RAPD primer screening as a preliminary study to analyze the genetic diversity of Citrus spp. in South Sulawesi, Indonesia

The typical citrus germplasm collection in South Sulawesi has not been thoroughly characterized, especially in several citrus development centers, which have begun to be promoted again after the decline in productivity due to CVPD infection. The study of citrus diversity is very important to support future citrus breeding programs. Random amplified polymorphic DNA (RAPD) has been widely used for the analysis of genetic diversity among species in populations. In this study, 23 RAPD primers were used on Citrus cultivated in Selayar and Pangkep Regencies, which are citrus development areas in South Sulawesi. A total of 19 primers (OPA-05, OPA-09, OPA-17, OPC-09, OPC-17, OPE-04, OPH-04, OPH-15, OPN-14, 0PN-16, OPR-08, OPR-20, OPW-06, OPW-09, OPX-07, OPX-11, OPX-17, UBC-18, and UBC-51) can form polymorphic bands in randomly selected DNA samples. Monomorphic bands were formed by OPA-12 and OPD-07 primer in 12 samples. The primers OPX-13 and OPX-16 produced unclear bands. These 19 primers can be used to amplify DNA and determine the genetic diversity of Citrus in further analysis.

Integrated Transcriptomics and Metabolomics Analyses Provide Insights Into the Response of Chongyi Wild Mandarin to Candidatus Liberibacter Asiaticus Infection

Candidatus Liberibacter asiaticus (CLas) is the causative agent of Huanglongbing (HLB), which has caused great economic losses to the citrus industry. The molecular mechanism of the host response to CLas in wild citrus germplasm has been reported less. Eighteen weeks after inoculation via grafting, all the CLas-inoculated Chongyi wild mandarin (Citrus reticulata) were positive and showed severe anatomical aberrations, suggesting its susceptibility to HLB. Transcriptomics and metabolomics analyses of leaves, barks, and roots from mock-inoculated (control) and CLas-inoculated seedlings were performed. Comparative transcriptomics identified 3,628, 3,770, and 1,716 differentially expressed genes (DEGs) between CLas-infected and healthy tissues in the leaves, barks, and roots, respectively. The CLas-infected tissues had higher transcripts per kilobase per million values and more genes that reached their maximal expression, suggesting that HLB might cause an overall increase in transcript accumulation. However, HLB-triggered transcriptional alteration showed tissue specificity. In the CLas-infected leaves, many DEGs encoding immune receptors were downregulated. In the CLas-infected barks, nearly all the DEGs involved in signaling and plant-pathogen interaction were upregulated. In the CLas-infected roots, DEGs encoding enzymes or transporters involved in carotenoid biosynthesis and nitrogen metabolism were downregulated. Metabolomics identified 71, 62, and 50 differentially accumulated metabolites (DAMs) in the CLas-infected leaves, barks and roots, respectively. By associating DEGs with DAMs, nitrogen metabolism was the only pathway shared by the three infected tissues and was depressed in the CLas-infected roots. In addition, 26 genes were determined as putative markers of CLas infection, and a hypothesized model for the HLB susceptibility mechanism in Chongyi was proposed. Our study may shed light on investigating the molecular mechanism of the host response to CLas infection in wild citrus germplasm.

An Eleven-Year Survey on Field Disease Susceptibility of Citrus Accessions to Colletotrichum and Alternaria Species

In the past decade Colletotrichum gloeosporioides, C. karstii, and Alternaria alternata represent emerging fungal pathogens on citrus in the Mediterranean basin. Selection of tolerant Citrus germplasm offers evaluable long-term solution and should be considered as promising alternative to limit synthetic fungicide application to manage Alternaria and Colletotrichum infections in Citrus groves. In this study, the high variability of pre-harvest disease symptoms among 37 Citrus accessions was investigated in Italy in the most representative Sicilian production districts covering 1500 ha and including 20 homogeneous areas over an eleven-year survey period (2010–2020). Early fruit drop, brown spot, and anthracnose on fruit and leaves, pre-harvest fruit drop, and twig blight and defoliation associated to Alternaria and Colletotrichum spp. were identified on oranges, mandarins, and lemons. Comprehensively, first extensive data obtained herein on field susceptibility within “Tarocco” blood orange group to above disease symptoms clearly indicate as “Tarocco Nucellare 57-1E-1,” “Tarocco Tapi,” “Tarocco Sant’Alfio,” and “Tarocco Catania” accessions should be preferred to remaining Tarocco ones. A broad degree of tolerance or susceptibility was also observed within other Citrus group as it happens for tolerant lemon “Femminello Zagara Bianca,” thus demonstrating a putative resource for further studies to employ in a breeding program for genetic improvement of Citrus.

Identification of Local Citrus Breeds Using the Start-Codon Targeted Polymorphism Technique Combined with Clonal Sequencing

This study explored the genetic diversity of 35 citrus accessions using start-codon targeted (SCoT) markers. Total 15 primers were used to amplify products ranging in length from 100 to 2000 bp. A total of 133 fragments were amplified and found that 126 (95%) were polymorphic. The genetic similarity coefficients among the 35 accessions ranged from 0.67 to 1.00, indicating that the SCoT markers could reveal high genetic diversity in the citrus germplasm. A cluster analysis showed that local citrus breeds of Sihui city fell into one cluster, possibly reflecting the geographical distribution of the tested samples. The sizesof the fragments of eight local citrus cultivars amplified by one of the primers ranged from 951 to 1001 bp, and the similitude was 95.17%high. Both single-base mutations, insertions and deletions were identified among the fragments and comparison with asequence database suggested that the amplified region was part of a ribosomal protein-coding sequence. Using Scot marks combined with clonal sequencing, each of the test samples had one or more mutation sites that could be used as markers to differentiate from the other seven test samples. Thus, the resolution of the genetic diversity among the local citrus breeds revealed by the SCoT technique was enhanced by the subsequent sequencing analysis of specific fragments. The experimental results also provide evidence that the relationship between CitrusnobilisL our. ‘gonggan’ and C. hanianaHort. ex Tseng ‘Sihuihanggan’ is that between parent and offspring hybrids and not between bud-mutation strains. The SCoT marker is a targeted gene molecular marker, based on the characteristic bands of primers, it can be used as a marker to isolate genes of local citrus varieties and also for investigating mutational hotspots of the segene.© 2021Friends Science Publishers

Optimizing Recovery of Hybrid Embryos from Interspecific Citrus Crosses of Polyembryonic Rough Lemon (Citrus jambhiri Lush.)

The prevalence of rough lemon rootstock in India is presently threatened by the spread of Phytophthora root rot. Developing an alternative Phytophthora-resistant rootstock that also withstands other abiotic constraints is a priority for a sustainable citrus industry in India. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Citrus and Poncirus, a closely related genus. The production of hybrids accumulating the desired dominant traits, irrespective of the heterozygosity level of the parents, is an effective way to develop new candidate rootstocks. Rough lemon was hybridized with Swingle citrumelo, X-639 citrandarin, and GouTou Cheng. A reliable method was developed for greater recovery of hybrid seedlings from these crosses on the basis of morphology and simple sequence repeat (SSR) markers. The greatest proportion of hybrid seedlings resulted from embryo rescue at 95 days after pollination (DAP) (48.5% in rough lemon × X-639 citrandarin and 56.7% in rough lemon × Swingle citrumelo) on the basis of multifoliate leaves. The proportion of hybrid seedlings decreased with increase in embryo development stage after 95 DAP until the last stage of observation (125 DAP). Significantly higher proportion of multifoliate seedlings was recovered by in vitro culture compared with in vivo germination from the mature seeds (26.9% vs. 5.2%, respectively). Swingle and X-639 citrandarin are not homozygous for the multifoliate allele from Poncirus, and GouTou Cheng lacks the multifoliate allele; thus, unifoliate hybrids were selected from all crosses on the basis of SSR marker genotypes.

Target-Genes Reveal Species and Genotypic Specificity of Anthocyanin Pigmentation in Citrus and Related Genera

Background: Anthocyanin pigmentation characterizes a number of tissues of Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species. Methods: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus, Murraya, and Severinia. A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3′-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species. Results: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids. Conclusion: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.

Phylogenetic relationships among accessions in Citrus and related genera based on the insertion polymorphism of the CIRE1 retrotransposon

A total of 145 accessions of the genus Citrus and related genera, maintained in the Conservation Garden for Citrus Germplasm at the Experimental Farm of Kindai University, Yuasa, Wakayama, Japan, were examined for their phylogenetic relationships. The present classification was conducted using an inter-retrotransposon amplified polymorphism (IRAP) method based on the insertion polymorphism of a retrotransposon, CIRE1, identified in C. sinensis. The objective of this study was to evaluate the applicability of the IRAP method for citrus classification. The constructed dendrogram showed that the 145 accessions and two outgroup species were successfully classified into five major clades. A large number of C. sinensis accessions were divided into three traditional groups, navel orange, sweet orange, and blood orange, almost corresponding to the sub-clades in the dendrogram. Several other accessions belonging to the same species, and also many hybrid cultivars from crossbreeding, were localized into the respective sub-clades or near positions in the dendrogram. Several unclassified accessions could also be located in the dendrogram, suggesting novel relationships with other accessions. It was concluded that the IRAP method based on CIRE1 insertion polymorphism was suitable for the classification of citrus from a molecular point of view.