2022 年 87 巻 3 号 p. 231-238
Longan is one of the most economically important fruits of Asian countries belonging to the family Sapindaceae. Essentially, there is a lack of fundamental karyological data on different cultivars in longan although the whole genome of longan has been successfully sequenced. This study is the first to report the chromosome numbers and karyotypes of nine cultivars of the genus Dimocarpus cultivated widely in the northern regions of Thailand. These cultivars are Chompoo, Haew, Phuangthong, Thao, Jumbo, Phuen-Mueang, Pumateenkong, Chuliang (China), Kohala (America), and one of them (Thao, Dimocarpus obtusus, Lithanatudom & Chaowasku, comb. et stat. nov.) is considered as a rare species found in Thailand. The somatic chromosome number of cells derived from the root tip collected from the air layering branch of all cultivars was determined as 2n=2x=30. All cultivars showed a predominance of metacentric chromosomes in each karyotype indicating their symmetric nature. The karyotype of these cultivars was placed into categories 1B and 2B based on Stebbins classification. In addition, we analyzed the scatter diagrams of asymmetry indices, UPGMA dendrogram with fruit morphology, and PCoA graph together with the karyotypic relationship, and overall, the results revealed that all cultivars of the genus Dimocarpus examined can be divided into two major groups.
The family Sapindaceae or soapberry family comprises 141 genera (∼1,900 species), mostly trees and shrubs, tropical or subtropical with four economically important species at a global level; Litchi chinensis (lychee or litchi), Dimocarpus longan (longan), Nephelium lappaceum (rambutan) and N. ramboutan-ake (pulasan) (Menzel 2003, Buerki et al. 2010). These four species are closely related, native to Asia, and similar in fruiting habits, but different in fruit morphology (Menzel 2003). However, longan is among the most popular members of Sapindaceae due to its sweet and juicy flesh, profitable for the industry, and satisfying the fondness of the population, especially in Southeast Asia (Huang et al. 2005a, b).
The genus Dimocarpus is primarily distributed in tropical South and Southeast Asia, ranging from Sri Lanka and India to East Malaysia and Australia. The well-recognized edible fruits derived from this genus known as longan are from Dimocarpus longan (Leenhouts 1973, Lithanatudom et al. 2017). Historical documents have revealed that longan originated from southern China. Longan trees from China were sent to various countries such as Thailand, India, Ceylon, Myanmar, the Philippines, Cuba, the West Indies, Madagascar, Australia, Hawaii, and Florida. It is believed that longan trees were brought to Thailand from China in 1896. These trees were the ancestors of the present commercial varieties, which have been selected from several generations of seedlings. (Menzel 2003, Huang et al. 2005a, b, Mishra et al. 2015, Mitra and Pan 2020). The main area where longan is usually planted is in the northern part of the country, especially in Chiang Mai province (Subhadrabandhu and Yapwattanaphun 2001, Anupunt and Sukhvibul 2005).
There are numerous longan cultivars in Thailand. Although the whole genome and DNA markers have been established in longan (Lin et al. 2017, Lithanatudom et al. 2017), however, karyological data are still scarce for different cultivars of longan particularly, in Thailand. In an earlier report, a karyological investigation of longan has been carried out only in China. Zhang et al. (2010) reported that the chromosome number of several longan cultivars was determined as 2n=2x=30 except for “Lieye” (one of the rare longan cultivars found in Guangxi province) was a haploid (2n=x=15). Paradoxically, Lu et al. (2014) reported that the chromosome number of “Lieye” longan was varied (2n=15, 18, 21, and 24), proving that ‘Lieye’ is a mixoploid. Moreover, Liuxin (1994) reported that the karyotype formula of longan is 2n=30=16m(2sat)+8sm+6st. In addition, according to the online chromosome databases, Index to Plant Chromosome Numbers (IPCN, http://www.tropicos.org/Project/IPCN) and Chromosome Counts Database (CCDB version 1.58, http://ccdb.tau.ac.il/home/), the diploid chromosome number was reported with 2n=24, 28, 30, and 32. The conflicting data of longan karyology may be due to the extremely small size of a chromosome resulting in difficulties in obtaining reproducibility by conventional methods. Thus, there are no scientifically validated reports on chromosome number and karyotype which might help explain the evolutionary analysis, genetic diversity, and morphological differences among cultivars found in Thailand.
In this study, we provide the first report of karyological data of six longan cultivars in Thailand with an addition of two cultivars from China and America, respectively, and another species D. obtusus (Lithanatudom & Chaowasku, comb. et stat. nov.) formerly regarded as a subspecies of D. longan (Boonsuk and Chantaranothai 2017, Lithanatudom et al. 2017), to determine the karyotypic relationship between different cultivars and to deposit karyological data to support the taxonomic and phylogenetic study of longan in future studies. Furthermore, the data obtained from this study could be used to assist in evolutionary analysis and plant breeding programs in longan
Plant specimens with authentication belonging to the genus Dimocarpus were obtained from Maejo University, Chiang Mai, Thailand, and were selected for this study (Table 1). Voucher specimens were deposited at Chiang Mai University Herbarium (CMUB), Thailand. The roots from individual cultivars were induced on each stem by air layering and collected for karyological observations using the modified method from Dyer (1979). The actively growing root tips were pretreated with a saturated solution of p-dichlorobenzene for 1 h at 4°C then fixed with Carnoy’s fixative (absolute ethanol : glacial acetic acid=3 : 1) for at least 24 h. Treated root tips were used for slide preparation by squash technique or preserved in 70% ethanol at 4°C until used. For slide preparation, the root tips were washed in distilled water, hydrolyzed in 1 M HCl for 7 min at 60°C, and washed again in distilled water. The root tips were then stained in carbol fuchsin solution for 15–20 min at room temperature and gently squashed on a glass slide. A minimum of five metaphase plates were observed to obtain karyological data and carefully counted to ensure the chromosome number in all cultivars. The best metaphase plates were photographed using an Olympus CX23 equipped with a Canon 600D digital camera (Canon, Japan).
| Species | Cultivar name | Locality | Origin | Collector (voucher number) |
|---|---|---|---|---|
| D. longan | Chomphoo | CMUB herbarium | Thailand | Jaroenkit T, Manochai P, Lithanatudom SK. #2 (CMUB) |
| D. longan | Haew | CMUB herbarium | Thailand | Jaroenkit T, Manochai P, Lithanatudom SK. #4 (CMUB) |
| D. longan | Phuangthong | CMUB herbarium | Thailand | Jaroenkit T, Manochai P, Lithanatudom SK. #5 (CMUB) |
| D. obtusus | Thao | CMUB herbarium | Thailand | Jaroenkit T, Manochai P, Lithanatudom SK. #6 (CMUB) |
| D. longan | Jumbo | CMUB herbarium | Thailand | Jaroenkit T, Manochai P, Lithanatudom SK. #14 (CMUB) |
| D. longan | Phuen-Mueang | CMUB herbarium | Thailand | Jaroenkit T, Manochai P, Lithanatudom SK. #30 (CMUB) |
| D. longan | Pumateenkong | CMUB herbarium | Thailand | Jaroenkit T, Manochai P, Lithanatudom SK. #35 (CMUB) |
| D. longan | Chuliang | CMUB herbarium | China | Jaroenkit T, Manochai P, Lithanatudom SK. #37 (CMUB) |
| D. longan | Kohala | CMUB herbarium | America | Jaroenkit T, Manochai P, Lithanatudom SK. #38 (CMUB) |
The basic parameters were measured to characterize the morphometrics of the chromosomes: the length of the long arm (L), short arm (S), the total length of the chromosome (CL=L+S), and total haploid length (THL). Karyotype formula was determined by arm ratio (AR=L/S) and centromeric index (CI%=S/CL×100) according to the classification of Levan et al. (1964) with the symbols m and sm designating metacentric and submetacentric chromosomes, respectively.
Karyotype analyses were conducted using KaryoType 2.0 software (Altınordu et al. 2016) to calculate different karyotypic parameters and generate idiograms. The karyotype asymmetry of each cultivar was estimated via various techniques including total form percent (TF%) (Huziwara 1962), asymmetric karyotype percent (AsK%) (Arano 1963), degree of karyotype asymmetry (A) (Watanabe et al. 1999), intra-chromosomal asymmetry index (A1) and inter-chromosomal asymmetry index (A2) (Zarco 1986), coefficient of variation of centromeric index (CVCI) (Paszko 2006, Zuo and Yuan 2011), coefficient of variation of chromosome length (CVCL) and asymmetry index (AI) (Paszko 2006), mean centromeric asymmetry (MCA) (Peruzzi and Eroğlu 2013) and Stebbins classification (Stebbins 1971).
The karyological relationship among cultivars was determined by UPGMA (unweighted pair group method with arithmetic) using the conclusion of THL, TF%, MCA, and CVCL indices with photographs of fruits morphology of the genus Dimocarpus (Gower 1971, Jaroenkit 2015). In addition, principal coordinate analysis (PCoA) was also performed using six uncorrelated parameters (2n, x, THL, MCA, CVCL, CVCI) as suggested by Peruzzi and Altınordu (2014), and to perform this analysis, the software Past 4.04 (Hammer et al. 2001) was used. Finally, scatter diagrams between asymmetry indices (A1 against A2, CVCI against CVCL, and MCA against CVCL) were drawn.
This study is the first karyological analysis of nine cultivars of the genus Dimocarpus including two cultivars from China and America, and a rare species D. obtusus (formerly regarded as a subspecies of D. longan), Lithanatudom & Chaowasku, comb. et stat. nov. All cultivars in the present study exhibited the same chromosome number 2n=30 (Fig. 1). Although all showed the same chromosome number (2n=30), their chromosome sizes and karyotype formulas were different (Figs. 2–4, Table 2). In terms of the total haploid chromosome length, two cultivars namely Jumbo and Chompoo showed a relatively longer chromosome in length (23.01 and 17.37 µm, respectively) as compared with other cultivars (ranging from 13.02 to 14.93 µm) (Table 2). Based on karyotype formulas, all cultivars were separated into two groups. Karyotype formula in the first group was determined as 30m which are Thao, Phuen-Mueang, Chuliang, and Kohala and the second group was determined as 26m+4sm which are Chompoo, Haew, Phuangthong, Jumbo, and Pumateenkong (Table 2). It is widely accepted that an increase in chromosome size and karyotype diversity is an evolutionary trend (Stebbins 1971, Liu et al. 2006). Additionally, two indices [asymmetry index (AI) and Stebbins classification] based on a combination of intra- and inter-chromosomal asymmetry revealed that the karyotype of the first group falls under category 1B whereas the second group was placed into 2B category based on Stebbins classification (Table 3). Therefore, the second group is more advanced or more asymmetric than the first group due to a shift of centromere position from median to submedian and an accumulation of differences in the relative size between the chromosomes of the complement (Stebbins 1971). However, Stebbins’s quali-quantitative method reveals that all cultivars in this study exhibited symmetrical karyotype in nature (predominance of m and sm chromosomes of approximately the same size). Furthermore, it was found that the AI of the first group (0.48–1.08) had significantly lower than the second group (2.07–2.79). According to these two indices, it is suggested that the karyotype of the first group is more homogenous than the second group (Paszko 2006). Nevertheless, AI and CVCI in the formula AI=CVCL×CVCI/100 were inadequate for karyological analysis in nine cultivars belonging to two species of the genus Dimocarpus as criticized by Peruzzi et al. (2008) and Zuo and Yuan (2011).
| Cultivar name | 2n | AR (L/S) | S (±SD) (µm) | L (±SD) (µm) | CL (±SD) (µm) | THL | CI% (±SD) | Karyotype formula |
|---|---|---|---|---|---|---|---|---|
| Chomphoo | 30 | 1.39 | 0.48 (±0.06) | 0.67 (±0.21) | 1.16 (±0.25) | 17.37 | 42.57 (±4.93) | 26m+4sm |
| Haew | 30 | 1.42 | 0.37 (±0.11) | 0.51 (±0.12) | 0.88 (±0.22) | 13.21 | 41.84 (±4.48) | 26m+4sm |
| Phuangthong | 30 | 1.44 | 0.39 (±0.10) | 0.55 (±0.13) | 0.94 (±0.22) | 14.13 | 41.32 (±3.71) | 26m+4sm |
| Thao | 30 | 1.17 | 0.44 (±0.06) | 0.52 (±0.11) | 0.96 (±0.17) | 14.46 | 46.22 (±2.20) | 30m |
| Jumbo | 30 | 1.39 | 0.65 (±0.14) | 0.89 (±0.20) | 1.53 (±0.30) | 23.01 | 42.41 (±4.87) | 26m+4sm |
| Phuen-Mueang | 30 | 1.16 | 0.46 (±0.09) | 0.53 (±0.12) | 1.00 (±0.20) | 14.93 | 46.30 (±2.42) | 30m |
| Pumateenkong | 30 | 1.37 | 0.37 (±0.10) | 0.50 (±0.14) | 0.87 (±0.23) | 13.02 | 42.74 (±4.52) | 26m+4sm |
| Chuliang | 30 | 1.12 | 0.42 (±0.10) | 0.48 (±0.10) | 0.90 (±0.20) | 13.50 | 47.15 (±1.03) | 30m |
| Kohala | 30 | 1.15 | 0.45 (±0.09) | 0.52 (±0.11) | 0.98 (±0.19) | 14.65 | 46.63 (±1.78) | 30m |
| Cultivar name | TF% | AsK% | A | A1 | A2 | CVCI | CVCL | AI | MCA | Karyotypic asymmetry |
|---|---|---|---|---|---|---|---|---|---|---|
| Chomphoo | 41.71 | 58.29 | 0.15 | 0.25 | 0.22 | 11.59 | 21.98 | 2.55 | 14.87 | 2B |
| Haew | 41.95 | 58.05 | 0.16 | 0.27 | 0.25 | 10.72 | 24.74 | 2.65 | 16.32 | 2B |
| Phuangthong | 41.47 | 58.53 | 0.17 | 0.29 | 0.23 | 8.98 | 23.05 | 2.07 | 17.36 | 2B |
| Thao | 45.94 | 54.06 | 0.08 | 0.14 | 0.17 | 4.76 | 17.29 | 0.82 | 7.56 | 1B |
| Jumbo | 42.26 | 57.74 | 0.15 | 0.25 | 0.19 | 11.48 | 19.28 | 2.21 | 15.19 | 2B |
| Phuen-Mueang | 46.20 | 53.80 | 0.07 | 0.13 | 0.21 | 5.22 | 20.64 | 1.08 | 7.40 | 1B |
| Pumateenkong | 42.65 | 57.35 | 0.15 | 0.24 | 0.26 | 10.58 | 26.37 | 2.79 | 14.52 | 2B |
| Chuliang | 47.19 | 52.81 | 0.06 | 0.11 | 0.22 | 2.18 | 21.83 | 0.48 | 5.70 | 1B |
| Kohala | 46.56 | 53.44 | 0.07 | 0.12 | 0.19 | 3.81 | 19.43 | 0.74 | 6.74 | 1B |
As Stebbins classification or AI alone is insufficient to conclude the karyotypic relationship between different cultivars in the genus Dimocarpus, Peruzzi and Eroğlu (2013) suggested that a scatter diagram between asymmetry indices is the best way to represent the karyotype asymmetry relationship among organisms. Here, three scatter diagrams were generated, comparing the indices of A1 against A2, CVCI against CVCL, and MCA against CVCL. These three scatter diagrams indicated that all cultivars from the genus Dimocarpus were separated into two major groups in which the second group shows comparatively high intra-chromosomal asymmetry indices than those in the first group. On the other hand, inter-chromosomal asymmetry indices in all cultivars indicated a small variation between groups (Fig. 4). Consistently, all the karyological analysis as described above is in agreement with the constructed UPGMA dendrogram based on Gower’s similarity matrix of four uncorrelated karyological parameters (THL, TF%, MCA, and CVCL) (Fig. 5). THL is a great proxy for genome size (the basic karyomorphological character) (Peruzzi et al. 2008) while MCA and CVCL are among the most reliable intra- and inter-chromosomal asymmetry parameters, respectively as suggested by Peruzzi and Altınordu (2014). Furthermore, TF% was used to construct the dendrogram instead of CVCI since it is more accurate than CVCI as criticized by Zuo and Yuan (2011) and it is an appropriate indicator for the symmetric type of karyotype which is the case observed in this study (Saha et al. 2020). Strikingly, the fruit morphology of the genus Dimocarpus is also related to karyological data because of the size of the fruit and seed (Fig. 5).
In the genus Dimocarpus, the morphology of the fruit is one of the attractive criteria due to the unique characteristic of each cultivar; Chompoo (faint pink tinge in the flesh), Haew (thick and rough fruit peel), Phuangthong (golden-yellow fruit peel), Thao (smell sulfur-like flesh), Jumbo (the largest flesh), Phuen-Mueang (the most symmetric fruit), Pumateenkong (smell fishy-like flesh but beautiful fruit), Chuliang (thick and firm flesh) and Kohala (sweet aroma and spicy flavor flesh) (Choo 2000, Jaroenkit 2015). According to the dendrogram, all cultivars are divided into two major groups (Gower distance=0.05) (Fig. 5). The first group consists of Thao, Phuen-Mueang, Chuliang, and Kohala while the second group is composed of Chompoo, Haew, Phuangthong, Jumbo, and Pumateenkong. Interestingly, the grouping of longan cultivars categorized by the four parameters mentioned above is consistent with morphological analyses of fruit size together with flesh and seed ratio. The cultivars in the first group have smaller fruit sizes and lower flesh/seed ratios than in the second group. Therefore, we hypothesized that the lower the flesh/seed ratio, the more primitive the longan cultivar based on karyological data and fruit morphology. Thao has been recently recognized as a rare species in Thailand and it is also known as the “wild longan” (Santisuk 2005), the only cultivar that is a scandent shrub and capable of flowering more than once a year (Boonsuk and Chantaranothai 2017, Lithanatudom et al. 2017). While Phuen-Mueang is a large tree (20–30 m), also known as “Bone longan” due to the smallest size of fruit, low fleshly part, and similar to the “wild longan”. According to fruit morphology and karyological analyses, both Thao and Phuen-Mueang are in the same group supporting that they are both considered “wild longan”. Historically, it is documented that Thai longan was brought to Thailand from China in 1896 (Jaroenkit 2015), therefore, it is likely that Chuliang native to China is closely related to “Phuen-Mueang” which is believed to be the original cultivar of longan in Thailand. In support of this notion, both cultivars are in the same group as shown in scatter diagrams, dendrogram, and PCoA (Figs. 4–6). Additionally, all longan cultivars in the second group, the relatively advanced longan cultivars as compared to the first group according to the chromosomal symmetry, are developed by local breeders in favor of taste, fruit size, and yield such as Jumbo is one of the commercial cultivars of longan in Thailand that has been registered on “Plant Varieties Protection Office” with the largest flesh (the smallest seed) (Jaroenkit 2015).
The PCoA graph illustrates two groups of the genus Dimocarpus. However, Jumbo and Chuliang were located at a distinct positions in each group. Therefore, this analysis needs more cultivars to clearly define the karyotypic relationship between different cultivars. Moreover, either the TF% or CVCI used in these analyses led to the same PCoA graph based on Gower’s similarity matrix of six uncorrelated karyological parameters (Fig. 6).
Overall, the karyological analysis and morphological data in this present study can be concluded that longan can be distinguished into two different groups. The first group (cultivars with large seeds and small fruit) is more primitive than the second group (cultivars with a small seeds and large fruit). Also, we believe that Thao and Phuen-Mueang in the first group are ancient cultivars found in Thailand. However, to warrant further chromosome investigation for higher resolution, fluorescence in situ hybridization would be more informative to pinpoint a relationship between cultivars in the genus Dimocarpus for validation of the proposed phylogenetic systematics of longan in our previous study.
The longan trees used in this study were kindly provided and maintained by Asst. Prof. Pawin Manochai and Assoc. Prof. Dr. Theeranuch Jaroenkit, Department of Horticulture, Faculty of Agricultural Production, Maejo University, Chiang Mai, Thailand. This work was supported by the Development and Promotion of Science and Technology Talents Project (DPST) (PP); Teaching Assistant and Research Assistant Scholarships for the Academic Year 2020, Chiang Mai University (NJ); Thailand Science Research and Innovation (TSRI) under Grant number DBG6180017 (SKL).
