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The Horticulture Journal 86 (2): 200–207. 2017. e Japanese Society for doi: 10.2503/hortj.OKD-013 JSHS Horticultural Science http://www.jshs.jp/

Genetic Background, Inbreeding, and Genetic Uniformity in the National Breeding Program,

Atsushi Imai1,2,3, Takeshi Kuniga1,4, Terutaka Yoshioka1,5, Keisuke Nonaka1,5, Nobuhito Mitani1,2, Hiroshi Fukamachi1,5, Naofumi Hiehata1,6, Masashi Yamamoto1,7 and Takeshi Hayashi3,8*

1Kuchinotsu Citrus Research Station, NARO Institute of Fruit Tree Science, Minamishimabara 859-2501, Japan 2NARO Institute of Fruit Tree and Tea Science, Tsukuba 305-8605, Japan 3Graduate School of Life and Environmental Sciences, Tsukuba University, Tsukuba 305-8666, Japan 4NARO Western Region Agricultural Research Center, Zentsuji 765-0053, Japan 5NARO Institute of Fruit Tree and Tea Science, Shimizu 424-0292, Japan 6Kenou Development Bureau, Nagasaki Prefectural Government, Isahaya 854-0071, Japan 7Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan 8NARO Institute of Crop Science, Tsukuba 305-8518, Japan

We analyzed the pedigree records (1995–2010) of the Kuchinotsu Citrus Breeding Program (KCBP) at the National Institute of Fruit Tree Science (NIFTS) in Japan, abbreviated as NIFTS-KCBP, to reveal the genetic background and current status of inbreeding and genetic uniformity of the parental /genotypes and their F1 breeding progenies. The founding genotypes mostly used for crossing in NIFTS-KCBP were satsuma mandarin ( Marcow.), sweet (C. sinensis [L.] Osbeck), king mandarin (C. nobilis Lour.), (C. clementina hort. ex Tanaka), mediterranean mandarin (C. deliciosa Ten.), dancy (C. tangerina hort. ex Tanaka), and (C. reticulata Blanco). The intensive use of these seven genotypes and their progenies as crossed parents has led to a high degree of inbreeding in the breeding population. Moreover, these seven genotypes have dominated about 80% of the genetic composition of the breeding population. Although further studies are needed to reveal the influence of inbreeding and genetic uniformity on agronomically important traits, these data offer useful information for the selection of cross combinations and breeding strategies in the ongoing NIFTS citrus breeding program, Japan.

Key Words: citrus hybrids, founding genotypes, inbreeding coefficients, pedigree, plant breeding.

genotypes and their progenies can lead to inbreeding Introduction depression and genetic uniformity in breeding materi- Inbreeding and genetic uniformity in breeding popu- als, resulting in the loss of breeding efficiency in the lations are major concerns for crop breeders. In most long term. Inbreeding depression downgrades fitness- fruit breeding programs, the number of high-quality related traits, such as tree vigor, whereas genetic uni- genotypes is restricted, and thus they are extensively formity causes genetic erosion and decreases the used as crossed parents (Kajiura and Sato, 1990; Noiton genetic gain per selection. Therefore, both inbreeding and Alspach, 1996; Scorza et al., 1985). The common and genetic uniformity prohibit the future progress in practice of using a limited number of genotypes with genetic performance. For the continuous development high quality as crossed parents contributes to the effi- of improved cultivars that meet market demands, the cient genetic improvement in the initial stages of a current status of inbreeding and genetic uniformity in breeding program. However, the recurrent use of few the existing breeding populations needs to be evaluated. Several studies have evaluated the inbreeding and genetic uniformity in breeding populations of fruit tree Received; May 2, 2016. Accepted; August 16, 2016. First Published Online in J-STAGE on September 24, 2016. crops. For instance, Kajiura and Sato (1990) reported * Corresponding author (E-mail: [email protected]). that only 15 cultivars out of the 1212 native varieties

© 2017 The Japanese Society for Horticultural Science (JSHS), All rights reserved. Hort. J. 86 (2): 200–207. 2017. 201 are used as crossed parents in Japanese pear breeding; Genetic background Scorza et al. (1985) demonstrated that the selection for The pedigree records of 126 parental cultivars and fruit quality in the freestone peach has led to a high de- their 12541 F1 breeding progenies that contained the gree of inbreeding and genetic uniformity in the eastern names of individual genotypes and their seed and pollen United States; and Yamada (1993) and Yamada et al. parents were obtained from NIFTS-KCBP. The pedi- (1994) found that repeated crosses over generations grees of all genotypes were traced back to genotypes within the narrow gene pool of persimmon have led to with unknown parents, which were regarded as found- inbreeding depression, which reduces tree vigor, pro- ing genotypes, and all the founding genotypes and an- ductivity, and fruit weight. cestral genotypes were added to the pedigree records to In Japan, the National Citrus Breeding Program is obtain the full pedigree charts that were constructed conducted at the National Institute of Fruit Tree Science using Pedimap (Voorrips et al., 2012). Such full pedi- (NIFTS) Kuchinotsu Citrus Research Station and gree information was used to reveal the genetic back- NIFTS Okitsu Citrus Research Station. The Kuchinotsu ground and founding genotypes of the parental cultivars

Citrus Breeding Program (KCBP) at the NIFTS, which and their F1 progenies. is abbreviated as NIFTS-KCBP, started in 1964 and has been recognized as one of the largest fruit breeding pro- Inbreeding and genetic uniformity grams in the world (Matsumoto and Takahara, unpub- The inbreeding coefficients of 126 parental cultivars lished). NIFTS-KCBP has successfully developed 21 and their 12541 F1 progenies, as well as the coefficients novel citrus cultivars such as ‘Shiranuhi’ (Matsumoto, of co-ancestry between them and their founding geno- 2001) and ‘’ (Matsumoto et al., 2003), which are types, were computed based on the assumption that the widely grown in Japan. founding genotypes were genetically unrelated and non- Since the 1970s, fruit quality traits, including high inbred. The inbreeding coefficient was defined as the sugar content, easy peeling, seedlessness, soft pulp probability that two alleles of an individual at a given firmness, and soft segment firmness, have been the locus were identical by descent (IBD) and reflected the main breeding targets of NIFTS-KCBP. These breeding genetic similarity of a mating pair to generate the indi- objectives narrowed down the candidate parental culti- vidual. To investigate the changes in the inbreeding co- vars, and thus may lead to inbreeding depression and efficients of F1 progenies, we evaluated the mean genetic uniformity in the breeding populations. How- inbreeding coefficients of F1 progenies obtained in each ever, the genetic background, as well as the degree of year from 1995 through 2010. Coefficient of co- inbreeding and genetic uniformity in NIFTS-KCBP ancestry between two individuals was defined as the populations, have not yet been investigated. probability that the two alleles sampled from respective The objectives of this study were to reveal the genet- individuals at a given locus were IBD. Thus, the coeffi- ic background and current status of inbreeding and cients of co-ancestry between parental cultivars or F1 genetic uniformity in NIFTS-KCBP parental cultivars/ progenies and the founding genotypes reflected the ge- genotypes (hereinafter referred to as parental cultivars) nome composition of a breeding population, each com- and their F1 breeding progenies using their pedigree in- ponent of which was originated from the genomes of formation. These estimates can offer useful information the founding genotypes. We calculated twice the coeffi- for the future selection of parental cultivars or plants for cient of co-ancestry as a numerator relationship coeffi- cross combinations and breeding strategies. cient between the parental cultivars or F1 progenies and the founding genotypes, which can be interpreted as the Materials and Methods genetic contributions of founding genotypes to individ- Plant materials uals in a breeding population (Bulmer, 1980).

Parental cultivars and their F1 breeding progenies ob- Inbreeding coefficients and coefficients of co- tained in NIFTS-KCBP from 1995 to 2010 were con- ancestry were calculated using Peditree (van Berloo and sidered in this study. Seventy-three seed parents and 69 Hutten, 2005) based on the assumption that the found- pollen parents, a total of 126 parental cultivars, pro- ing genotypes were unrelated and non-inbred, and that duced 12541 F1 progenies from 466 cross combinations breeding selections had no influence on the genetic con- in NIFTS-KCBP during the period. About 1000 F1 tribution of descendant genotypes. In addition, all mu- progenies from 20–40 cross combinations were planted tants included in the breeding population were regarded in breeding fields in each year from 1995 through 2010. to have the same genetic composition as the original

From these 12541 F1 progenies, we selected a promi- genotypes. nent early-maturated ‘Mihaya’ (Nonaka et al., Results 2012), and two prominent genotypes were selected and are evaluated in a national trial as of June 2016. They Genetic background were also used as parental cultivars, and were included The pedigree chart of 126 parental cultivars used for in the 126 parental cultivars in this study. crossing between 1995 and 2010 along with their founding genotypes is shown in Figure 1. The 126 pa- 202 A. Imai, T. Kuniga, T. Yoshioka, K. Nonaka, N. Mitani, H. Fukamachi, N. Hiehata, M. Yamamoto and T. Hayashi Pedigree chart of parental cultivars used in the Kuchinotsu Citrus Breeding Program at the National Institute of Fruit Tree Science (NIFTS-KCBP) from 1995 to 2010. Pedigrees are oriented in genera- in oriented are Pedigrees 2010. to 1995 from (NIFTS-KCBP) Science Tree Fruit of Institute National the at Program Breeding Citrus Kuchinotsu the in used cultivars parental of chart Pedigree dancy tangerine, and ponkan. Genotypes in the yellow box represent the other 14 founding genotypes. Red lines represent seed parents. Blue lines represent pollen parents. Cross symbols connect parents to parents connect symbols Cross parents. pollen represent lines Blue parents. seed represent lines Red genotypes. founding 14 other the represent box yellow the in Genotypes ponkan. and tangerine, dancy offspring. tions from left to right. The founding genotypes mostly used for crossing in NIFTS-KCBP are shown in the green box: satsuma mandarin, sweet orange, king mandarin, clementine, mediterranean mandarin, mediterranean clementine, mandarin, king orange, sweet mandarin, satsuma box: green the in shown are NIFTS-KCBP in crossing for used mostly genotypes founding The right. to left from tions Fig. 1. Hort. J. 86 (2): 200–207. 2017. 203 rental cultivars were traced back to 21 founding geno- nies. In total, the seven predominant founding geno- types, which were as follows: satsuma mandarin (Citrus types dominated 88.7% of the genetic composition of unshiu Marcow.), clementine (C. clementina hort. ex F1 progenies. Therefore, an apparent genetic uniformity Tanaka), sweet orange (C. sinensis [L.] Osbeck), iyo was observed in both the parental cultivars and F1 prog- (C. iyo hort. ex Tanaka), hyuga-natsu (C. tamurana enies of NIFTS-KCBP. hort. ex Tanaka), (C. paradisi Macfad.), Discussion dancy tangerine (C. tangerina hort. ex Tanaka), king mandarin (C. nobilis Lour.), mediterranean mandarin The evaluation of inbreeding and genetic uniformity (C. deliciosa Ten.), ponkan (C. reticulata Blanco), is essential for ongoing fruit tree breeding programs. ‘Mukaku-kishu’ (C. kinokuni hort. ex Tanaka), hassaku This study investigated the genetic background and cur- (C. hassaku hort. ex Tanaka), ‘Tanikawa-buntan’, rent status of inbreeding and genetic uniformity in the

‘Ogon-kan (C. flavicarpa hort. ex Tanaka)’, ‘Kawachi- NIFTS-KCBP parental cultivars and their F1 breeding bankan’, ‘San-jacinto’ , ‘Soren-tangelo’, tankan progenies and revealed a relatively narrow genetic (C. tankan Hayata), ‘’, yuge-hyokan (C. yuge- background and high levels of inbreeding and genetic hyokan hort. ex Yu. Tanaka), and ‘’ . We uniformity. generated 12541 F1 progenies from crosses with the 126 We used the pedigree information to calculate in- parental cultivars in NIFTS-KCBP between 1995 and breeding and co-ancestry coefficients. However, pedi- 2010 as described in Materials and Methods; therefore, gree information may not be an accurate predictor of these 21 founding genotypes composed the genetic inbreeding and genetic contribution in artificially se- background of NIFTS-KCBP parental cultivars and lected populations (Culp, 1998). Our parental cultivars their F1 breeding progenies. were selected as the superior genotypes in NIFTS- KCBP and other citrus breeding programs, and thus the Inbreeding and genetic uniformity estimates of inbreeding and genetic uniformity may in- Inbreeding coefficients ranged from 0 to 0.250 with clude some degree of bias. Additionally, a recent study the mean and standard deviation of 0.029 ± 0.062 in the using molecular markers indicated high probabilities of 126 parental cultivars (Table 1), of which 30 were esti- incorrect parentage for several parental cultivars mated to have inbreeding coefficients greater than 0, (Ninomiya et al., 2015). Molecular markers also indi- including 13 parental cultivars with inbreeding cated genetic relationships between the founding geno- coefficients higher than 0.10. Parental cultivars released types, which were assumed to be genetically unrelated in later generations tended to have higher inbreeding in this study; for instance, clementine is a be- coefficients, indicating the upward trend in the inbreed- tween mediterranean mandarin and sweet orange ing coefficient with the progress of NIFTS-KCBP. (Ollitrault et al., 2012), and satsuma mandarin is a puta-

In F1 progenies, inbreeding coefficients ranged from tive hybrid between ‘Kunenbo’ (C. nobilis Lour) and 0 to 0.500 (data not shown). The mean values of in- kishu-mikan (C. kinokuni hort. ex Tanaka) (Shimizu breeding coefficients in F1 progenies obtained in each et al., 2016). Although these issues may slightly bias year were relatively high and ranged from 0.076 to the estimates, our study provided a practical evaluation 0.169 in 1995–2010 (Table 2). The grand mean value of of the current status of inbreeding and genetic uniformi- inbreeding coefficients in all F1 progenies was 0.116 ty in NIFTS-KCBP parental cultivars and their F1 (± 0.079). breeding progenies. Of the 21 founding genotypes, satsuma mandarin, The intensive use of the seven predominant founding sweet orange, king mandarin, clementine, genotypes and their descendants as parental cultivars in mediterranean mandarin, dancy tangerine, and ponkan NIFTS-KCBP could eventually result in the loss of ge- were extensively used as predominant parents for cross- netic diversity, which could limit the genetic improve- ing in NIFTS-KCBP and dominated 77.1% of the ge- ment and development of new citrus cultivars in the netic composition of parental cultivars. Satsuma near future. A narrow genetic background and high de- mandarin showed the highest mean genetic contribution gree of genetic uniformity were also observed in other (25.7%), followed by sweet orange (17.3%), king man- fruit tree crops, such as the Japanese pear (Kajiura and darin (8.1%), clementine (7.9%), mediterranean man- Sato, 1990), apple (Bannier, 2011; Noiton and Alspach, darin (7.3%), dancy tangerine (5.8%), and ponkan 1996; Son et al., 2012), freestone peach (Scorza et al., (4.9%) in terms of numerator relationship coefficients 1985), almond (Lansari et al., 1994), and sweet cherry (Table 1). (Choi and Kappel, 2004), which could lead to the loss

In F1 progenies, satsuma mandarin showed the high- of potential gain from selection and genetic vulnerabili- est mean genetic contribution (28.5%–45.2%) in every ties to epidemic insects, diseases, and stress conditions. year of crossing (Table 2). Although the rank between In NIFTS-KCBP, the influence of a high degree of ge- the other six predominant founding genotypes slightly netic uniformity on agronomic traits has not yet been changed through the years, they steadily dominated a investigated in detail. However, some citrus industries total of 54.5% of the genetic composition of F1 proge- have pointed out that the fruit appearance of some Table204 1. InbreedingA. Imai,coefficient T. Kuniga, and T.genome Yoshioka, composition K. Nonaka, of parental N. Mitani, cultivars H. Fukamachi, in the Kuchinotsu N. Hiehata, Citrus M. YamamotoBreeding Program and T. Hayashiat the National Institute of Fruit Tree Science (NIFTS-KCBP) from 1995 to 2010. Table 1. Inbreeding coefficient and genome composition of parental cultivars in the Kuchinotsu Citrus Breeding Program at the National Institute of Fruit Tree Science (NIFTS-KCBP) from 1995 to 2010. Genetic contribution of founding genotypes (twice as coefficient of co-ancestry) Genotypez Inbreeding 14 other coefficient satsuma sweet king clementine mediterranean dancy ponkan founding mandarin mandarin tangerine genotypes satsuma mandarin 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 sweet orange 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 king mandarin 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 clementine 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 mediterranean mandarin 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 dancy tangerine 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 ponkan 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 iyo 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 hyuga-natsu 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 grapefruit 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Mukaku-kishu 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 hassaku 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Tanikawa-buntan 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Ogon-kan 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Kawachi-bankan 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 San-jacinto 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Soren-tangelo 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 tankan 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Haruka 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 yuge-hyokan 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Murcott 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Nankou 0.000 0.500 0.000 0.000 0.500 0.000 0.000 0.000 0.000 HF15 0.000 0.500 0.500 0.000 0.000 0.000 0.000 0.000 0.000 HF17 0.000 0.500 0.500 0.000 0.000 0.000 0.000 0.000 0.000 HF20 0.000 0.500 0.500 0.000 0.000 0.000 0.000 0.000 0.000 HF21 0.000 0.500 0.500 0.000 0.000 0.000 0.000 0.000 0.000 HF9 0.000 0.500 0.500 0.000 0.000 0.000 0.000 0.000 0.000 Aki Tangor 0.000 0.500 0.500 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.000 0.000 0.000 0.000 0.000 0.000 Minneola 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.000 0.500 Orlando 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.000 0.500 Encore 0.000 0.000 0.000 0.500 0.000 0.500 0.000 0.000 0.000 Wilking 0.000 0.000 0.000 0.500 0.000 0.500 0.000 0.000 0.000 Ariake 0.000 0.000 0.500 0.000 0.500 0.000 0.000 0.000 0.000 Sweet Spring 0.000 0.500 0.000 0.000 0.000 0.000 0.000 0.000 0.500 No. 1087 0.000 0.000 0.500 0.000 0.000 0.000 0.000 0.000 0.500 Awa Orange 0.000 0.000 0.500 0.000 0.000 0.000 0.000 0.000 0.500 JHG 0.000 0.500 0.000 0.000 0.000 0.000 0.000 0.000 0.500 Kara 0.000 0.500 0.000 0.500 0.000 0.000 0.000 0.000 0.000 Hayaka 0.000 0.500 0.000 0.000 0.000 0.000 0.000 0.500 0.000 Kankitsu Chukanbohon Nou 6 Gou 0.000 0.000 0.000 0.500 0.000 0.000 0.000 0.000 0.500 Southern Yellow 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 Nishinokaori 0.250 0.250 0.750 0.000 0.000 0.000 0.000 0.000 0.000 Osceola 0.000 0.000 0.000 0.000 0.500 0.000 0.250 0.000 0.250 Lee 0.000 0.000 0.000 0.000 0.500 0.000 0.250 0.000 0.250 Robinson 0.000 0.000 0.000 0.000 0.500 0.000 0.250 0.000 0.250 KyAo37 0.250 0.750 0.250 0.000 0.000 0.000 0.000 0.000 0.000 KyOw14 0.250 0.750 0.250 0.000 0.000 0.000 0.000 0.000 0.000 KyOw21 0.250 0.750 0.250 0.000 0.000 0.000 0.000 0.000 0.000 EnOw20 0.000 0.500 0.000 0.250 0.000 0.250 0.000 0.000 0.000 EnOw21 0.000 0.500 0.000 0.250 0.000 0.250 0.000 0.000 0.000 EnOw7 0.000 0.500 0.000 0.250 0.000 0.250 0.000 0.000 0.000 EnOw8 0.000 0.500 0.000 0.250 0.000 0.250 0.000 0.000 0.000 Shiranuhi 0.000 0.250 0.250 0.000 0.000 0.000 0.000 0.500 0.000 Setomi 0.000 0.250 0.250 0.000 0.000 0.000 0.000 0.500 0.000 Harumi 0.000 0.250 0.250 0.000 0.000 0.000 0.000 0.500 0.000 Youkou 0.000 0.250 0.250 0.000 0.000 0.000 0.000 0.500 0.000 Page 0.000 0.000 0.000 0.000 0.500 0.000 0.250 0.000 0.250 No. 2681 0.000 0.250 0.250 0.000 0.000 0.000 0.000 0.000 0.500 Tsunonozomi 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 KyEn4 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 Hort. J. 86 (2): 200–207. 2017. 205

Table 1. Continued KyEn5 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 Amaka 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 Tamami 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 F118 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 Okitsu 45 Gou 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 Benibae 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 HF9·En29 0.000 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 A7 0.000 0.250 0.500 0.000 0.000 0.000 0.000 0.000 0.250 Okitsu 46 Gou 0.000 0.250 0.500 0.000 0.000 0.000 0.000 0.000 0.250 Hareyaka 0.000 0.000 0.000 0.250 0.000 0.250 0.000 0.500 0.000 M4 0.000 0.250 0.250 0.000 0.000 0.000 0.500 0.000 0.000 M5 0.000 0.250 0.250 0.000 0.000 0.000 0.500 0.000 0.000 2700·OIy25 0.000 0.125 0.375 0.000 0.000 0.000 0.000 0.000 0.500 E647 0.000 0.250 0.250 0.000 0.250 0.000 0.125 0.000 0.125 KyOw21·D4 0.000 0.375 0.125 0.000 0.000 0.000 0.500 0.000 0.000 KyOw21·D49 0.000 0.375 0.125 0.000 0.000 0.000 0.500 0.000 0.000 Kuchinotsu 28 Gou 0.000 0.375 0.125 0.000 0.000 0.000 0.500 0.000 0.000 EnOw21·Youkou21 0.063 0.375 0.125 0.125 0.000 0.125 0.000 0.250 0.000 Kuchinotsu 27 Gou 0.063 0.375 0.125 0.125 0.000 0.125 0.000 0.250 0.000 Seinannohikari 0.063 0.375 0.125 0.125 0.000 0.125 0.000 0.250 0.000 Amakusa 0.000 0.375 0.125 0.000 0.250 0.000 0.125 0.000 0.125 Kuchinotsu 33 Gou 0.000 0.375 0.125 0.250 0.000 0.250 0.000 0.000 0.000 Tsunokagayaki 0.000 0.375 0.125 0.250 0.000 0.250 0.000 0.000 0.000 Kuchinotsu 18 Gou 0.000 0.375 0.125 0.250 0.000 0.250 0.000 0.000 0.000 Kuchinotsu 35 Gou 0.000 0.375 0.125 0.250 0.000 0.250 0.000 0.000 0.000 Kuchinotsu 38 Gou 0.000 0.375 0.125 0.000 0.250 0.000 0.125 0.000 0.125 No. 1408 0.063 0.375 0.125 0.125 0.000 0.125 0.000 0.000 0.250 Kankitsu Chukanbohon Nou 5 Gou 0.000 0.000 0.000 0.000 0.250 0.000 0.125 0.000 0.625 990343 0.188 0.250 0.250 0.250 0.000 0.250 0.000 0.000 0.000 0.188 0.250 0.500 0.000 0.000 0.000 0.000 0.250 0.000 Okitsu 57 Gou 0.094 0.250 0.375 0.000 0.000 0.000 0.000 0.250 0.125 Asumi 0.094 0.250 0.375 0.000 0.000 0.000 0.000 0.250 0.125 Okitsu 59 Gou 0.094 0.250 0.375 0.125 0.000 0.125 0.000 0.000 0.125 Setoka 0.000 0.125 0.125 0.125 0.000 0.125 0.000 0.000 0.500 KyEn4·Min26 0.000 0.125 0.125 0.125 0.000 0.125 0.250 0.000 0.250 KyEn4·Mur26 0.000 0.125 0.125 0.125 0.000 0.125 0.000 0.000 0.500 KyEn5·En6 0.250 0.125 0.125 0.375 0.000 0.375 0.000 0.000 0.000 Reikou 0.000 0.125 0.125 0.125 0.000 0.125 0.000 0.000 0.500 Kuchinotsu 36 Gou 0.000 0.125 0.125 0.125 0.000 0.125 0.000 0.000 0.500 KyOw21·CC33 0.000 0.375 0.125 0.000 0.500 0.000 0.000 0.000 0.000 KyOw21·CC4 0.000 0.375 0.125 0.000 0.500 0.000 0.000 0.000 0.000 KyOw21·Mur13 0.000 0.375 0.125 0.000 0.000 0.000 0.000 0.000 0.500 KyOw21·Ariake22 0.063 0.375 0.375 0.000 0.250 0.000 0.000 0.000 0.000 Kuchinotsu 40 Gou 0.063 0.375 0.375 0.000 0.000 0.000 0.000 0.000 0.250 Haruhi 0.125 0.125 0.500 0.000 0.000 0.000 0.000 0.000 0.375 LeeAo25 0.000 0.500 0.000 0.000 0.250 0.000 0.125 0.000 0.125 LeeAo30 0.000 0.500 0.000 0.000 0.250 0.000 0.125 0.000 0.125 LeeAo35 0.000 0.500 0.000 0.000 0.250 0.000 0.125 0.000 0.125 LeeAo9 0.000 0.500 0.000 0.000 0.250 0.000 0.125 0.000 0.125 P4 0.000 0.250 0.250 0.125 0.000 0.125 0.000 0.250 0.000 No. 1010 0.031 0.313 0.188 0.000 0.250 0.000 0.000 0.000 0.250 No. 1011 0.031 0.313 0.188 0.000 0.250 0.000 0.000 0.000 0.250 Harehime 0.125 0.625 0.125 0.000 0.125 0.000 0.063 0.000 0.063 Kuchinotsu 51 Gou 0.094 0.375 0.125 0.063 0.000 0.063 0.250 0.125 0.000 No. 1051 0.125 0.313 0.438 0.000 0.000 0.000 0.000 0.000 0.250 Ehime Kashi No. 28 0.156 0.438 0.063 0.000 0.375 0.000 0.063 0.000 0.063 Kuchinotsu 52 Gou 0.031 0.188 0.313 0.125 0.250 0.125 0.000 0.000 0.000 960203 0.094 0.313 0.188 0.125 0.000 0.125 0.000 0.250 0.000 Kuchinotsu 49 Gou 0.094 0.375 0.125 0.063 0.125 0.063 0.063 0.000 0.188 Mihaya 0.156 0.313 0.188 0.188 0.000 0.188 0.000 0.000 0.125 980389 0.125 0.438 0.313 0.063 0.000 0.063 0.000 0.000 0.125 Okitsu 56 Gou 0.000 0.125 0.125 0.125 0.125 0.125 0.063 0.000 0.313 040381 0.000 0.156 0.094 0.000 0.125 0.000 0.000 0.000 0.625 031045 0.063 0.313 0.063 0.000 0.563 0.000 0.031 0.000 0.031 031060 0.063 0.313 0.063 0.000 0.563 0.000 0.031 0.000 0.031 Grand mean 0.029 0.257 0.173 0.081 0.079 0.073 0.058 0.049 0.229 z Names in bold represent the founding genotypes. Table206 2. InbreedingA. Imai, coefficient T. Kuniga, and T. genomeYoshioka, composition K. Nonaka, of N. F1 Mitani,breeding H. progeniesFukamachi, in N.the Hiehata, Kuchinotsu M. Yamamoto Citrus Breeding and T. HayashiProgram at the National Institute of Fruit Tree Science (NIFTS-KCBP) from 1995 to 2010.

Table 2. Inbreeding coefficient and genome composition of F1 breeding progenies in the Kuchinotsu Citrus Breeding Program at the National Institute of Fruit Tree Science (NIFTS-KCBP) from 1995 to 2010.

Genetic contribution of founding genotypes (twice as coefficient of co-ancestry) Crossing year Mean 14 other of F1 breeding inbreeding satsuma king mediterranean dancy sweet orange clementine ponkan founding progenies coefficient mandarin mandarin mandarin tangerine genotypes 1995 0.088 0.338 0.209 0.026 0.103 0.026 0.001 0.050 0.247 1996 0.089 0.287 0.242 0.092 0.129 0.092 0.057 0.082 0.019 1997 0.169 0.452 0.216 0.059 0.008 0.059 0.052 0.081 0.074 1998 0.139 0.357 0.194 0.169 0.009 0.169 0.030 0.007 0.064 1999 0.126 0.324 0.177 0.139 0.035 0.139 0.021 0.051 0.114 2000 0.130 0.379 0.210 0.096 0.061 0.096 0.053 0.057 0.048 2001 0.135 0.335 0.216 0.103 0.035 0.103 0.049 0.007 0.150 2002 0.099 0.350 0.222 0.102 0.047 0.102 0.004 0.117 0.055 2003 0.104 0.306 0.216 0.067 0.117 0.067 0.017 0.134 0.076 2004 0.124 0.365 0.205 0.078 0.049 0.078 0.010 0.061 0.153 2005 0.100 0.308 0.184 0.079 0.083 0.062 0.013 0.180 0.091 2006 0.110 0.316 0.215 0.111 0.049 0.109 0.011 0.115 0.073 2007 0.102 0.331 0.179 0.079 0.083 0.065 0.029 0.082 0.151 2008 0.094 0.348 0.183 0.082 0.036 0.044 0.009 0.087 0.211 2009 0.100 0.335 0.226 0.052 0.079 0.036 0.044 0.089 0.139 2010 0.076 0.285 0.144 0.097 0.170 0.047 0.005 0.058 0.195 Grand mean 0.116 0.343 0.199 0.090 0.064 0.080 0.027 0.084 0.113

newly developed cultivars is fairly similar to the exist- also unclear at present. However, Frost (1943) reported ing cultivars, probably due to the high degree of genetic that crosses of ‘Ruby’ × ‘Valencia’ oranges and uniformity in the breeding materials (Takahara, ‘Eureka’ × ‘Lisbon’ produced progenies with a Personal communication). The uniform appearance is a weak tree vigor. These two crosses are extreme exam- critical disadvantage for citrus marketing; therefore, we ples because they produced progenies with an inbreed- need to elucidate the influence of a high degree of ge- ing coefficient of 0.5. However, they indicate that netic uniformity on agronomic traits using our accumu- inbreeding depression can occur in citrus and that the lated phenotypic records. sweet orange has genetic factors that cause inbreeding The mean inbreeding coefficient in parental cultivars depression. Sweet orange was the second most fre- (0.029 ± 0.062) was as low as that in the sweet cherry quently used founding genotype in NIFTS-KCBP, and (0.033) (Choi and Kappel, 2004), apple (0.01–0.04) as a result, its influence of inbreeding on agronomic (Noiton and Alspach, 1996), Japanese-type plum traits, including tree vigor, needs to be taken into seri- (0.016–0.054) (Byrne, 1989), and almond (0.022) ous consideration if the upward inbreeding coefficient (Lansari et al., 1994), in which inbreeding depression trend continues in NIFTS-KCBP. has a negligible influence on agronomic traits. Howev- Although the NIFTS germplasm collection includes er, our results showed that the inbreeding coefficients of more than 3000 citrus accessions, composed of local 13 parental cultivars in advanced generations exceeded varieties, wild species, and related species from all 0.10, revealing an upward trend with the progress of around the world, the genetic background of parental

NIFTS-KCBP. Additionally, the grand mean of the in- cultivars and their F1 breeding progenies is very limit- breeding coefficient in F1 progenies was as high (0.116) ed. It is, therefore, necessary to broaden the genetic as that in the blueberry (0.13) (Hancock and Siefker, base of cross breeding and use diverse citrus accessions 1982) and raspberry (0.12) (Dale et al., 1993): These as parents to enable future NIFTS citrus breeding pro- values were close to 0.125, which is expected in half- gram to stably progress. sib mating, suggesting the potential threat of inbreeding Acknowledgements depression. Although the susceptibility to inbreeding is different among crops, careful consideration to inbreed- We would like to thank all the parties that support ing depression may have to be given regarding the NIFTS-KCBP, Japan. breeding materials of NIFTS-KCBP. The influence of inbreeding on citrus is not well known, and in the Literature Cited breeding materials of NIFTS-KCBP, associations be- Bannier, H. J. 2011. Modern apple breeding: genetic narrowing tween inbreeding coefficients and agronomic traits are and inbreeding tendencies. Erwerbs-Obstbau. 52: 85–110. Hort. J. 86 (2): 200–207. 2017. 207

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