “offspring” from a supplied dataset. To confirm the 1 allele was transmitted from the parents to their off- assumed parent–offspring relationship, we defined the spring without any discrepancies. Thus, we concluded 33 cultivars developed in the citrus breeding program that the 19 CAPS markers were each amplified from a by the national institute of Japan as “offspring”, and the single locus, and could be effectively used in cultivar cultivars or selections reported as parents in previous identification and parentage analysis. records as “parents”. The software then revealed wheth- er the inheritance pattern based on the genetic markers Cultivar identification supported the assumed parentage or revealed a different The genotyping data based on the 19 CAPS markers one. We judged that a discrepancy in the parent– for the 59 accessions (Table 3) were analyzed with offspring relationship existed if neither of the two al- MinimalMarker software (Fujii et al., 2013a) to esti- leles in the offspring existed in the candidate parent. mate the minimal number of markers that could be used to identify the 33 cultivars developed by the citrus Validation of the putative parent–offspring relationships breeding program in the national institute. We found a using SNP genotyping data set of 8 CAPS markers that was sufficient to discrimi- We used the 116 SNPs randomly selected from the nate among 57 accessions, except for ‘Mato Buntan’ 268 reliable SNPs obtained from genotyping data of (C. grandis Osbeck) and ‘Hirado Buntan’ (C. grandis 103 citrus accessions analyzed by Fujii et al. (2013b) to Osbeck), resulting in 7 subsets of the 19 CAPS mark- validate the parent–offspring relationships and to identi- ers: fy the putative parentage predicted based on the CAPS Set 1: Al0413/MspI, Bf0029/StyI, Bf0036/MspI, marker analysis performed in MARCO. SNP genotyp- Cp1624/MspI, If0208/HinfI, Tf0062/RsaI, Tf0150/ ing data for accessions that were not analyzed by Fujii HinfI, Tf0326/HhaI et al. (2013b) were acquired by using the same genotyp- Set 2: Al0413/MspI, Bf0028/HaeIII, Bf0036/MspI, ing method (i.e., the GoldenGate assay). Cp1624/MspI, Mf0097/DraI, Tf0150/HinfI, Tf0271/ RsaI, Tf0326/HhaI Results Set 3: Al0413/MspI, Bf0028/HaeIII, Bf0036/MspI, Selection and evaluation of 19 CAPS markers for culti- Cp1624/MspI, Tf0150/HinfI, Tf0235/HaeIII, Tf0271/ var identification and parentage analysis RsaI, Tf0326/HhaI We selected 19 CAPS markers from the frame mark- Set 4: Al0413/MspI, Bf0036/MspI, Bf0158/PvuII, ers in the linkage map of Shimada et al. (2014) and Cp1624/MspI, If0208/HinfI, Tf0062/RsaI, Tf0150/ evaluated their use for cultivar identification and par- HinfI, Tf0326/HhaI entage analysis using the 59 accessions (Table 3). The Set 5: Al0413/MspI, Bf0036/MspI, Cp1624/MspI, sample genotypes for each CAPS marker were decided If0208/HinfI, Tf0062/RsaI, Tf0079/StyI, Tf0150/HinfI, from the fragment patterns obtained from images of the Tf0326/HhaI Table6 3. The geneotypes for the 19K. CAPS Nonaka, markers H. Fujii, and theM. Kita,associated T. Shimada, embryo T. types Endo, of T.the Yoshioka 59 cultivars and andM. Omuraselections used in the parentage analysis. The underlined genotypes represent discrepancies with the assumed parent-offspring relationship. Table 3. The geneotypes for the 19 CAPS markers and the associated embryo types of the 59 cultivars and selections used in the parentage analysis. The underlined genotypes represent discrepancies with the assumed parent-offspring relationship.

CAPS marker Observed heterozygosity (Ho)/polymorphic information contents (PIC) Embryo Code Sample name typez Al0326/NdeII Al0413/MspI Al0636/EcoRI Bf0028/HaeIII Bf0029/StyI Bf0036/MspI Bf0145/MspI Bf0158/PvuII Cp1624/MspI 0.475/0.326 0.508/0.358 0.559/0.322 0.576/0.326 0.322/0.278 0.492/0.364 0.254/0.265 0.559/0.322 0.492/0.351 1 ‘Akemi’ p aa ab ab ab aa ab aa bb ab 2 ‘Amaka’ m aa aa bb ab aa ab aa ab bb 3 ‘Amakusa’ p ab ab ab aa ab ab aa ab ab 4 ‘Ariake’ m aa ab bb aa ab ab ab ab bb 5 ‘Asumi’ m ab aa ab aa aa ab ab ab aa 6 ‘Benibae’ m ab ab ab aa aa aa aa bb ab 7 ‘Benimadoka’ m aa bb ab aa bb bb bb bb aa 8 ‘Harehime’ m ab ab ab ab aa ab aa ab ab 9 ‘Hareyaka’ p aa aa bb ab aa bb aa bb ab 10 ‘Harumi’ p ab ab ab ab aa ab aa ab aa 11 ‘Hayaka’ p aa aa bb ab aa ab aa ab bb 12 ‘Kankitsu Chukanbohon Nou 5 Gou’ m ab ab ab ab aa ab aa ab bb 13 ‘Kankitsu Chukanbohon Nou 6 Gou’ m aa ab bb ab aa ab aa bb bb 14 ‘Kiyomi’ m ab ab ab ab ab ab aa ab ab 15 ‘May Pummelo’ m aa bb ab aa ab bb ab bb ab 16 ‘Mihaya’ m ab aa bb aa aa ab aa bb bb 17 ‘Miho-core’ p ab aa bb ab aa bb aa bb bb 18 ‘Nankou’ m ab aa bb ab ab ab aa ab bb 19 ‘Nishinokaori’ m ab ab ab ab aa aa aa ab aa 20 ‘Reikou’ p ab ab ab ab aa ab aa ab bb 21 ‘Seinannohikari’ p aa aa bb ab aa ab aa ab bb 22 ‘Setoka’ p aa ab ab aa ab ab ab bb ab 23 ‘Shiranuhi’ p ab aa ab aa aa aa aa ab ab 24 ‘Southern Red’ m aa aa bb aa aa bb aa bb bb 25 ‘Southern Yellow’ m aa ab ab ab aa bb ab ab ab 26 ‘Summer Fresh’ p aa bb bb aa ab bb ab bb ab 27 ‘Sweet Spring’ m aa aa bb ab ab bb ab bb bb 28 ‘Tamami’ m bb aa ab ab aa aa aa ab bb 29 ‘Tsunokagayaki’ m ab aa ab ab aa ab aa ab ab 30 ‘Tsunokaori’ p ab ab ab ab ab ab aa ab ab 31 ‘Tsunonozomi’ m ab ab ab aa aa aa aa bb ab 32 ‘Yellow Pummelo’ m aa ab ab aa ab bb ab bb ab 33 ‘Youkou’ p aa aa ab aa aa aa aa ab ab 34 Clementine m ab ab ab ab ab aa aa ab ab 35 ‘E-647’ m ab bb ab aa aa aa aa ab ab 36 ‘Encore’ m ab aa bb ab aa ab aa bb bb 37 ‘EnOw No.21’ m ab aa bb ab aa bb aa bb bb 38 Hassaku m aa ab ab aa ab bb ab bb ab 39 ‘HF9’ m ab ab ab ab aa ab aa ab ab 40 ‘Hirado’ buntan m aa bb ab aa bb bb bb bb aa 41 ‘Kara’ p aa aa bb ab aa bb aa ab bb 42 ‘Kawano’ Natsudaidai p aa ab ab ab ab bb ab bb ab 43 King p aa ab bb ab aa ab aa ab bb 44 ‘KyEn No.5’ m bb aa bb ab ab bb aa ab bb 45 ‘KyOw No.14’ m ab aa ab ab ab ab aa ab ab 46 ‘Lee’ m bb ab ab aa aa aa aa ab bb 47 ‘Mato’ buntan m aa bb ab aa bb bb bb bb aa 48 Mukaku kishu m aa aa bb ab aa ab aa ab ab 49 ‘Murcott’ p aa ab bb aa aa bb ab bb bb 50 ‘No.1408’ m aa ab bb ab aa bb aa bb bb 51 ‘Okitsu 46 gou’ m ab aa ab aa ab ab ab bb ab 52 ‘Okitsu Wase’ p aa aa bb ab aa bb aa ab bb 53 ‘Page’ p ab ab bb aa ab ab ab ab ab 54 ‘Seminole’ p ab ab bb ab aa bb ab ab bb 55 ‘Tanikawa’ buntan m aa bb ab aa ab bb bb bb ab 56 ‘Trovita’ orange p ab ab ab aa ab ab ab ab ab 57 ‘Wilking’ m ab ab bb ab aa ab aa bb ab 58 ‘Yoshida’ ponkan p ab ab bb ab aa ab aa ab ab 59 ‘Osceola’ m aa ab bb aa aa ab aa bb bb z Embryo types: “p”, polyembryony; “m”, monoembryony. Table 3. Continued. Hort. J. Preview 7

Table 3. Continued. CAPS marker

Observed heterozygosity (Ho)/polymorphic information contents (PIC) Code Sample name Gn0043/HincII If0208/HinfI Mf0097/DraI Tf0062/RsaI Tf0079/StyI Tf0150/HinfI Tf0168/RsaI Tf0235/HaeIII Tf0271/RsaI Tf0326/HhaI 0.559/0.356 0.475/0.341 0.441/0.326 0.322/0.322 0.847/0.369 0.475/0.317 0.492/0.330 0.407/0.317 0.271/0.207 0.525/0.375 1 ‘Akemi’ aa ab ab aa ab bb aa ab aa bb 2 ‘Amaka’ ab bb ab aa ab ab aa ab aa aa 3 ‘Amakusa’ ab bb ab ab ab bb aa aa ab bb 4 ‘Ariake’ ab ab ab aa ab bb aa ab aa ab 5 ‘Asumi’ ab ab ab aa ab ab ab ab aa bb 6 ‘Benibae’ aa bb bb ab ab bb aa aa aa bb 7 ‘Benimadoka’ bb aa aa bb ab bb aa bb aa aa 8 ‘Harehime’ ab ab ab ab ab ab ab ab ab ab 9 ‘Hareyaka’ aa bb bb aa ab ab ab aa aa ab 10 ‘Harumi’ aa bb bb aa ab bb aa aa aa bb 11 ‘Hayaka’ ab bb bb aa aa ab ab aa aa bb 12 ‘Kankitsu Chukanbohon Nou 5 Gou’ aa ab ab aa aa bb ab ab aa bb 13 ‘Kankitsu Chukanbohon Nou 6 Gou’ ab bb bb ab ab ab ab aa ab ab 14 ‘Kiyomi’ ab ab ab aa ab ab aa ab aa ab 15 ‘May Pummelo’ ab ab aa bb ab bb aa bb aa ab 16 ‘Mihaya’ aa bb bb aa ab bb aa aa aa aa 17 ‘Miho-core’ aa bb bb aa ab bb ab aa aa ab 18 ‘Nankou’ ab aa bb aa ab ab ab aa aa aa 19 ‘Nishinokaori’ ab ab ab aa ab ab aa ab aa ab 20 ‘Reikou’ aa bb bb aa ab bb aa aa aa bb 21 ‘Seinannohikari’ ab bb bb ab ab ab ab aa ab ab 22 ‘Setoka’ ab bb bb aa ab bb aa aa aa aa 23 ‘Shiranuhi’ ab bb ab aa ab bb aa ab aa ab 24 ‘Southern Red’ aa ab bb aa ab bb ab aa aa ab 25 ‘Southern Yellow’ ab ab ab ab aa ab ab ab aa ab 26 ‘Summer Fresh’ aa ab ab ab ab bb ab ab aa ab 27 ‘Sweet Spring’ ab bb ab ab ab ab bb ab ab ab 28 ‘Tamami’ aa ab bb aa aa bb aa aa aa ab 29 ‘Tsunokagayaki’ ab bb bb aa ab aa aa aa aa aa 30 ‘Tsunokaori’ aa ab ab ab ab ab aa ab ab bb 31 ‘Tsunonozomi’ ab bb bb aa ab ab aa aa aa ab 32 ‘Yellow Pummelo’ ab ab ab bb ab bb ab ab aa ab 33 ‘Youkou’ ab bb bb aa ab ab aa aa aa bb 34 Clementine aa ab bb aa aa bb aa aa aa ab 35 ‘E-647’ aa ab ab aa ab bb aa ab aa ab 36 ‘Encore’ aa bb bb aa ab ab ab aa aa ab 37 ‘EnOw No.21’ ab ab bb ab ab ab bb aa ab aa 38 Hassaku ab ab ab bb ab ab ab ab ab ab 39 ‘HF9’ ab bb bb ab ab ab ab aa aa bb 40 ‘Hirado’ buntan bb aa aa bb ab ab aa bb aa aa 41 ‘Kara’ ab bb bb aa ab ab ab aa aa aa 42 ‘Kawano’ Natsudaidai ab ab ab ab ab ab aa ab ab ab 43 King ab bb bb ab ab ab ab aa ab aa 44 ‘KyEn No.5’ aa ab ab aa aa ab ab ab aa aa 45 ‘KyOw No.14’ bb bb ab ab ab ab ab aa ab ab 46 ‘Lee’ ab ab ab aa aa bb ab ab aa bb 47 ‘Mato’ buntan bb aa aa bb ab ab aa bb aa aa 48 Mukaku kishu aa bb bb aa aa bb bb aa aa bb 49 ‘Murcott’ ab bb ab ab ab bb ab ab ab ab 50 ‘No.1408’ aa ab bb aa ab bb ab aa aa aa 51 ‘Okitsu 46 gou’ ab ab ab ab ab ab ab ab ab ab 52 ‘Okitsu Wase’ ab ab bb ab ab ab ab aa ab ab 53 ‘Page’ ab ab bb aa ab bb aa aa aa ab 54 ‘Seminole’ ab ab ab aa ab bb ab ab aa ab 55 ‘Tanikawa’ buntan bb ab ab bb ab aa ab ab ab aa 56 ‘Trovita’ orange ab ab ab ab ab bb aa ab aa ab 57 ‘Wilking’ aa bb bb ab ab bb ab aa ab ab 58 ‘Yoshida’ ponkan aa bb bb aa aa bb ab aa aa bb 59 ‘Osceola’ aa aa bb aa ab bb aa aa aa ab 8 K. Nonaka, H. Fujii, M. Kita, T. Shimada, T. Endo, T. Yoshioka and M. Omura

Set 6: Al0413/MspI, Bf0036/MspI, Cp1624/MspI, ‘Tsunonozomi’ and the pollen parent ‘Murcott’. In this If0208/HinfI, Tf0062/RsaI, Tf0150/HinfI, Tf0168/RsaI, parent–offspring relationship, there were discrepancies Tf0326/HhaI at Cp0336/STS, Cp2223/NdeII, and Tf0250/HindIII Set 7: Al0413/MspI, Bf0036/MspI, Cp1624/MspI, (Fig. 1), and the result for Cp2223/NdeII suggested that If0208/HinfI, Tf0062/RsaI, Tf0150/HinfI, Tf0271/RsaI, ‘Tsunonozomi’ could not be the seed parent. ‘Murcott’ Tf0326/HhaI. was compatible with being the pollen parent because Both ‘Mato’ buntan and ‘Hirado’ buntan are pumme- both alleles at all 37 CAPS marker loci were trans- lo (C. grandis Osbeck) local varieties and have the mitted without any discrepancies (Table 4). Among the same genotypes at all 19 CAPS markers. Pummelo has candidate parent accessions, ‘KyEn No. 4’ appears high homozygosity at most loci (Moore, 2001), so it more likely to be the seed parent than ‘Tsunonozomi’. may be difficult to find polymorphisms among pumme- ‘Southern Red’ is registered as being derived from a lo cultivars. ‘Mato’ buntan and ‘Hirado’ buntan were cross between the seed parent ‘Kara’ and the pollen par- not distinguished by the seven subsets of the CAPS ent ponkan. In this parent–offspring relationship, there markers, but this may not be important because both were discrepancies at Fb0159/HincII, Gn0071/PvuII, cultivars are local varieties with no breeders’ rights to and If0211/PvuII (Fig. 2), and the result for Fb0159/ protect, and other developed pummelo cultivars such as HincII indicated that ponkan could not be the pollen par- ‘Benimadoka’ (C. grandis Osbeck) could be distin- ent. ‘Kara’ was compatible with being the seed parent guished from them by the marker sets. because at least one of the two alleles for each of the 34 CAPS markers loci was transmitted without any dis- Parentage analysis crepancies (Table 5). Among the candidate parent ac- We compared previously reported parent–offspring cessions, ‘Osceola’ was predicted to be the pollen relationships with the parentage predicted using the 19 parent instead of ponkan. CAPS markers within the 33 citrus cultivars in In contrast, we could not confirm the seed parent for MARCO software. The reported relationships were ‘Reikou’, which was registered as being derived from a validated for 30 of the cultivars, but not for ‘Setoka’, cross between the seed parent ‘KyEn No. 5’ and the ‘Southern Red’, or ‘Reikou’ (Table 1). Every allele at pollen parent ‘Murcott’, because there were discrepan- the 19 CAPS marker loci within the 30 cultivars was cies at Al0636/EcoRI, Bf0036/MspI, Tf0326/HhaI, and the previously reported parent allele. However, Tf0300/DraI (Table 6). ‘Murcott’ was compatible with ‘Setoka’, ‘Southern Red’, and ‘Reikou’ had a discrep- being the pollen parent because at least one of two al- ant allele at one or more loci compared with their re- leles for each of the 34 CAPS markers’ loci was trans- ported parents: there were discrepancies at Bf0029/StyI mitted without any discrepancies (Table 6). in ‘Setoka’; at If0208/HinfI in ‘Southern Red’; and at Al0636/EcoRI, Bf0036/MspI, and Tf0326/HhaI in Validation of the putative parent–offspring relationships ‘Reikou’. In the case of ‘Reikou’, ‘Murcott’ was cor- using SNP genotyping data rectly described as the pollen parent, but the discrepan- To further evaluate the putative parentage of ‘Setoka’ cy at Tf0326/HhaI showed that ‘KyEn No. 5’ was not and ‘Southern Red’, we used 116 reliable SNPs from correctly described as the seed parent. However, it was the analysis by Fujii et al. (2013b). We obtained the not clear which parent was discrepant in ‘Setoka’ and genotyping data for ‘KyEn No. 4’, which was not in- ‘Southern Red’ solely on the basis of the genotyping cluded in their analysis, by following their method; data from the 19 CAPS markers. thus, we used genotype data from a total of 104 citrus To solve this problem, we applied an additional 15 accessions for the parentage validation. CAPS markers to the parentage analysis for ‘Southern We confirmed discrepancies in the parentage of Red’ and ‘Reikou’, and an additional 18 (including the ‘Setoka’ at six SNP markers (SI116, SI145, SI209, aforementioned 15 markers) for ‘Setoka’ (Table 2). Fur- SI269, SI282, and SI363), and the ‘Tsunonozomi’ al- thermore, we included ‘KyEn No. 3’, ‘KyEn No. 4’, leles had discrepancies at three of these (SI145, SI209, ‘KyEn No. 86’, ‘Minneola’, and ‘Osceola’ as potential and SI282; Table 4). In contrast, there were no discrep- true parents, since these cultivars were commonly uti- ancies in the inheritance of the alleles at any marker lized as parents in the cross-breeding program at the when the parents for ‘Setoka’ were ‘KyEn No. 4’ and time when the three discrepant cultivars were produced. ‘Murcott’. Furthermore, when ‘Murcott’ was fixed as ‘KyEn No. 3’, ‘KyEn No. 4’, ‘KyEn No. 5’, and ‘KyEn the pollen parent, MARCO indicated ‘KyEn No. 4’ as No. 86’ were full siblings of ‘Tsunonozomi’ (that is, the only seed parent. These results strongly support our they were selected from seedlings that resulted from a finding that ‘Setoka’ is the offspring of a cross between cross between ‘Kiyomi’ and ‘Encore’). ‘KyEn No. 4’ and ‘Murcott’. The genotyping data in the parentage analysis for We also confirmed discrepancies in the parentage of ‘Setoka’ and ‘Southern Red’ are summarized in Tables ‘Southern Red’, which was reported as being the result 4 and 5, respectively. ‘Setoka’ is registered as being of a cross between ‘Kara’ and ponkan, at seven SNP derived from a cross between the seed parent markers (SI173, SI199, SI228, SI270, SI322, SI348, Hort. J. Preview 9

Table 4. CAPS and SNP genotypes for the reported and putative parents of ‘Setoka’. The underlined CAPS genotypes represent discrepancies between the reported parent–offspring relationship, in which ‘Setoka’ resulted from a cross between ‘Tsunonozomi’ and ‘Murcott’, and the marker results. A discrepancy ex- Table 4. CAPS and SNPists genotypes if neither for of thethe reportedtwo alleles and in putative the offspring parents exist of in‘Setoka’. the candidate The underlined parent. The CAPS first genotypes19 CAPS markersrepresent discrepancies between the reportedwere parent–offspringused in the overall relationship, parentage inanalysis which for‘Setoka’ all 33 resulted NIFTS cultivars;from a cross the between next 18 ‘Tsunonozomi’markers were used and to‘Murcott’, and the marker results. clarify any parentage discrepancies revealed by the first 19 markers. SNP marker results are shown only for loci with discrepancies.

Cultivar Marker name ‘Setoka’ ‘Tsunonozomi’ ‘Murcott’ ‘KyEn No. 4’ CAPS marker Al0326/NdeII aa ab aa aa Al0413/MspI ab ab ab ab Al0636/EcoRI ab ab bb ab Bf0028/HaeIII aa aa aa ab Bf0029/StyI ab aa aa ab Bf0036/MspI ab aa bb ab Bf0145/MspI ab aa ab aa Bf0158/PvuII bb bb bb bb Cp1624/MspI ab ab bb ab Gn0043/HincII ab ab ab aa If0208/HinfI bb bb bb bb Mf0097/DraI bb bb ab bb Tf0062/RsaI aa aa ab aa Tf0079/StyI ab ab ab ab Tf0150/HinfI bb ab bb bb Tf0168/RsaI aa aa ab ab Tf0235/HaeIII aa aa ab aa Tf0271/RsaI aa aa ab aa Tf0326/HhaI aa ab ab aa Bf0136/HhaI bb bb ab bb Cp0975/HinfI ab ab ab ab Fb0159/HincII bc ab ac ab Gn0040/HaeIII bb bb ab bb Gn0071/PvuII bb bb bb bb If0211/PvuII aa aa aa aa Ov0305/MspI ab aa bb aa Tf0069/MspI bb ab ab ab Tf0165/DraI bb bb bb bb Tf0300/DraI ab ab bb aa Tf0348/HinfI ab ab ab ab Tf0350/DraI ab ab ab ab Tf0364/HinfI ab aa ab aa Tf0386/MspI ab ab ab ab Wy0003/MspI aa ab ab ab Cp0336/STS ab aa aa ab Cp2223/NdeII aa bb ab ab Tf0250/HindIII ab aa aa ab SNP marker SI116 CG GG GG CG SI145 TT AA AT AT SI209 GG AA AG GG SI269 AG AA AA AG SI282 AA GG AG AG SI363 AT AA AA AT 10 K. Nonaka, H. Fujii, M. Kita, T. Shimada, T. Endo, T. Yoshioka and M. Omura

Table 5. CAPS and SNP genotypes for the reported and putative and SI363), and the ponkan alleles had discrepancies at parents of ‘Southern Red’. The underlined CAPS genotypes two of these (SI270 and SI348; Table 5). In contrast, represent discrepancies between the reported parent– there were no discrepancies in the inheritance of the al- offspring relationship, in which ‘Southern Red’ resulted from a cross between ‘Kara’ and ponkan, and the marker re- leles at any marker when the parents for ‘Southern Red’ sults. A discrepancy exists if neither of the two alleles in the were ‘Kara’ and ‘Osceola’. Furthermore, when ‘Kara’ offspring exist in the candidate parent. The first 19 CAPS was fixed as the seed parent, MARCO predicted markers were used in the overall parentage analysis for all Table 5. CAPS and SNP genotypes for the reported and putative parents of ‘Southern‘Osceola’ Red’. as The the underlined only possible CAPS genotypes pollen represent parent. discrepancies These re- 33 NIFTS cultivars; the next 15 markers were used to clarify sults strongly suggest that ‘Southern Red’ is the off- anybetween parentage the reported discrepancies parent–offspring revealed byrelationship, the first 19 in mark-which ‘Southern Red’ resulted from a cross between ‘Kara’ and ponkan, and the ers.marker SNP results. marker results are shown only for loci with dis- spring of a cross between ‘Kara’ and ‘Osceola’. crepancies. We also confirmed discrepancies in the parentage of ‘Reikou’, which was reported as being the result of a Cultivar Marker name cross between ‘KyEn No. 5’ and ‘Murcott’, at nine SNP ‘Southern Red’ ‘Kara’ Ponkan ‘Osceola’ markers (SI124, SI156, SI158, SI190, SI220, SI251, CAPS marker SI313, SI334, and SI373), and the ‘KyEn No. 5’ alleles Al0326/NdeII aa aa ab aa had a discrepancy at one of these (SI190; Table 5). Al0413/MspI aa aa ab ab However, when ‘Murcott’ was fixed as the pollen par- Al0636/EcoRI bb bb bb bb ent, MARCO could not select a compatible seed parent Bf0028/HaeIII aa ab ab aa with no discrepancies. Bf0029/StyI aa aa aa aa Bf0036/MspI bb bb ab ab Discussion Bf0145/MspI aa aa aa aa Intellectual property protection for plant breeders has Bf0158/PvuII bb ab ab bb become an important issue, and techniques for cultivar Cp1624/MspI bb bb ab bb identification and parentage confirmation based on mo- Gn0043/HincII aa ab aa aa lecular markers have been developed to solve this prob- If0208/HinfI ab bb bb aa lem in various plants. In Japanese citrus breeding, Mf0097/DraI bb bb bb bb Ninomiya et al. (2015) developed genotyping data Tf0062/RsaI ab ab aa ab based on nine CAPS markers for 33 representative cul- Tf0079/StyI aa aa aa aa tivars and selections, and used this to develop protec- Tf0150/HinfI bb ab bb bb tion for citrus cultivars developed in the Ehime Tf0168/RsaI ab ab ab aa Prefecture breeding program. Our result complement Tf0235/HaeIII aa aa aa aa this previous work. However, additional genotyping Tf0271/RsaI aa aa aa aa data will be required to protect breeders’ rights for the Tf0326/HhaI ab aa bb ab major citrus cultivars distributed in Japan. This is be- Bf0136/HhaI bb bb bb bb cause the CAPS markers we used are mainly bi-allelic Cp0975/HinfI ab bb ab ab markers and have a lower PIC than would be provided Fb0159/HincII bb ab aa bb by multi-allelic markers such as SSRs. In addition, su- Gn0040/HaeIII bb bb bb bb perior cultivars, their offspring, and their full siblings Gn0071/PvuII ab bb bb ab are frequently used as breeding parents in the current If0211/PvuII ab aa aa bb breeding program. This makes it difficult to develop Ov0305/MspI ab ab bb ab methods for cultivar identification and to find discrep- Tf0069/MspI ab ab ab aa ancies in the reported parentage, since the developed Tf0165/DraI bb bb bb bb cultivars and selections have similar genetic back- Tf0300/DraI bb ab bb bb grounds. Tf0348/HinfI ab ab bb bb In strawberry breeding (Fragaria × ananassa Duch.), Tf0350/DraI ab ab aa ab CAPS markers have contributed greatly to preventing Tf0364/HinfI bb ab ab ab infringement of breeders’ rights (Kunihisa, 2011). Tf0386/MspI ab ab ab aa Packed strawberry fruits imported from Korea that were Wy0003/MspI bb ab bb bb labeled as ‘Nyohou’ were identified as a mix of SNP marker ‘Redpearl’ and ‘Sachinoka’ by using CAPS markers SI173 AG AA AA AG (Kunihisa et al., 2005). After warnings and legal action SI199 AC AA AA CC against the brokers, the amount of illegally imported SI228 CG CC CC GG strawberry fruits decreased sharply. In other infringe- SI270 GG CG CC CG ment cases, DNA profiling methods were developed for SI322 AT AA AA AT the sweet cherry (Prunus avium L.), rush (Juncus SI348 AA AG GG AA effuses L.), kidney beans (Phaseolus vulgaris L.), and SI363 AT AA AA AT adzuki beans (Vigna angularis (Willd.) Ohwi & Ohashi), among others (Shoda et al., 2012). Our results Hort. J. Preview 11

Fig. 1. CAPS markers fragment patterns that show a discrepancy in the parentage of ‘Setoka’. Results are for the markers (A) Cp0336/STS, (B) Cp2223/NdeII, and (C) Tf0250/HindIII. Identities of the attached band patterns: LM, 200-bp DNA ladder marker; S, ‘Setoka’, T: ‘Tsunonozomi’; M, ‘Murcott’; and K, ‘KyEn No. 4’.

Fig. 2. CAPS marker fragment patterns that show a discrepancy in the parentage of ‘Southern Red’. Results are for (A) Fb0159/HincII, (B) Gn0071/PvuII, and (C) If0211/PvuII. Identities of the band patterns: LM, 200-bp DNA ladder marker; S, ‘Southern Red’; K, ‘Kara’; P, ponkan; and O, ‘Osceola’. show that it is possible to identify 59 citrus cultivars, monoembryony and high susceptibility to citrus canker local varieties, and selections, including new cultivars disease (Kobayashi, 1995b), but the offspring of pon- recently developed by the citrus breeding program in kan frequently exhibit polyembryony (Parlevliet and the national institute of Japan and that are now com- Cameron, 1959) and relatively low susceptibility to mercially produced and frequently used as citrus breed- citrus canker disease (Matsumoto and Okudai, 1990). ing parents in Japan, on the basis of their genotypes by On the other hand, the parentage of ‘Setoka’ was not using eight selected CAPS markers. Therefore, a culti- suspected as being incorrect on the basis of its morpho- var identification method based on these CAPS markers logical traits or its disease resistance. This is reasonable and genotype data will contribute greatly to protecting because the putative seed parent, ‘KyEn No. 4’, is part breeders of the major citrus cultivars in Japan. of the full-sib family of ‘Tsunonozomi’, which was pre- These results also show that the actual parentage of viously reported as the seed parent, and has a similar ‘Setoka’, ‘Southern Red’, and ‘Reikou’ differed from genetic background to ‘Tsunonozomi’. their reported parentage, and that the candidate true pa- Genetic assessment is important to validate the pedi- rents of ‘Setoka’ and ‘Southern Red’ could be estimated gree of the promising cultivar ‘Setoka’ because it has by using DNA markers. Since citrus cultivars have superior fruit quality and has been frequently used as a great diversity in their morphological and physiological parent in citrus breeding programs. However, the cor- characteristics, an incorrect recording of the parentage rect parentage of ‘Reikou’ could not be estimated in of a cultivar may be detected from phenotypic data. For this study. It is difficult to preserve all cultivars and se- example, the parentage of ‘Southern Red’ appeared for lections used as parents in fruit tree breeding programs a long time to be incorrect on the basis of its morpho- owing to the high labor costs and limitations on the logical traits and disease resistance. ‘Southern Red’ has amount of usable land in Japan. Therefore, it is possible 12 K. Nonaka, H. Fujii, M. Kita, T. Shimada, T. Endo, T. Yoshioka and M. Omura

Table 6. CAPS and SNP genotypes for the reported parents of that the actual parent of ‘Reikou’ has already been fell- ‘Reikou’. The underlined CAPS genotypes represent dis- ed to make room for new accessions. crepancies between the reported parent–offspring relation- Analysis based on the CAPS markers that we have ship, in which ‘Reikou’ resulted from a cross between ‘KyEn No. 5’ and ‘Murcott’, and the marker results. A dis- developed will be useful because it does not demand crepancy exists if neither of the two alleles in the offspring expensive devices such as DNA sequencers; accessions exist in the candidate parent. The first 19 CAPS markers can be genotyped by means of electrophoresis. Howev- Table 6. wereCAPS used and in SNP the overallgenotypes parentage for the analysis reported for and all putative 33 NIFTS parents of ‘Reikou’. The underlined CAPS genotypes represent discrepancies between the reported parent–offspring relationship, in which ‘Reikou’er, resulted there arefrom advantages a cross between and ‘KyEn disadvantages No. 5’ and ‘Murcott’, for each and type the cultivars; the next 15 markers were used to clarify any par- of DNA marker (Kumar et al., 2009). In order to make entagemarker discrepanciesresults. revealed by the first 19 markers. SNP marker results are shown only for loci with discrepancies. DNA markers available in a range of situations, genotyping data for the DNA markers must be accumulated. Cultivar Marker name CAPS marker data for the 59 cultivars and selections ‘Reikou’ ‘KyEn No. 5’ ‘Murcott’ analyzed here will help us to protect breeders’ rights CAPS marker and to better understand the parentage, genetic diversi- Al0326/NdeII ab bb aa ty, and origins of the cultivars. Together, these benefits Al0413/MspI ab aa ab will help us to conduct more efficient citrus breeding Al0636/EcoRI ab bb bb programs. Bf0028/HaeIII ab ab aa Bf0029/StyI aa ab aa Literature Cited Bf0036/MspI ab bb bb Botstein, D., R. L. White, M. Skolnick and R. W. Davis. 1980. Bf0145/MspI aa aa ab Construction of a genetic linkage map in man using restric- Bf0158/PvuII ab ab bb tion fragment length polymorphisms. Amer. J. Hum. Genet. Cp1624/MspI bb bb bb 32: 314–331. Gn0043/HincII aa aa ab Cai, Q., C. L. Guy and G. A. Moore. 1994. Extension of the linkage map in citrus using random amplified polymorphic DNA If0208/HinfI bb ab bb (RAPD) markers and RFLP mapping of cold-acclimation- Mf0097/DraI bb ab ab responsive loci. Theor. Appl. Genet. 89: 606–614. Tf0062/RsaI aa aa ab Chen, C., K. D. Bowman, Y. A. Choi, P. M. Dang, M. N. Rao, S. Tf0079/StyI ab aa ab Huang, J. R. Soneji, T. G. McCollum and F. G. Gmitter, Jr. Tf0150/HinfI bb ab bb 2008. EST-SSR genetic maps for Citrus sinensis and Tf0168/RsaI aa ab ab Poncirus trifoliata. Tree Genet. Genomes 4: 1–10. Tf0235/HaeIII aa ab ab Close, T. J., P. R. Bhat, S. Lonardi, Y. Wu, N. Rostoks, L. Ramsay, A. Druka, N. Stein, J. T. Svensson, S. Wanamaker, Tf0271/RsaI aa aa ab S. Bozdag, M. L. Roose, M. J. Moscou, S. Chao, R. K. Tf0326/HhaI bb aa ab Varshney, P. Szűcs, K. Sato, P. M. Hayes, D. E. Matthews, Bf0136/HhaI ab bb ab A. Kleinhofs, G. J. Muehlbauer, J. DeYoung, D. F. Marshall, Cp0975/HinfI ab bb ab K. Madishetty, R. D. Fenton, P. Condamine, A. Graner and Fb0159/HincII aa ab ac R. Waugh. 2009. Development and implementation of high- Gn0040/HaeIII ab ab ab throughput SNP genotyping in barley. BMC Genomics 10: 582. Gn0071/PvuII bb bb bb Fujii, H., T. Ogata, T. Shimada, T. Endo, H. Iketani, T. Shimizu, If0211/PvuII aa ab aa T. Yamamoto and M. Omura. 2013a. Minimal marker: an Ov0305/MspI ab ab bb algorithm and computer program for the identification of Tf0069/MspI bb ab ab minimal sets of discriminating DNA markers for efficient Tf0165/DraI bb ab bb variety identification. J. Bioinform. Comput. Biol. 11: Tf0300/DraI ab bb bb 1250022. Tf0348/HinfI ab ab ab Fujii, H., T. Shimada, K. Nonaka, M. Kita, T. Kuniga, T. Endo, Y. Ikoma and M. Omura. 2013b. High-throughput genotyping Tf0350/DraI ab aa ab in citrus accessions using an SNP genotyping array. Tree Tf0364/HinfI aa aa ab Genet. Genomes 9: 145–153. Tf0386/MspI ab ab ab Fujii, H., H. Yamashita, F. Hosaka, S. Terakami and T. Wy0003/MspI ab bb ab Yamamoto. 2010. Development of a software MARCO to SNP marker presume the parental-child relationship using the result of DNA marker typing. Hort. Res. (Japan) 9 (Suppl. 1): 34 (In SI124 AG AA AA Japanese). SI156 AG GG GG Hodgson, R. W. 1967. Horticultural varieties of citrus. p. 431– SI158 AC AA AA 591. In: W. Reuther, H. J. Webber and L. D. Batchelor (eds.). SI190 CC AA CC The citrus industry. Vol. 1. University of California Press, SI220 AG GG GG Berkeley, USA. SI251 CG CC CC Imai, A., T. Takahara, H. Fukamachi, K. Nonaka, R. Matsumoto, SI313 AG AA AA T. Yoshioka, T. Kuniga, N. Mitani and N. Hiehata. 2008. A new citrus cultivar ‘Tsunokagayaki’. Hort. Res. (Japan) 7 SI334 AG GG GG (Suppl. 1): 43 (In Japanese). SI373 AT AA AA Hort. J. Preview 13

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