Tree Genetics & Genomes (2017) 13: 58 DOI 10.1007/s11295-017-1133-0 ORIGINAL ARTICLE Genetic diversity and population structure of pummelo (Citrus maxima) germplasm in China Huiwen Yu1 & Xiaoming Yang 1 & Fei Guo1 & Xiaolin Jiang1 & Xiuxin Deng1 & Qiang Xu1 Received: 31 July 2016 /Revised: 11 March 2017 /Accepted: 19 March 2017 /Published online: 26 April 2017 # Springer-Verlag Berlin Heidelberg 2017 Abstract Pummelo (Citrus maxima) is one of the basic spe- Keywords Pummelo . Genetic diversity . Population cies of Citrus. It has been cultivated for about 4000 years in structure . Nuclear simple sequence repeat (nSSR) China, and therefore, there are abundant germplasm during the long time of culture. However, there is still a lack of a detailed study of the genetic characteristics of pummelo pop- Introduction ulation. In this study, genetic diversity and population struc- ture among 274 pummelo accessions collected in China were Citrus is one of the most important fruit crops in the world. analyzed using 31 nuclear simple sequence repeat (nSSR) The genetic background of citrus is very complicated because markers. The observed heterozygosity was calculated as of its biological characteristics, such as wide sexual compati- 0.325 and genetic differentiation Fst as 0.077. Genetic struc- bility on interspecies and intergenus levels. The complex ge- ture analysis divided the whole germplasm into three subpop- netic background has hindered the genetic studies in citrus. ulations, Pop-a, Pop-b, and Pop-c. Pop-a was composed of Exploring genetic variation within a single species will facil- accessions mostly from Southeast China, Pop-b was com- itate genetic analysis such as genome-wide association studies posed of accessions from the central region of South China, (GWAS) of important traits. According to Swingle (1967) and Pop-c was composed of accessions from Southwest system, the genus Citrus was classified into three basic spe- China. Meanwhile, the analysis of principal coordinate analy- cies: citron (Citrus medica L.), mandarin (C. reticulata sis and neighbor-join tree supported the viewpoint of three Blanco), and pummelo (C. maxima (Burm.) Merrill) subpopulations, and then the possible dispersal routes of (Swingle 1967;Scora1975; Barrett and Rhodes 1976). The pummelos in China were proposed. This study provides an mandarins are of complex background because of introgres- insight into the genetic diversity, facilitates future genome- sion of pummelo elements (Wu et al. 2014b;Uzunand wide association studies, and promotes the breeding program Yesiloglu 2012). Most mandarins reproduce through apomix- of pummelo as well. is, an asexual reproduction via nucellar embryo, and propa- gate clonally by grafting. These characteristics result in low genetic diversity in cultivated mandarins. In contrast, the cit- ron and pummelo produce seeds through sexual reproduction Communicated by V. Decroocq and are candidate species promising for genetic variant explo- Electronic supplementary material The online version of this article ration and application. (doi:10.1007/s11295-017-1133-0) contains supplementary material, Pummelo is believed to be native to Southeast Asia (Li which is available to authorized users. 1970;Ye1996). It is widely cultivated in tropical and subtrop- ical regions in China, showing particularly high genetic diver- * Qiang Xu [email protected] sity compared with other types of citrus (Liu et al. 2005;Li et al. 2006). This may be due to three main reasons. Firstly, 1 Key Laboratory of Horticultural Plant Biology (Ministry of there is a long history of pummelo cultivation in China, as Education), Huazhong Agricultural University, Wuhan 430070, pummelo was recorded as one of the fruits used as tribute to China the emperor 4000 years ago in the Chinese book Tribute of Yu 58 Page 2 of 10 Tree Genetics & Genomes (2017) 13: 58 (Scora 1975). Secondly, Chinese culture custom may also To investigate the genetic diversity and population struc- contribute to the wide genetic variation. People in Southern ture of pummelo, 274 pummelo accessions were collected China usually grow pummelo in front- or backyards from through a national survey of citrus germplasm across China seeds. This seed-propagation mode under natural pollination by our group in the past 4 years. These accessions were eval- conditions may introduce introgressions during the long his- uated with the evenly distributed 31 nSSR markers. The re- tory of cultivation. This is distinct from other citrus types such sults not only are helpful to reveal the genetic variation, pop- as mandarins and sweet oranges which exhibit strict nature of ulation properties, and dispersal history of pummelo but also asexual reproduction and depend on vegetative propagation. can provide new gene pools of pummelo for citrus breeding Thirdly, the diversified climates in tropical and subtropical programs. regions enable these areas to produce abundant germplasm due to the adaptation to the environments. Pummelo is also an important gene pool for breeding new types of citrus, and a Materials and methods dozen of pummelo-derived cultivars have been released in the past years such as grapefruits, tangelos, and hybrids (Nicolosi Pummelo germplasm collection et al. 2000; Barkley et al. 2006;Garcia-Loretal.2012;Guo et al. 2015). Leaves and mature fruits of 274 accessions (Table S1)and9 Genetic variation of the pummelo has been noticed in dif- relatives were collected. It took 4 years (2010–2013) to obtain ferent countries (Paudyal and Haq 2008; Nartvaranant and the whole germplasm from 12 provinces of Southern China. Nartvaranant 2011), and a number of studies have investigated Meanwhile, the accessions with the same name collected from the variety of pummelos in China from different aspects, such different places were labelled by different accession numbers as GOT isozymes and chromosomal karyotypes evaluation on (AN) to avoid losing homonym of samples. The nine relatives, 22 pummelo accessions (Chen and Lai 1993), assessment of Daoxian mandarin (C. reticulata), Bendizao mandarin the genetic diversity of 120 accessions using AFLP and SSR (C. reticulata), Ponkan mandarin (C. reticulata), Citron markers (Liu et al. 2005), and selection and improvement of (C. medica), Ichang papeda (C. ichangensis), pummelo in Nepal (Paudyal and Haq 2008). Pummelo in Mangshanyegan (Citrus nobilis Lauriro) and Trifoliate southern Zhejiang Province and Hunan Province were ana- (Poncirus trifoliata), and Hongkong kumquat (Fortunella lyzed by sequence-related amplified polymorphism (SRAP) hindsii), were sampled from the National Center of Citrus markers (Chen et al. 2012;Lietal.2013), which showed the Breeding (NCCB), Huazhong Agricultural University high diversity of Chinese pummelos. A comprehensive and (HZAU), Wuhan, China, and used as outgroups. detailed investigation of pummelo population is still very lim- ited, and the genetic structure of this species has not been fully Molecular marker analysis characterized. Simple sequence repeat (SSR), a useful and efficient DNA was extracted from leaves via the CTAB methods molecular marker based on PCR, is co-dominant, usually (Cheng et al. 2003). To study the genetic diversity of pumme- multiallelic and hypervariable, abundantly and randomly lo, 31 polymorphic simple sequence repeat (SSR) markers distributed in genomes, and had been widely used to study spreading over the chromosomes were selected from Citrus the genetic diversity and population analysis in many spe- sinensis genome (Xu et al. 2013)(TableS2). Polymerase cies (Barthe et al. 2012). Many previous studies have uti- chain reaction (PCR) was performed in a volume of 10 μl lized SSR markers to analyze the genetic diversity of citrus containing 50 ng of genomic DNA, 5 μl 2×TaqPCR ingeneral(Barkleyetal.2006; Jannati et al. 2009;Amar GREEN MIX (Dingguo, Beijing, China), and 0.5 μMofeach et al. 2011;Biswasetal.2011; Garcia-Lor et al. 2012, primer pair. Amplification was carried out as follows: 7 min at 2013; Nematollahi et al. 2013),andinparticular,lemons 95 °C, followed by 33 cycles of 30 s at 95 °C, 30 s at 57 °C (Gulsen and Roose 2001;Curketal.2016), sweet orange and 45 s at 72 °C, and a final extension at 72 °C for 10 min in a (Novelli et al. 2006), mandarin (Li et al. 2006; Kacar et al. MJ-PTC_200 thermal controller (MJ Research, Waltham 2013; Garcia-Lor et al. 2015), as well as grapefruit Mass). Products were separated by polyacrylamide gel elec- (Corazza-Nunes et al. 2002), and pummelo (Liu et al. trophoresis for further analysis (Ruiz et al. 2000). 2005; Barkley et al. 2006;Liuetal.2006). These studies also indicated greater genetic diversity of pummelo when Data analysis compared with other species. Further broadening the ge- netic background and including more accessions of pum- The statistics such as allele number per locus and polymor- melo will provide a comprehensive evaluation of the ge- phism information content (PIC) values were determined by netic parameters, which constitutes a theoretic basis for PowerMarker version 3.25 (Liu and Muse 2005). genome-wide association studies in Citrus. STRUCTURE version 2.3.4 (Pritchard et al. 2000) was used Tree Genetics & Genomes (2017) 13: 58 Page 3 of 10 58 to analyze the genetic structure of pummelo, and software distribution to study the diversity of this germplasm collec- STRUCTURE HARVESTER was used to obtain the autofit tion. Totally, 147 alleles were detected with a mean of 4.7 value of K for the population clustering according to Evanno’s alleles for each marker. Observed heterozygosity (Ho) was protocol (Earl 2012;Evannoetal.2005). To calculate the Nm ranged from 0.038, as is the case for marker Ma3_8, to (gene flow), Ho (observed heterozygosity), and He (expected 0.689 of Csin.0278 marker, and with a mean value of 0.325. heterozygosity), program POPGENE version 1.32 (Yeh and Meanwhile, the maximum value of expected heterozygosity Boyle 1997; Chung et al. 2003;Ranaetal.2015) was applied. (He) was 0.766 for Csin.0078, and the minimum value was The principal coordinate analysis (PCoA), the analysis of mo- 0.043 for marker Ma3_176, with an average value at 0.430.