Graft Compatibility Classification within Aurantioideae Based on Biometric Traits and the Anatomy of Graft Union

Citrus relatives are a relevant source of valuable traits for use in citrus breeding, including resistance to diseases such as Huanglongbing (HLB). Resistant rootstocks may impact tree responses to HLB. This requires graft compatibility, which has been poorly investigated within the Aurantioideae. In this study, the biometric characteristics and the anatomy of the graft union of 86 scion/rootstock combinations were assessed. This comprised 18 genotypes/species and 8 genera from Citrinae, Balsamocitrinae, and Clauseninae subtribes sensu Swingle and Reece. Most graft combinations were found to be noncompatible. Phylogenetic proximity did not ensure successful grafting as, for example, Orange jasmine autografts failed, whereas some intergeneric grafts were successful (>60% of graft-take). Plant scion height was directly related to graft-take, but the correlation between the scion and rootstock stem diameters was not a reliable indicator of graft compatibility. Rangpur/Tabog, Tabog/Rangpur, Wampee/Rangpur, Wampee/Pomeroy, Wampee/Swingle, Pomeroy/Wampee, and Swingle/Wampee were the most compatible intergeneric graft combinations. Graft-take success for this was at similar levels to those of sweet orange grafted on common citrus rootstocks. The position as a scion or rootstock in the combination affected the performance and was specific to the genotypes tested. The lack of differentiation between xylem-derived calli and the accumulation of phenolic compounds at the graft union were clear anatomical and biochemical markers, respectively, of incompatibility for most Aurantioideae combinations. In the field, within a set of the ten most promising combinations, Hamlin/Rangpur (control) was the only one that became infected by ‘Candidatus Liberibacter asiaticus’. This was first observed 12 months after planting. Overall, the assessment of biometric traits and anatomy of the graft union allowed Aurantioideae genotypes to be divided into four clusters, with respect to their graft compatibility, as follows: fully compatible with high graft-take and plant growth; potentially compatible with high graft-take but lower plant growth; partially incompatible with lower graft-take and poor plant growth; and fully incompatible with a complete absence of graft-take.

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.

Results of the Biochemical Study of Mandarin (Citrus reticulata Blanco) Mutants

Background: Induced mutagenesis has been used in citrus breeding, in Georgia, since 1960, resulting in many interesting and promising mutant forms that require morphological, physiological and biochemical studies. The present paper presents the results of biochemical research of some promising mutants. Methods: During the period 2017-2019 investigate on 13 promising mutant forms of mandarin has been carried out. In the current study, phenolic composition content in the fruits of mandarin mutants were determined using Ultraviolet Spectrophotometer and High Performance Liquid Chromatography (HPLC) and their antioxidant capacities were evaluated using 2.2-diphenyl-1-picrylhydrazyl (DPPH) radicals.Result: The results have shown that different concentrations of mutagen effect on mandarin seeds and, accordingly, on the biochemical components which they include. In most cases, this leads to a positive result. The impact of the mutagen significantly increases the range of changes in biochemical components and varies over a wide range. Analyzing the data, we can conclude that the solutions of all concentrations of the Nash mutagen effect a change in the biochemical indicators in mutant forms. However, a wide range of variability is largely associated with the use of medium and low concentrations of the mutagen. In most cases, the majority of mutant forms significantly outperform the control variants in the content of biochemical components. The study has revealed a variety of mutant forms (gene pool), which characterized a whole complex of positive signs. Their use in selection allows getting perspective forms with positive features.

Allelic composition of carotenoid metabolic genes in 13 founders influences carotenoid composition in juice sac tissues of fruits among Japanese citrus breeding population

To enrich carotenoids, especially β-cryptoxanthin, in juice sac tissues of fruits via molecular breeding in citrus, allele mining was utilized to dissect allelic variation of carotenoid metabolic genes and identify an optimum allele on the target loci characterized by expression quantitative trait (eQTL) analysis. SNPs of target carotenoid metabolic genes in 13 founders of the Japanese citrus breeding population were explored using the SureSelect target enrichment method. An independent allele was determined based on the presence or absence of reliable SNPs, using trio analysis to confirm inheritability between parent and offspring. Among the 13 founders, there were 7 PSY alleles, 7 HYb alleles, 11 ZEP alleles, 5 NCED alleles, and 4 alleles for the eQTL that control the transcription levels of PDS and ZDS among the ancestral species, indicating that some founders acquired those alleles from them. The carotenoid composition data of 263 breeding pedigrees in juice sac tissues revealed that the phenotypic variance of carotenoid composition was similar to that in the 13 founders, whereas the mean of total carotenoid content increased. This increase in total carotenoid content correlated with the increase in either or both β-cryptoxanthin and violaxanthin in juice sac tissues. Bayesian statistical analysis between allelic composition of target genes and carotenoid composition in 263 breeding pedigrees indicated that PSY-a and ZEP-e alleles at PSY and ZEP loci had strong positive effects on increasing the total carotenoid content, including β-cryptoxanthin and violaxanthin, in juice sac tissues. Moreover, the pyramiding of these alleles also increased the β-cryptoxanthin content. Interestingly, the offset interaction between the alleles with increasing and decreasing effects on carotenoid content and the epistatic interaction among carotenoid metabolic genes were observed and these interactions complexed carotenoid profiles in breeding population. These results revealed that allele composition would highly influence the carotenoid composition in citrus fruits. The allelic genotype information for the examined carotenoid metabolic genes in major citrus varieties and the trio-tagged SNPs to discriminate the optimum alleles (PSY-a and ZEP-e) from the rest would promise citrus breeders carotenoid enrichment in fruit via molecular breeding.

Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection

Alternaria alternata is a fungus that causes a serious disease in susceptible genotypes of citrus, particularly in mandarins. The Alternaria citri toxin (ACT) produced by the pathogen induces necrotic lesions on young leaves and fruits, defoliation and fruit drop. Here, we describe two methods of marker-assisted selection (MAS) that could be used for the early identification of Alternaria brown spot (ABS)-resistant mandarin hybrids. The first method is based on a nested PCR coupled to high resolution melting (HRM) analysis at the SNP08 locus, which is located at 0.4 cM from the ABS resistance locus, and was previously indicated as the most suitable for the selection of ABS-resistant hybrids. The method was validated on 41 mandarin hybrids of the CREA germplasm collection, and on 862 progenies generated from five crosses involving different susceptible parents. Four out of five populations showed Mendelian segregation at the analyzed locus, while a population involving Murcott tangor as male parent showed distorted segregation toward the susceptible hybrids. The second method is based on a cleaved amplified polymorphic sequences (CAPS) marker that was developed using the same primers as the nested PCR at the SNP08 locus, coupled with BccI restriction enzyme digestion. To verify the reliability of the two genotyping methods, in vitro leaf phenotyping was carried out by inoculating A. alternata spores onto young leaves of 101 hybrids, randomly chosen among the susceptible and resistant progenies. The phenotyping confirmed the SNP08 genotyping results, so the proposed method of selection based on HRM or CAPS genotyping could be routinely used as an alternative to KBioscience competitive allele specific polymerase chain reaction (KASPar) single nucleotide polymorphism (SNP) genotyping system to improve citrus breeding programs. While the study confirmed that the SNP08 marker is a reliable tool for MAS of new citrus hybrids with different genetic backgrounds, it also identified a small group of genotypes where the resistance mechanism requires further investigation.

Seed and Seedling Nursery Characteristics for 10 USDA Citrus Rootstocks

Six new hybrid rootstocks, ‘US-1279’, ‘US-1281’, ‘US-1282’, ‘US-1283’, ‘US-1284’, and ‘US-1516’, were released from the U.S. Department of Agriculture (USDA) citrus breeding program to provide improved tree tolerance to huanglongbing (HLB), the most destructive disease facing the citrus industry in the United States and many other parts of the world. Five of these new rootstocks were released based on field performance in trials with the rootstocks propagated by stem cuttings, rather than the traditional propagation using nucellar seedlings. In this study, we evaluated the fruit, seed, and seedling characteristics of these new rootstocks, along with four other USDA rootstocks of commercial importance. The study included a determination of the percentage of true-to-type and off-type seedlings by both plant morphology and simple sequence repeat (SSR) markers. All 10 rootstocks produced an acceptable number of seeds and good seedling emergence from those seeds. The rootstocks ‘Swingle’, ‘US-802’, ‘US-812’, ‘US-1283’, ‘US-1284’, and ‘US-1516’ had a high percentage of true-to-type seedlings and correspondingly good potential to be propagated by seeds. However, no true-to-type plants were observed among seedlings from the rootstocks ‘US-1279’, ‘US-1281’, and ‘US-1282’, indicating that economical seed propagation will be impossible for these cultivars. The 10 SSR marker sets used in this study were observed to easily differentiate the 10 rootstocks studied, and readily distinguished true-to-type and off-type seedlings among progeny from all 10 rootstock clones. This study presents information of significant value for commercial nurseries involved in propagation of citrus rootstocks, and those involved in citrus rootstock breeding and development around the world. We propose the use of these 10 SSR marker sets as readily applicable for accurate identification of most citrus rootstock cultivars and their true-to-type seedlings.

Citrus Polyamines: Structure, Biosynthesis, and Physiological Functions

Polyamines (PAs) are ubiquitous biogenic amines found in all living organisms from bacteria to Archaea, and Eukaryotes including plants and animals. Since the first description of putrescine conjugate, feruloyl-putrescine (originally called subaphylline), from grapefruit leaves and juice, many research studies have highlighted the importance of PAs in growth, development, and other physiological processes in citrus plants. PAs appear to be involved in a wide range of physiological processes in citrus plants; however, their exact roles are not fully understood. Accordingly, in the present review, we discuss the biosynthesis of PAs in citrus plants, with an emphasis on the recent advances in identifying and characterizing PAs-biosynthetic genes and other upstream regulatory genes involved in transcriptional regulation of PAs metabolism. In addition, we will discuss the recent metabolic, genetic, and molecular evidence illustrating the roles of PAs metabolism in citrus physiology including somatic embryogenesis; root system formation, morphology, and architecture; plant growth and shoot system architecture; inflorescence, flowering, and flowering-associated events; fruit set, development, and quality; stomatal closure and gas-exchange; and chlorophyll fluorescence and photosynthesis. We believe that the molecular and biochemical understanding of PAs metabolism and their physiological roles in citrus plants will help citrus breeding programs to enhance tolerance to biotic and abiotic stresses and provide bases for further research into potential applications.

Effects of Gamma Irradiation on Seedlessness and Fruit Quality of Ortanique Tangor

The existence of a large amount of seeds in citrus fruits results as a major impediment to customer acceptability, even if the fruits have high organoleptic properties. Irradiation, which mainly reduces seed number of varieties, is a faster way than hybridization. The use of irradiation in citrus breeding programs is now quite widespread with most programs in the major citrus producing countries actively developing new selections. The present study reports the primarily results of gamma irradiation on seed number and fruit quality of Ortanique tangor mutant population. The shoots of scion were irradiated with gamma rays from 60Co at the dose of 50 Gy (gray) in April of 2014. All the treated buds were then immediately budded onto rootstocks and the survival rate was recorded as 60.34%. In order to stabilize the mutation, mV3 plants were developed by re-budding and plants at mV3 generation were transplanted in the orchard in 2017. Within the following year, 68 mV3 plants out of 852 grown in the field bore sufficient amount of fruit and were screened in terms of 19 fruit quality characters such as seed number per fruit, fruit diameter and ripening index. Fruit diameters of mV3 population varied from 56.72 mm to 84.79 mm, and fruit weight ranged between 90.00 g and 287.60 g. The number of seeds per fruit ranged between 0.6 and 13.1 whereas seed number of non-irradiated Ortanique tangor was recorded as 10.7 in the same fruit crop year. In general, fruit characteristics such as fruit weight, fruit height and fruit diameter which describe fruit size of a big part of the population, were similar to Ortanique tangor. According to primarily results, 18 plants have been described as low seeded (≤4). In addition, a cluster analysis was performed by using Euclidean similarity coefficient and similarity index ranged between 29.29% and 93.10% regarding variables related to fruit. The stability of mutations detected is being evaluated and new commercial field trials will be established with the selected materials.