Construction of a high density genetic linkage map to define the locus conferring seedlessness from Mukaku Kishu mandarin.

Mukaku Kishu ('MK'), a small sized mandarin, is an important source of seedlessness in citrus breeding. Identification and mapping the gene(s) governing 'MK' seedlessness will expedite seedless cultivar development. In this study, two 'MK'-derived mapping populations- LB8-9 Sugar Belle® ('SB') × 'MK' (N=97) and Daisy ('D') × 'MK' (N=68) were genotyped using an Axiom_Citrus56 Array encompassing 58,433 SNP probe sets, and population specific male and female parent linkage maps were constructed. The parental maps of each population were integrated to produce sub-composite maps, which were further merged to develop a consensus linkage map. All the parental maps (except 'MK_D') had nine major linkage groups, and contained 930 ('SB'), 810 ('MK_SB'), 776 ('D') and 707 ('MK_D') SNPs. The linkage maps displayed 96.9 ('MK_D') to 98.5% ('SB') chromosomal synteny with the reference Clementine genome. The consensus map was comprised of 2588 markers including a phenotypic seedless (Fs)-locus and spanned a genetic distance of 1406.84 cM, with an average marker distance of 0.54 cM, which is substantially lower than the reference Clementine map. For the phenotypic Fs-locus, the distribution of seedy and seedless progenies in both 'SB' × 'MK' (55:42, χ2 = 1.74) and 'D' × 'MK' populations (33:35, χ2 = 0.06) followed a test cross pattern. The Fs-locus mapped on chromosome 5 with SNP marker 'AX-160417325' at 7.4 cM in 'MK_SB' map and between two SNP markers 'AX-160536283' and 'AX-160906995' at a distance of 2.4 and 4.9 cM, respectively in 'MK_D' map. The SNPs 'AX-160417325' and 'AX-160536283' correctly predicted seedlessness of 25-91.9% progenies in this study. Based on the alignment of flanking SNP markers to the Clementine reference genome, the candidate gene for seedlessness hovered in a ~ 6.0 Mb region between 3.97 Mb (AX-160906995) to 10.00 Mb (AX-160536283). This region has 131 genes of which 13 genes (belonging to seven gene families) reportedly express in seed coat or developing embryo. The findings of the study will prove helpful in directing future research for fine mapping this region and eventually underpinning the exact causative gene governing seedlessness in 'MK'.

Association of T2/S-RNase With Self-Incompatibility of Japanese Citrus Accessions Examined by Transcriptomic, Phylogenetic, and Genetic Approaches.

Several citrus varieties show gametophytic self-incompatibility (GSI), which can contribute to seedless fruit production in several cultivars. This study investigated the genes regulating this trait through RNA-seq performed using styles collected from the flowers of Japanese citrus cultivars 'Hyuganatsu,' 'Tosabuntan,' 'Hassaku,' 'Banpeiyu,' and 'Sweet Spring'. We screened the transcripts of putative T2 RNases, i.e., the protein family including all S-RNases from S-RNase-based GSI plants, and constructed a phylogenetic tree using the screened T2 RNases and S-RNases retrieved from citrus genome databases and a public database. Three major clusters (class I-III) were formed, among which, the class III cluster contained family specific subclusters formed by S-RNase and a citrus-specific cluster monophyletic to the S-RNase clusters. From the citrus class III cluster, six transcripts were consistent with the S haplotypes previously determined in Japanese citrus accessions, sharing characteristics such as isoelectric point, extracellular localization, molecular weight, intron number and position, and tissue-specific expression with S-RNases. One T2 RNase gene in self-incompatible Hyuganatsu was significantly down-regulated in the styles of a self-compatible mutant of Hyuganatsu in RNA-seq and qPCR analyses. In addition, the inheritance pattern of some T2 RNase genes was consistent with the pattern of the S haplotype in the progeny population of Hyuganatsu and Tosabuntan. As all results supported citrus self-incompatibility being based on S-RNase, we believe that six T2 RNase genes were S-RNases. The homology comparison between the six T2 RNases and S-RNases recently reported in Chinese citrus revealed that three out of six T2 RNases were identical to S-RNases from Chinese citrus. Thus, the other three T2 RNases were finally concluded to be novel citrus S-RNases involved in self-incompatibility.

Single-pollen genotyping to estimate mode of unreduced pollen formation in Citrus tamurana cv. Nishiuchi Konatsu.

KEY MESSAGE: 2n pollen formed by FDR in citrus. The Japanese local citrus cultivar, Nishiuchi Konatsu (Citrus tamurana hort. ex Tanaka; NK hereafter), has the ability to produce unreduced 2n pollen grains, allowing generation of polyploid progenies via sexual polyploidization. In this study, we developed a method of single-pollen genotyping for citrus and applied it to the analysis of transmission of heterozygosity in NK 2n pollen grains. Heterozygosity transmission was expressed as the percentage inheritance of a set of heterozygous alleles from the parent to the 2n gamete. The pathway of 2n pollen development was investigated by applying the observed heterozygosity transmission and genetic distance to two different map functions, for first division restitution (FDR) and second division restitution (SDR). The fit of the values observed for both functions was calculated, while virtually moving the centromere position. We screened for six heterozygous SSR (codominant microsatellite marker loci) in NK, all of which were expected to lie within the same linkage group. Pollen germination prior to DNA extraction was essential for this work, and 6-h incubation proved to be optimal for subsequent PCR amplification. Single-pollen genotyping unreduced NK 2n pollen grains revealed that heterozygosity transmission exceeded 50 % in all six alleles, and fitness tests indicated that the FDR map function better fitted the heterozygosity transmission observed rather than the SDR function. Our data thus strongly indicate that 2n pollen in NK is a result of first division restitution.