Integrated Karyotypes of Diploid and Tetraploid Carrizo Citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf.) as Determined by Sequential Multicolor Fluorescence in situ Hybridization With Tandemly Repeated DNA Sequences.

Honghong Deng,Guolu Liang,Dong Liang,Jin Wang,Zhijian Gao,Guohao Tang,Hui Xia,Zexi Cai,Xiulan Lv,Lijin Lin,Qunxian Deng,Nuo Xu

doi:10.3389/fpls.2020.00569

Abstract

Carrizo citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf., CC] is one of the most widely used rootstocks in citriculture worldwide, but its cytogenetic study has been hampered by its inherent small size, morphological similarity to mitotic chromosomes, and lack of accessible cytological landmarks. In our previous study, a spontaneously occurring tetraploid CC seedling was discovered. The main goals of this study were to elucidate the chromosome constitution and construct the karyotypes of diploid CC rootstock and its corresponding spontaneously occurring tetraploid. To accomplish these, the chromosomal characteristics were investigated by sequential multicolor fluorescence in situ hybridization (FISH) with eight properly labeled repetitive DNA sequences, including a centromere-like repeat, four satellite repeats, two rDNAs, and an oligonucleotide of telomeric (TTTAGGG)n repeat. The results nicely demonstrated that these repetitive DNAs are reliable cytogenetic markers that collectively facilitate simultaneous and unequivocal identification of homologous chromosome pairs. Based on chromosome size and morphology together with FISH patterns of repetitive DNAs, an integrated karyotype of CC rootstock was constructed, consisting of 2n = 2x = 12m (1sat) + 6sm with karyotype asymmetry degree being divided into 2B category. Cytogenetically speaking, the variable and asymmetric distribution patterns of these repetitive DNAs were fully confirmed the hybrid nature of CC rootstock. In addition, comparative distribution patterns and chromosomal localizations of these repetitive DNAs convincingly showed that this tetraploid CC material arose from somatic chromosome doubling of diploid CC rootstock. This study revealed, for the first time, the integrated karyotype and chromosomal characteristics of this important citrus rootstock as well as its spontaneously occurring tetraploid plant. Furthermore, this study is a good prospective model for study species with morphologically indistinguishable small chromosomes.

Highlights

Citrus is one of the most horticulturally and economically important fruit crops globally (Wu et al, 2018), extensively cultivated in more than 140 countries and regions in the tropics and subtropics (Cuenca et al, 2018)
In order to precisely identify each pair of homologous chromosomes and obtain detailed karyotypes of diploid and tetraploid CC plants, the physical distribution of highly repetitive DNAs on somatic chromosomes was determined by sequential multicolor Fluorescence in situ hybridization (FISH) on the mitotic metaphase chromosomes coming from the same metaphase spreads (Figures 1, 2)
FISH with a CL1 probe revealed an asymmetrical pattern that the CL1 loci were localized in the subtelomeric regions of the long arms of the chromosome pairs 2, 3, 4, 5, and 6, and only one short arm of the chromosome pair 3 (Figures 1B, 2B)

Summary

Introduction

Citrus is one of the most horticulturally and economically important fruit crops globally (Wu et al, 2018), extensively cultivated in more than 140 countries and regions in the tropics and subtropics (Cuenca et al, 2018). Citrus cultivars are grafted on the rootstock for commercial production worldwide (Oustric et al, 2017, 2019; Bowman and Joubert, 2020). Citrus scion cultivars may respond differently to vegetative growth, canopy size, fruit quality and production, resistance to pests and diseases, as well as tolerance to various abiotic and biotic stresses when grown on diverse rootstocks (Warschefsky et al, 2016; Wang et al, 2017; Oustric et al, 2019; Bowman and Joubert, 2020). The multibillion-dollar citrus industry is facing a century-old conundrum from Huanglongbing (HLB) (Clark et al, 2018). Albrecht et al (2012) evaluated the influence of 15 different rootstock varieties on HLB disease development in Florida field-grown Valencia and Early Gold sweet orange [C. sinensis (L.) Osb.], and they found that the highest fruit production, under high HLB pressure, were obtained from the combinations of citrus scion cultivars on US-802 and CC rootstock

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