ORIGINAL RESEARCH published: 25 October 2019 doi: 10.3389/fpls.2019.01341 Two Loci, RiAF3 and RiAF4, Contribute to the Annual-Fruiting Trait in Rubus Rubina Jibran 1, Jessica Spencer 2, Gina Fernandez 2, Amparo Monfort 3,4, Mourad Mnejja 3,4, Helge Dzierzon 1, Jibran Tahir 1, Kevin Davies 1, David Chagné 1 and Toshi M. Foster 1* 1The New Zealand Institute for Plant & Food Research Limited, Palmerston North Research Centre, Palmerston North, New Zealand, 2 Department of Horticultural Science, North Carolina State University, Raleigh, NC, United States, 3 IRTA (Institut de Recerca I Tecnologia Agroalimentàries), Barcelona, Spain, 4 Centre for Research in Agricultural Genomics (CRAG) CSIC- IRTA-UAB-UB, Barcelona, Spain Most Rubus species have a biennial cycle of flowering and fruiting with an intervening period of winter dormancy, in common with many perennial fruit crops. Annual-fruiting (AF) varieties of raspberry (Rubus idaeus and Rubus occidentalis L.) and blackberry (Rubus subgenus Rubus) are able to flower and fruit in one growing season, without the intervening dormant period normally required in biennial-fruiting (BF) varieties. We Edited by: used a red raspberry (R. idaeus) population segregating for AF obtained from a cross Lara Giongo, between NC493 and ‘Chilliwack’ to identify genetic factors controlling AF. Genotyping by Fondazione Edmund Mach, Italy sequencing (GBS) was used to generate saturated linkage maps in both parents. Trait Reviewed by: Tae Kyung Hyun, mapping in this population indicated that AF is controlled by two newly identified loci Chungbuk National University, (RiAF3 and RiAF4) located on Rubus linkage groups (LGs) 3 and 4. The location of these South Korea loci was analyzed using single-nucleotide polymorphism (SNP) markers on independent Maria Manuela Rigano, University of Naples Federico II, red raspberry and blackberry populations segregating for the AF trait. This confirmed that Italy AF in Rubus is regulated by loci on LG 3 and 4, in addition to a previously reported locus *Correspondence: on LG 7. Comparative RNAseq analysis at the time of floral bud differentiation in an AF Toshi M. Foster Toshi.Foster@plantandfood.co.nz and a BF variety revealed candidate genes potentially regulating the trait. Keywords: marker-assisted selection, primocane, floricane, comparative mapping, RNA sequencing, Specialty section: annual-fruiting, biennial-fruiting This article was submitted to Crop and Product Physiology, a section of the journal INTRODUCTION Frontiers in Plant Science Received: 11 June 2019 The Rosoideae subfamily of Rosaceae contains many economically important soft berry crops, Accepted: 26 September 2019 including red and black raspberry (Rubus idaeus and Rubus occidentalis L. respectively), blackberry Published: 25 October 2019 (Rubus subgenus Rubus) and strawberry (Fragaria species), which are renowned for their taste and Citation: health properties (Potter et al., 2007; Hummer and Janick, 2009; Shi et al., 2013; Simpson, 2018). Jibran R, Spencer J, Fernandez G, Raspberry (Rubus sp.) is a shrub that initiates shoots (canes) from a perennial root system (Keep, Monfort A, Mnejja M, Dzierzon H, 1988; Carew et al., 2000; Sønsteby and Heide, 2009; Heide et al., 2013; Graham and Simpson, 2018). Tahir J, Davies K, Chagné D and Biennial-fruiting (BF) raspberry varieties (also called floricane-fruiting or summer-fruiting) initiate Foster TM (2019) Two Loci, RiAF3 and RiAF4, Contribute to the axillary floral buds toward autumn of the first year of growth, but these do not develop into fruit Annual-Fruiting Trait in Rubus. until spring/summer of the following year. Annual-fruiting (AF) varieties (also called primocane- Front. Plant Sci. 10:1341. fruiting or autumn-fruiting) initiate flowers in late spring/early summer that develop into fruit from doi: 10.3389/fpls.2019.01341 summer until late autumn of the same year. In both AF and BF varieties, flowering and fruiting Frontiers in Plant Science | www.frontiersin.org 1 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus initiate from the shoot tip and develop basipetally after vegetative 2013; Perrotte et al., 2016; Hackett et al., 2018), whereas AF in growth has stopped. The key developmental difference between blackberry and red raspberry was suggested to be controlled by the two flowering phenologies is that AF floral buds are initiated a recessive monogenic trait (Lewis, 1939; Haskell, 1960; Lopez- earlier and progress directly to fruit set, whereas floral initiation Medina et al., 2000). Castro et al. (2013) reported that in auto- is normally followed by dormancy in BF types (Keep, 1988; tetraploid blackberry, this recessive locus was located on LG7. Carew et al., 2000; Sønsteby and Heide, 2009; Heide et al., 2013). Lewis (1939) demonstrated that the AF trait is controlled by a Flowering time is controlled by complex interactions among single recessive locus, later named “af ” (Haskell, 1960). However, endogenous factors, such as developmental pathways and trait segregation analysis performed on various AF populations hormones, as well as environmental cues, such as temperature suggested an alternative possibility of multiple loci having minor and day length (Simpson and Dean, 2002; Song et al., 2018; Tabas- effects on the expression of AF (Slate, 1940; Waldo and Darrow, Madrid et al., 2018; Kinmonth-Schultz et al., 2019; Kozlov et al., 1941; Oberle and Moore, 1952; Ourecky, 1976; Fejer, 1977; 2019). A number of genes that integrate specific signals and either Barrientos and Rodriguez, 1980). For example, Barrientos and repress or activate flowering have been identified and characterized Rodriguez (1980) suggested the possibility of partial dominance for in model species, such as Arabidopsis and Antirrhinum (Simpson the AF cultivar ‘Malling Exploit.’ Similarly, Fejer (1977) reported and Dean, 2002; Wigge et al., 2005; Khan et al., 2014; Blumel that inheritance of AF in mapping populations raised from a et al., 2015; Sasaki et al., 2015). Among these, CONSTANS series of diallelic crosses among seven red raspberry cultivars was (CO), FLOWERING LOCUS T (FT), SUPPRESSOR OF predominately additive and proposed that the genetic control for OVEREXPRESSION OF CONSTANS 1 (SOC1), FLOWERING the trait could not be recessive. Thus, the genetic regulation of AF LOCUS C (FLC), AGAMOUS1 (AG1), and LEAFY (LFY) are in raspberry is still unclear. the best characterized flowering integrators (Yanofsky et al., 1990; To address this issue, we constructed saturated linkage maps Blazquez et al., 1997; Kardailsky et al., 1999; Kobayashi et al., 1999; for the AF accession NC493 and the BF cultivar ‘Chilliwack’ to Michaels and Amasino, 1999; Yoo et al., 2007; Deng et al., 2011; map genetic loci associated with control of AF. Trait mapping Pin and Nilsson, 2012; Song et al., 2018; Tabas-Madrid et al., 2018). in this population indicated that AF is controlled by two newly For example, CO activates flowering under long days in both identified loci (RiAF3 and RiAF4) located on Rubus LGs 3 and FLC-dependent and independent manners by activating flower 4. The location of these loci was verified in independent red promoters FT and SOC1 (Kim et al., 2008; Michaels and Amasino, raspberry and blackberry populations segregating for AF. In 2001). FLC regulates floral transition by repressing the key genes of addition, we compared the transcriptomes of AF and BF axillary flowering pathway, for example, FT and SOC1 (Simpson and Dean, buds to identify candidate genes involved with the transcriptional 2002; Crevillen and Dean, 2011). FLC antagonizes the flowering regulation of the AF trait. pathway in a dose-dependent manner, with FLC abundance being regulated by an interplay between epigenetic factors and RNA- processing factors, such as polyadenylation and splicing (Simpson, MATERIALS AND METHODS 2004). Recently, it was shown that CO accelerates flowering under long days but represses flowering under very short days (3 hours Plant Material and Assessment of light) by regulating FT expression (Luccioni et al., 2019). Fruiting Phenotype Although there is little information on the genes controlling A segregating population of 131 F1 individuals was developed from flowering in raspberry, more is known about the environmental a controlled cross made in 2004 between AF accession NC493 cues that stimulate flowering. For example, floral induction in BF (R. parvifolius x R. idaeus ‘Cherokee’) and BF ‘Chilliwack’ (CW) varieties is triggered by a combination of decreased temperatures (R. idaeus). The seedling population was planted in 2006 at the and shorter photoperiod (Fejer and Spangelo, 1974; Dale Sandhills Research Station, Jackson Springs, NC, USA. In 2008, and Daubeny, 1987; Carew et al., 2000; Sønsteby and Heide, the 131 NC493 x CW progeny were assessed biweekly from July 2009; Hodnefjell et al., 2018). Although there is no absolute to September for the presence or absence of AF by determining requirement for AF varieties to experience chilling in the prior whether flowers or fruits were present on the primocanes (canes season to initiate flowering, as newly initiated canes can progress initiated that season). The population was again evaluated in 2009 through fruiting in a single season, the expression of AF in terms for AF on a weekly to biweekly basis from June to August, except of floral consistency across canes and the total number of flowers for two individuals that died over the winter. is strongly influenced by chilling (Sønsteby and Heide, 2009). Three families of red raspberry (R. idaeus), x16.093, x16.109, Modern AF varieties of red and black raspberry have complex and x16.111 of 47, 55, and 49 individuals, respectively, were pedigrees because of interspecific hybridization with other developed from controlled crosses between AF and BF parents Rubus species during their development, including R. arcticus, within the Plant & Food Research (PFR) breeding program and R. odoratus, and R. spectabilis (Keep, 1988; Lewers et al., 2005; planted in 2017 at the PFR site located at Motueka, New Zealand. Dossett et al., 2012). Many studies have been conducted to study These populations were phenotyped for the presence of AF in 2018. the genetic inheritance of AF in raspberry and blackberry, along A tetraploid blackberry (R. subgenus Rubus) mapping population with an analogous continuous flowering trait in strawberry. (C1) was generated from a cross between BF RM44 and AF RM63 Continuous or perpetual flowering in commercial strawberry from the IRTA-PLANASA breeding program. The population was (Fragaria × ananassa) is controlled by quantitative trait loci planted in 2015 at Cartaya, Spain. The parents and progeny were (QTLs) on linkage groups (LGs) 3, 4, and 7 (Gaston et al., phenotyped for the presence of AF in 2016 and 2017. Frontiers in Plant Science | www.frontiersin.org 2 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus Genotyping by Sequencing v5.0® (Van Ooijen and Voorrips, 2001). As the phenotypes in High molecular weight DNA was extracted from 100 mg of leaf the CW x NC493 population were scored as a presence-absence tissue from each individual in the 131 progeny in the NC493 x of AF, which is not quantitative and cannot be used for QTL CW family using a standard CTAB protocol (Doyle and Doyle, mapping with methods such as interval mapping, a chi-square 1987). The genotyping by sequencing (GBS) method of Elshire test was performed on all GBS markers that were heterozygous et al. (2011) was used to obtain reduced representation of the in only one parent to identify markers linked to AF. Chi-square genomes for the two parents and progeny of 83 individuals. The values for the significant differences between the expected allelic GBS library preparation protocol was first optimized for the red frequencies and the observed allelic frequencies were calculated raspberry genome by digesting DNA from a few individuals with with the formula: ApeK1, as described by Elshire et al. (2011). GBS libraries were ( ) 2 then constructed for 83 individuals and the two parents. The Χ 2 = Σ k i = 1 [ observed valuei − expected valuei / expected valuei ] libraries from 83 individuals were combined to make the final pooled library. The quantity and quality checks of the individual GBS markers with chi-square test values between 5 and 20 and libraries and the pooled library were performed using a Qubit p values <0.05 were selected for identification of QTLs controlling Fluorometer and a Fragment analyser, respectively. The pooled to the trait. This filtering criterion yielded 26,925 abxaa markers DNA library was dried and sent to the Australian Genome that are heterozygous for the NC493 parent and 6,571 aaxab Research Facility for sequencing on two lanes of the Illumina markers that are heterozygous for the CW parent. The markers HiSeq2500 platform using single-end sequencing chemistry. that are heterozygous for one parent and homozygous for other The sequencing reads were demultiplexed based on GBS parent were used for QTL mapping. library preparation bar codes using the ea-utils.1.1.2-537 Further mapping was performed using abxaa markers package (Richardson, 2013), and those reads starting with the (heterozygous for AF parent) located around the AF loci on the approved bar code immediately followed by the remnant of the NC493 parental map. The phase of these markers was calculated ApeK1 cleavage site sequence were retained for further analysis. using Joinmap v5.0® (Van Ooijen and Voorrips, 2001), and they The bar coded reads meeting the initial read quality criteria were ordered according to their physical location on the ORUS were aligned to the R. occidentalis genome assembly of ORUS 4115-3 v3.0 R. occidentalis genome assembly (Vanburen et al., 4115-3 v3.0 (Vanburen et al., 2018) (https://www.rosaceae.org/ 2018). Bins of 10 to 12 markers within focal points spanning no analysis/268) using Burrows-Wheeler Aligner (bwa/0.7.17) (Li more than 100 kb physical intervals were manually inspected. and Durbin, 2009). Single-nucleotide polymorphism (SNP) Focal points were evenly spaced throughout the region flanking calling and GBS data filtering were performed using the GATK the RiAF4 locus at 0.1, 2, 3.1, 3.6, 3.8, 4.1, 4.5, 6, and 8 megabase pipeline (gatk/3.8.0) (McKenna et al., 2010) using default pairs (Mb). Likely genotypic errors due to allelic dropout, a parameters. The GBS pipeline used to create a set of markers is common feature in GBS data for heterozygous species and available on Github at https://jupyterhub.powerplant.pfr.co.nz/ detectable as a single change in phase within a linked focal user/cfprxj/notebooks/cfprxj/bioinf_Braspberry_GBS/Variant_ point, were manually corrected. A consensus genotype was then calls_Braspberry_GATK_pipeline.ipynb. GATK_GBS analysis imputed for each focal point and compared to the neighboring yielded a total of 284,146 SNPs between the two parents. focal points. The linkage between each focal point and the AF phenotype was examined to delimitate the most likely genomic Preparation of GBS Markers for interval flanking the AF loci. Linkage Analysis The SNP data were filtered and formatted for appropriate genetic High-Resolution Melting Marker segregation codes using MS Excel (Microsoft Corporation, USA). Development Markers segregating abxaa, aaxab, and abxab were selected for SNPs that were closely associated with the AF loci were selected each parent using GATK and MS Excel. Homozygous SNP calls, from the GBS data set for the NC493 x CW population for such as A/A, G/G, T/T, and C/C, were converted into aa marker transformation into high-resolution melting (HRM) quantitative type, whereas heterozygous SNP calls (such as A/G, A/T, C/T, PCR markers. PCR primer pairs were designed to span amplicons etc.) were converted into the ab marker type. Joinmap v5.0® (Van ranging from 70 to 150 basepairs (bp) flanking the selected Ooijen and Voorrips, 2001) was used to develop genetic linkage SNPs using Primer3 (http://frodo.wi.mit.edu/primer3/). The maps for each parent of the NC493 x CW population. A LOD following criteria were employed for primer pair design: max score >6 was employed for grouping. Due to the high number of self-complementarity and max 3’ self-complementarity were markers on each linkage group, the markers were then filtered set to 4 and 1, respectively; GC content of the primers ranged based on chi-square values ranging from 0.1 to 7.0, and these from 40 to 55%. SNP analysis (Liew et al., 2004) was performed selected loci were subsequently used to reconstruct the maps on a LightCycler480 instrument (Roche Diagnostics), and using regression mapping (Kosambi mapping function). amplifications were performed using the PCR mix and conditions described in Guitton et al. (2011). Outputs were analyzed using Trait Mapping the LightCycler480 SW1.5 software. Heterozygous genotypes Trait mapping was initially performed by including the AF were identified as having a lower melting temperature in phenotypes of both years in the GBS data set using Joinmap comparison with homozygous genotypes and a shoulder in Frontiers in Plant Science | www.frontiersin.org 3 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus the melting peaks. HRM markers, which were heterozygous for AF : BF phenotypes were 55:76 and 66:65 in 2008 and and homozygous for the AF and BF parent, respectively, were 2009, respectively. Thirty-three phenotypes were inconsistent screened over the x16.093, x16.109, and x16.111 populations, between years. The subset of the population with consistent and association between the HRM genotypes and the presence of AF and BF phenotypes between years was used for GBS the AF trait was assessed using a chi-square test. analysis. Out of 98 individuals sampled, five did not yield sufficient DNA for GBS and could not be analyzed further. Simple Sequence Repeat Marker The final set (93 individuals) used for GBS library preparation Development and Screening contained 42 and 51 with AF and BF phenotypes, respectively PCR primers for simple sequence repeats (SSRs) from Rubus were (Table S1). developed close to the chromosomal regions associated with the loci of interest identified in the NC493 x CW raspberry population, GBS of the NC493 x ‘Chilliwack’ as well as the LG 7 locus identified in blackberry by Castro et al. Segregating Population (2013). SSRs were screened over the C1 blackberry population Two lanes of Illumina HiSeq2500 single-end 100 bp reads using a Hitachi ABI3500 Applied Biosystems genetic analyzer generated a total amount of 50,515,918,100 bp sequences and (Foster City, CA, USA). Association between the SSR alleles and 505,159,381 total reads. The removal of adapters and filtering the presence of the AF trait was determined using a chi-square test. of low-quality reads yielded 46,822,022,484 bp (92% of the total data). The GBS libraries for eight individuals failed to produce RNAseq and Differential Gene any sequencing data. In total, 85 GBS libraries from the progeny Expression Analysis and two duplicates of each parent yielded an average number Total RNA was extracted from axillary buds 5–10 nodes below of ~5.8 million reads per individual that were used for read the apex of ‘Heritage’ and ‘Wakefield,’ which are AF and BF, alignment against the R. occidentalis genome assembly ORUS respectively. The cultivars were grown together in the field under 4115-3 v3.0 (Vanburen et al., 2018) (https://www.rosaceae. standard conditions at the PFR orchard at Motueka, New Zealand. org/analysis/268). SNP calling identified 284,146 SNPs in Bud samples were collected on November 5 (spring/early summer). total (Table 1). Further filtering of the SNP data was applied Tissue was snap-frozen in liquid nitrogen, and total RNA was to remove loci that had more than 10% missing data and were extracted from three biological replicates of each cultivar, as described monomorphic or ambiguous. These filtering criteria yielded in Janssen et al. (2008). The quality and concentration of the RNA a total of 48,002 abxaa and 16,440 aaxab SNPs heterozygous samples were assessed using a Fragment Analyzer (Agilent, Santa for NC493 and CW, respectively. An additional 5,821 abxab Clara, CA, USA), and only samples with an RNA Integrity Number type markers were generated between the parents, resulting in higher than 8 were sequenced. Library preparation was completed at 70,263 SNP markers in total. the Australian Genome Research Facility using the TruSeq Stranded kit, and subsequent paired-end Illumina® sequencing employed the Map Construction Linkage maps were constructed for both parents (Figures 1 and 2; NovaSeq6000 platform, with the S2 flow cell. An average of ~19 Table 2). The NC493 map comprised 473 markers that spanned million, 150-bp paired-end reads was retrieved for each sample the seven LGs, and extended over 378.1 cM, with an average (~6 Gb of data). Read sequences of low-quality ribosomal RNA distance of 0.8 cM between markers (Figure 1). LG4 had the and adaptors were filtered out using Trimmomatic (Bolger et al., greatest number of markers (80), LG5 had the fewest number 2014) and SortMeRna (Kopylova et al., 2012). RNAseq reads were of markers (33), and LG4 was the longest (108.7 cM). The CW aligned to the R. occidentalis reference gene models (Vanburen et al., parental map was constructed from 419 markers that spanned the 2018) using Spliced Transcripts Alignment to a Reference (STAR), seven Rubus LGs and covered 251.6 cM, with an average distance and differential expression analysis was performed using DESeq2 of 0.6 cM between markers (Figure 2). LG5 had the greatest (Love et al., 2014). All RNAseq data, read statistics, and differentially number of markers (74), and LG7 had fewest markers (27). LG5 expressed genes (DEGs) are deposited in NCBI’s Gene Expression was the longest group, with 74 markers covering 75.96 cM. Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE135907 (https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc = GSE135907). Significant DEGs were selected using a threshold of α< -0.005 with an adjusted p value of <0.01 and TABLE 1 | Summary of single-nucleotide polymorphism (SNP) markers obtained a |log2 fold change | > 1. Arabidopsis orthologues were determined by genotyping by sequencing of a mapping population derived from NC493 x by BLAST against the TAIR database. ‘Chilliwack’ (CW). Parents Total SNPs Monomorphic abxaa abxab RESULTS identified by SNPs + markers SNPs SNPs GBS between with 10% Phenotypic Segregation for AF parents missing data in Raspberry NC493 284,146 230,323 48,002 5,821 The segregating population of 131 F1 individuals from the CW 261,885 16,440 NC493 x CW (AF x BF) cross was assessed for the AF trait abxaa type markers are heterozygous in one parent and homozygous in the other over two consecutive years. The observed segregation ratio parent. abxab type markers are heterozygous for both parents. Frontiers in Plant Science | www.frontiersin.org 4 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus FIGURE 1 | Rubus idaeus genetic map for Annual-Fruiting (AF) parent NC493. The vertical bars represent linkage groups, and the lines across the bars represent genotyping by sequencing (GBS) marker position in the map. The scale on the left represents the genetic distance in centiMorgans (cMs). Only markers that segregated according to the Mendelian ratio at p < 0.005 were employed for the map construction with Joinmap v5.0® (Van Ooijen and Voorrips, 2001). Each SNP marker was named according its physical position (right-hand side) in the genome. Frontiers in Plant Science | www.frontiersin.org 5 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus FIGURE 2 | Rubus idaeus linkage map for Biennial-Fruiting (BF) parent ‘Chilliwack’. Each linkage group (represented by a vertical bar) was constructed using SNP loci generated by genotyping by sequencing (GBS). The lines across the bars represent marker positions in the map, and the scale on the right represents the genetic distance in centiMorgan (cM). All markers segregated according to the Mendelian ratio at p < 0.005, and markers were named according to their physical positions in the genome (right-hand side of each LG). The maps were generated in Joinmap v5.0® (Van Ooijen and Voorrips, 2001). Frontiers in Plant Science | www.frontiersin.org 6 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus TABLE 2 | Summary of the linkage groups (LG) constructed for NC493 (Annual- NC493 identified two genomic regions, located on LGs 3 and 4, Fruiting, AF) and ‘Chilliwack’ (CW, Biennial-Fruiting, BF) parents and the number that were significantly associated (LOD > 4) with AF (Figure 3). of markers identified per LG. These two new loci were named RiAF3 and RiAF4 for R. idaeus AF, Linkage groups Number of loci mapped in Number of loci mapped located on LGs 3 and 4 of NC493, respectively. The GBS markers the NC493 map in the CW map chr3_41,124,650 and chr4_4,076,592 are those most significantly 1 75 58 linked to RiAF3 and RiAF4, respectively. A third locus on LG5 may 2 64 53 be present; however, none of the markers were associated with the 3 73 83 trait with a LOD score greater than 4. No linkage with phenotype 4 80 64 was found for the markers that were informative for CW. 5 33 74 6 76 60 Examination of the genotypes of 85 individuals over segments 7 72 27 of chromosomes 3 and 4 spanning the SNP markers with the most Total markers 473 419 significant LOD scores enabled us to determine the genotypes of The LGs were constructed by using the SNP markers obtained from a NC493 x the population between 35.1 to 43.7 Mb of chromosome 3 and ‘Chilliwack’ mapping population. 0 to 8 Mb of chromosome 4. The mapping analysis using a bin map based on the number of recombinants over a window of 8 Mb indicated that RiAF4 is located in an interval between 3.50 Trait Locus Mapping for AF Mb and 4.38 Mb (Figure 4). We were unable to perform similar The AF trait was not significantly associated with any markers on the mapping analysis for RiAF3 because the order of markers on the saturated CW (BF) linkage map when the AF phenotypes for both linkage map was not colinear with the ORUS 4115-3 v3.0 genome. years were included in the GBS data set used in map construction. Because the phenotypes were scored qualitatively as presence– Analysis of QTLs in Three Independent absence of AF, they could not be used for interval mapping of Populations of Red Raspberry QTLs. Hence, the chi-square test was performed on heterozygous The phenotypes of parents and number of progeny in the three GBS markers for each parent to identify markers linked to the AF independent populations segregating for the AF trait are shown in phenotype (Table S2). The analysis of markers heterozygous for Table 3. HRM-based markers were developed by designing PCR FIGURE 3 | Manhattan plot showing genomic regions associated with Annual-Fruiting (AF) in Rubus idaeus. Genotyping by sequencing (GBS)-based single- nucleotide polymorphism (SNP) markers significantly linked to the trait was identified by the chi-square test using markers that were heterozygous in NC493 (AF) and homozygous in ‘Chilliwack’ (Biennial-Fruiting, BF). The x-axis shows LOD scores, and the y-axis indicates the physical position of markers (black points) in the genome divided into seven linkage groups (LGs). The names and the physical position of the markers associated with control of AF are given in Supplementary Table S2. CW = ‘Chilliwack’. Frontiers in Plant Science | www.frontiersin.org 7 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus FIGURE 4 | Fine mapping the RiAF4 locus. A bin map was created by analyzing the genotypes of individuals over small genomic regions located within 0–8 Mb of LG4. Each column represents genotypic data from a single individual, either Annual-Fruiting (AF) or Biennial-Fruiting (BF). The allele of single-nucleotide polymorphisms (SNPs) in the genomic region of LG4 listed in the first column is indicated as a or b. The “a” allele (in pink) and “b” allele (in green) are linked to AF and BF, respectively. Recombination breakpoints are visible as a change in color and narrow the RiAF4 locus to the region located between 3.6 and 4.1Mb indicated by black horizontal lines. TABLE 3 | Summary of red raspberry (R. idaeus) populations segregating for the Annual-Fruiting (AF) trait. Population Female parent Male parent Total progeny AF individuals BF individuals in Chi-square value P value size in progeny progeny x16.093 Z12041-2 (AF) Z12022-7 (BF) 47 23 24 .01 .950 x16.109 Z12027-13 (AF) Z12022-7 (BF) 55 32 23 .73 .75 x16.111 Z12027-13 (AF) Z12011-8 (BF) 49 30 19 1.23 .75 These populations were used for validating RiAF3 and RiAF4 loci. Seedlings were planted in spring of 2017 at the Plant & Food Research site located at Motueka, New Zealand, and were scored for AF in the summer of 2018. BF, Biennial-Fruiting. primer pairs that flanked the most closely linked SNPs on LG3 (Table S5). A number of genes in these intervals are involved and LG4 and near the Rubus285a maker on LG7 (Table S3). These with flowering time. markers were screened over the three populations (Figure 5). In x16.093 and x16.109 populations, the chr3-9,188,040 marker is DEGs Between AF and BF Axillary Buds associated with the AF trait, with chi-square tests significant at 95% To identify genes potentially involved with the transcriptional and 99% (d.f. = 1), respectively. In population x16.111, ch4-7,738,811 regulation of AF, we compared gene expression in axillary buds of and chr7-4,042,651 markers are associated with the AF trait with AF ‘Heritage’ and BF ‘Wakefield’ in late spring. Of the DEG genes chi-square test significance of 95% and 99% (d.f. = 1), respectively. with a |log2fold change| > 1, 443 (2.22%) were upregulated, and 363 (1.8%) were downregulated in AF compared with BF. The Screening of SSR Markers for RiAF3, majority of the DEGs are orthologues of genes identified in other RiAF4, and the LG7 locus in a Blackberry species as being involved in reproduction, flower development, (R. subgenus Rubus) Population and defence responses against biotic and abiotic stress. To verify the two newly identified loci, we analyzed markers linked Table S6 shows the relative expression of all genes within the to AF in a tetraploid blackberry population (C1) segregating for genomic intervals spanning the RiAF3 and RiAF4 loci. The genomic the AF trait. In this population, 76 progenies were scored over locations and biological functions of some of the most promising two consecutive years as 12 AF and 64 BF (Table S4). The C1 differentially expressed candidate genes are listed in Table 4. Within population was screened with SSR markers from R. idaeus LGs 3, 4, the RiAF3 interval, a gene orthologous to Arabidopsis JUMONJI and 7 (Castro et al., 2013), and only the LG3 marker Rubus285a was 14 (JMJ14) was expressed 2-fold higher in AF relative to BF. In associated with AF (Figure 6). The data show that the 244-bp allele the RiAF4 region, PHYTOCHROME AND FLOWERING TIME of Rubus285a from BF parent is linked to the BF phenotype. This 1 (PFT1), FLOWERING LOCUS A (FCA), and AGAMOUS-LIKE allele is not present in the AF parent. No association between marker 24 (AGL24) genes were all upregulated in AF, with AGL24 having and phenotype was identified for markers located on LGs 4 and 7. 5-fold higher transcript levels in AF relative to BF (Figure 7). Candidate Genes Underlying RiAF3 DISCUSSION and RiAF4 The region of the ORUS 4115-3 v3.0 genome spanning from 35.1 We report that AF is a complex genetic trait regulated by at least to 43.7 Mb on LG3 contains 1,276 predicted gene models, and two loci on R. idaeus LGs 3 and 4, RiAF3 and RiAF4. These novel the region from 0 to 8 Mb on LG4 contains 1,399 gene models loci are syntenic to QTLs identified in strawberry for control of Frontiers in Plant Science | www.frontiersin.org 8 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus FIGURE 5 | Histograms showing allelic frequencies of SNP-based markers underlying quantitative trait loci in three independent red raspberry populations segregating for the Annual-Fruiting trait. The marker names indicate their chromosome and physical positions (in bp) on the Rubus occidentalis genome (Vanburen et al., 2018). The * and ** represent chi-square test significance of 95% and 99% (d.f. = 1), respectively. The marker chr7-4,042,651 was monomorphic in the x16.109 population. Frontiers in Plant Science | www.frontiersin.org 9 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus FIGURE 6 | Rubus285a marker segregation in a tetraploid blackberry mapping population segregating for Annual-Fruiting (AF). (A) Table showing the presence or absence of alleles in parents and the numbers of AF and Biennial-Fruiting (BF) progeny with each allele and the percentage in parentheses. The AF parent has three alleles with PCR product sizes of 197, 201, and 265 bp. The BF parent has four alleles with sizes of 195, 197, 201, and 244 bp. (B) Histogram showing allelic frequencies of the Rubus285a marker in the C1 blackberry population. The 244-bp allele is linked with BF trait with chi-square test significance of 99% (d.f. = 1) indicated by **. recurrent flowering, suggesting conserved function across the in a mapping population segregating for AF (Castro et al., 2013) Rosoideae subfamily. Markers linked to the newly discovered loci was employed to identify three markers linked to AF. However, one and to a locus previously identified on LG7 (Castro et al., 2013) marker was 71cM from AF and, hence, unlinked. Furthermore, were tested in independent raspberry and blackberry populations none of their LG7 markers mapped on the black raspberry genetic segregating for AF. In addition, we identified DEGs that may be map of Bushakra et al. (2012) and the blackberry LG7 was assigned involved in regulating the AF trait in Rubus. by default. It is possible that one of the other six LGs could have split and been mistakenly designated LG7 by Castro et al. (2013). Development of a High-Density Genetic Map of R. idaeus AF Is a Complex Genetic Trait GBS technology has greatly facilitated SNP discovery and Castro et al. (2013) proposed that AF in blackberry was genotyping for crop genetics (Crossa et al., 2013; He et al., 2014; controlled by a single locus located on LG7. We were unable to İpek et al., 2016; Hackett et al., 2018). We used GBS-based SNP map the AF phenotype as a single locus in the NC493 x CW markers to develop a high-density genetic map of red raspberry. population. A chi-square test of the thousands of markers This new map is constructed with 70,263 SNP markers and is detected by GBS identified two novel loci on LGs 3 and 4 for aligned with the genome assembly of black raspberry. In a previous control of AF in red raspberry. The previous single locus study, a blackberry genetic map developed with 119 SSR markers hypothesis was largely based on the 3:1 (BF : AF) phenotypic Frontiers in Plant Science | www.frontiersin.org 10 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus TABLE 4 | Differentially expressed candidate genes that underlie RiAF3 and RiAF4 loci. Rubus gene model Location in genome (bp) Log2Foldchange (BF/AF) Arabidopsis hom Description ologue Ro04_G19915 8,594,128 -0.28 AT4G16280 Flowering Control Locus A (FCA) Ro04_G02642 4,485,423 -0.51 AT1G25540 Phytochrome and Flowering Time 1 Ro04_G36356 8,736,093 -1.75 AT4G24540 Agamous-like 24 (AGL24) Ro03_G05776 35,959,751 -0.001 AT3G12680 Hua1, Enhancer of Ag-4 1 Ro03_G15781 37,388,564 -1.08 AT4G20400 Jumonji 14 (JMJ14) Ro03_G33037 38,575,123 -0.88 AT1G30330 Auxin Response Factor 6 (ARF6) Ro03_G13396 39,438,396 1.43 AT1G29390 Cold regulated thylakoid membrane Ro03_G13391 39,478,286 -0.03 AT4G18130 Phytochrome E Ro03_G05247 40,252,833 0.50 AT2G42610 Light Sensitive Hypocotyls 10 Ro03_G06544 40,834,857 -0.5 AT1G28330 Dormancy-Associated Protein- like 1 Ro03_G06488 41,189,726 -0.16 AT4G04920 Sensitive to Freezing 6 RNAseq was performed on axillary buds from three biological replicates each of ‘Heritage’ (Annual-Flowering) and ‘Wakefield’ (Biennial-Flowering). The physical location of each R. occidentalis gene model is given in bps (ORUS 4115-3 v3.0, Vanburen et al., 2018). The log2 fold change of ‘Wakefield’ relative to that of ‘Heritage’ is presented. FIGURE 7 | Expression of flowering time genes located in RiAF3 and RiAF4 mapping intervals. RNA was collected from axillary buds in late spring from ‘Heritage’ (Annual-Fruiting, AF) and ‘Wakefield’ (Biennial-Flowering, BF). The x-axis shows RPKM (Reads Per Kilobase Million) for Ro03_G15781 (orthologous to JUMONJI 14, JMJ14), Ro04_G02642 (orthologous to PHYTOCHROME AND FLOWERING TIME 1, PFT1), Ro04_G19915 (orthologous to FLOWERING CONTROL LOCUS A, FCA), and Ro04_G36356 (orthologous to AGAMOUS-LIKE 24, AGL24). The columns represent the means of three biological replicates, and error bars are standard error of the mean. * and **indicate statistically significant differences (P < 0.05 and P < 0.01, respectively) between AF and BF using Student’s t-test. ratio observed in several populations but is unsupported by In this study, we found that LGs 3, 4, and 7 HRM markers molecular marker data (Oberle and Moore, 1952; Keep, 1961). were linked to the AF trait in red raspberry (Figure 5). An LG3 On the basis of the phenotypic data collected from intercrossing SSR marker (Rubus285a) was linked to the BF trait in blackberry or selfing AF individuals, some studies have suggested a complex (Figure 6). We were unable to verify all three loci in all of the genetic basis for the trait (Lewis, 1939; Waldo and Darrow, 1941; populations, which could be due to one or more loci being fixed Haskell, 1960; Fejer and Spangelo, 1974; Fejer, 1977). Various in a homozygous state in the parents, hence preventing detection studies concluded that AF is controlled by a number of minor of polymorphic markers linked to AF. genes with predominant evidence that AF is a complex genetic Our GBS data indicated that RiAF3 is located at the bottom trait controlled by loci on three LGs (RiAF3, RiAF4, and LG7). of chromosome 3 (Figure 3). However, the HRM marker that Recently, linkage analysis of ‘Glen Moy’ x ‘Latham’ raspberry was developed from the most closely linked SNP is located population identified flower development QTLs on LGs 3, 5, and on the upper arm of chromosome 3 (chr3-9,188,040). This 7 (Hackett et al., 2018). These QTLs harbored genes involved discrepancy is likely due to errors in the assembly, and it is in regulating flowering time. For example, FKF1, a regulator possible that the HRM marker position is incorrect. The of CO expression, was mapped to LG7. Similarly, FT, EFL7 (a populations used for the QTL analysis were relatively small, regulator of FLC levels), and COL9 (a regulator of CO levels) which would reduce the possibility of detecting several were mapped to LG3. loci. This analysis should be repeated in one or more large Frontiers in Plant Science | www.frontiersin.org 11 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus populations to more precisely identify the genomic intervals expression and a decrease in H3K4me3 at the FLC locus that cause linked to AF. FLC repression. It has also been shown that JMJ14 is required for gene silencing (Searle et al., 2010). Similarly, Zheng et al. (2019) RiAF3 and RiAF4 Are Syntenic With Two found that JMJ13 is a floral repressor that regulates Arabidopsis Loci for Control of Recurrent Flowering flowering timing in a temperature- and light-dependent manner. in Strawberry The genomic region underlying the RiAF4 locus contains three To extend the fruiting season, many international strawberry key genes that promote flowering, PFT1 (Ro04_G02642), FCA breeding programs are focusing on developing early, late, and (Ro04_G19915), and AGL24 (Ro04_G36356) (Simpson et al., 2003; perpetual (continuous) flowering cultivars. Comparative genomic Turck et al., 2008; Michaels et al., 2003; Torti and Fornara, 2012). studies between strawberry and raspberry have revealed a high PFT1 is a nuclear protein that integrates various environmental degree of synteny between the genomes of two species (Bushakra cues into plant flowering pathway both in a CO-dependent and et al., 2012; Jibran et al., 2018). Results from molecular studies also -independent manner (Liu et al., 2017). PFT1 regulates flowering support a common molecular mechanism for control of flowering by increasing the transcript abundance of CO and FT (Iñigo time among different Rosaceae species (Gaston et al., 2013; Honjo et al., 2012). FCA is an RNA binding protein that downregulates et al., 2016; Perrotte et al., 2016; Samad et al., 2017). Floral repressors, expression of the floral repressor FCA by methylating central such as Perpetual Flowering 1 and Terminal Flower 1, control perpetual parts of the FLC gene (Liu et al., 2007). Reduced FCA function flowering habits in strawberry. Seasonal flowering (SF) strawberry results in late flowering due to increased FLC activity, whereas plants produce flowers only in autumn, whereas perpetual flowering overexpression of FCA causes early flowering, both in long and (PF) plants flower over an extended time. The strawberry PF habit short day photoperiods (Liu et al., 2007). AGL24 is a MADS- is under the control of a major QTL named FaPFRU, located on the box transcription factor that regulates flower timing by inducing lower arm of LG4 called LG4b-F (Gaston et al., 2013). This mapped expression of the floral integrator SOC1 (Liu et al., 2008). region contains a floral activator gene orthologous to Flowering Time AGL24 loss-of-function mutants and plants with reduced AGL24 (FT) (Yoo et al., 2005). Perrotte et al. (2016) investigated the effect of transcript levels showed delayed flowering phenotypes, whereas FaPFRU on PF habit in 28 strawberry genotypes and found that the overexpression of AGL24 resulted in early flowering phenotypes locus was linked to PF when analysis was carried out in both PF and (Michaels et al., 2003; Yu et al., 2002). It was suggested that AGL24 SF genotypes. However, the analysis involving only PF genotypes controls flower timing in a dosage-dependent manner (Yu et al., did not detect linkage of FaPFRU with the PF trait; instead, a QTL 2002). Our finding that the raspberry orthologues of PFT1, FCA, located on LG3c-F (LG3c), associated with a late PF-intense phase, and AGL24 are upregulated in AF buds relative to BF makes each was identified. Hence, the authors concluded that PF in strawberry of them promising candidate genes in the control of AF. is regulated by multiple loci. It was postulated that FaPFRU regulates the switch between PF and SF, whereas the LG3c locus controls the intensity of flowering. Furthermore, Albani et al. (2004) suggested CONCLUSION that the PF trait in woodland strawberry (Fragaria vesca L.) and We have presented the first evidence of loci linked to control of cultivated strawberry are regulated by different genetic components. AF in R. idaeus. Our results suggest that two major loci RiAF3 In woodland strawberry, two early flowering QTLs were identified and RiAF4 and a region located on the upper arm of LG7 control on the upper and lower arm of LG4 (Samad et al., 2017). AF in Rubus. Additionally, we identified putative flowering The two novel loci RiAF3 and RiAF4 identified from the NC493 time genes as candidates for functional validation. The genetic x CW population colocalize with the previously identified QTLs loci identified will be of value for marker-assisted selection of related to PF in strawberry. FaPFRU is located on the lower arm AF raspberries and blackberries following further validation in of LG4, whereas RiAF4 locus is located at the upper arm of LG4. breeding germplasm. The difference in the genome positions between species might well be because parts of LG4 are inverted in raspberry compared to strawberry (Vanburen et al., 2018). DATA AVAILABILITY STATEMENT Candidate Genes Underlying RiAF3 All datasets for this study are included in the article/ and RiAF4 Supplementary Material. JMJ14 is the best candidate for RiAF3. JMJ14 is a histone H3 lysine 4 (H3K4) demethylase, and H3K4 methylation is linked to AUTHOR CONTRIBUTIONS transcription of key flowering time genes (Lu et al., 2010; Lu et al., 2011; Cui et al., 2016). The Arabidopsis jmj14-1 mutant flowers early RJ, substantial contributions to the conception or design of the under short day conditions and has elevated levels of LFY, FT, and work; or the acquisition, analysis or interpretation of data for the AP1 transcripts (Jeong et al., 2009). Previous studies have indicated work. JS, contribution to labwork and fieldwork. GH, acquisition, that demethylases are involved with the regulation of flowering analysis or interpretation of data for the work. AM, acquisition, time. Yang et al. (2012) found that Arabidopsis plants overexpressing analysis or interpretation of data for the work. MM, contribution JMJ15, a member of the H3K4 demethylase JARID1 family, had to labwork and fieldwork. HD, contribution to data analysis. JT, accelerated flowering time. The early flowering phenotypes of contribution to data analysis. KD, comments to the manuscript. DC, the overexpression lines were associated with an increased FT substantial contributions to the conception or design of the work; or Frontiers in Plant Science | www.frontiersin.org 12 October 2019 | Volume 10 | Article 1341 Jibran et al. Genetic Control of Annual-Flowering in Rubus the acquisition, analysis or interpretation of data for the work. TF, Fund (SSIF) allocated to Plant & Food Research as part of the substantial contributions to the conception or design of the work; or Discovery Science programs. the acquisition, analysis or interpretation of data for the work. SUPPLEMENTARY MATERIAL ACKNOWLEDGMENTS The Supplementary Material for this article can be found online This project was funded by New Zealand Ministry of Business at: https://www.frontiersin.org/articles/10.3389/fpls.2019.01341/ Innovation and Employment (MBIE) Strategic Science Investment full#supplementary-material REFERENCES Fejer, S. (1977). Inheritance of yield, yield components, and fall-fruiting habit in red Albani, M., Battey, N., and Wilkinson, M. (2004). The development of ISSR-derived raspberry diallel crosses. Can. J. Genet. Cytol. 19, 1–13. doi: 10.1139/g77-001 SCAR markers around the SEASONAL FLOWERING LOCUS (SFL) in Fragaria Fejer, S., and Spangelo, L. (1974). Three generations of inbreeding and S2 factorial vesca. Theor. Appl. Genet. 109, 571–579. doi: 10.1007/s00122-004-1654-4 test crosses in red raspberry cultivars. Can. J. Genet. Cytol. 16, 419–432. doi: Barrientos, F., and Rodriguez, J. A. 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The remaining authors declare that the research A near complete, chromosome-scale assembly of the black raspberry (Rubus was conducted in the absence of any commercial or financial relationships that occidentalis) genome. GigaScience 7, giy094. doi: 10.1093/gigascience/giy094 could be construed as a potential conflict of interest. Waldo, G. F., and Darrow, G. M. (1941). “Breeding autumn-fruiting raspberries under Oregon conditions”, in: Proceedings of the American Society for Copyright © 2019 Jibran, Spencer, Fernandez, Monfort, Mnejja, Dzierzon, Horticultural Science), 263-267. Tahir, Davies, Chagné and Foster. This is an open-access article distributed Wigge, P. A., Kim, M. C., Jaeger, K. E., Busch, W., Schmid, M., Lohmann, J. U., et al. under the terms of the Creative Commons Attribution License (CC BY). The (2005). Integration of spatial and temporal information during floral induction use, distribution or reproduction in other forums is permitted, provided the in Arabidopsis. Science 309, 1056–1059. doi: 10.1126/science.1114358 original author(s) and the copyright owner(s) are credited and that the original Yang, H., Mo, H., Fan, D., Cao, Y., Cui, S., and Ma, L. (2012). Overexpression of a publication in this journal is cited, in accordance with accepted academic histone H3K4 demethylase, JMJ15, accelerates flowering time in Arabidopsis. practice. No use, distribution or reproduction is permitted which does not comply Plant cell Rep. 31, 1297–1308. doi: 10.1007/s00299-012-1249-5 with these terms. Frontiers in Plant Science | www.frontiersin.org 15 October 2019 | Volume 10 | Article 1341