Key flowering genes including FT-like genes are upregulated in the vasculature of apple dwarfing rootstocks

The Malling 9 (M.9) dwarfing rootstock is widely used in apple breeding and commercial cultivation to shorten the juvenile period, reduce vegetative growth and increase flowering of the scion. A segment of M.9 stem (interstock) or M.9 bark grafted into a compound tree can cause significant dwarfing of the scion, suggesting that the dwarfing signal may be vascular derived. To better understand how the M.9 rootstock alters the growth and development of the scion, we compared gene expression in vascular-enriched tissue from dwarfing and vigorous rootstocks. RNA sequencing indicated that key flowering genes were upregulated in M.9 relative to a vigorous rootstock, Malling 793 (M.793). An in-depth analysis of the apple FT/TFL1 gene family identified four new members: MdMFTa, MdMFTb, MdBFTa and MdBFTb. Quantitative RT-PCR analysis confirmed the higher expression of MdFT1/2, MdBFTa/b, MdCO, MdGI, and MdSOC1 in two different dwarfing rootstocks (M.9 and Malling 27 (M.27)) relative to M.793. Both MdFT1/2, and MdBFTa/b were expressed at higher levels in multiple dwarfing rootstock accessions relative to more vigorous genotypes. In perennial species, FT promotes flowering, and has additional roles in accelerating the transition from juvenility to maturity, and regulating cycles of seasonal growth and termination. Apple dwarfing rootstocks reduce the juvenile phase and promote both flowering and early shoot termination. Our work supports a role for MdFT in promoting flowering and earlier shoot termination. We suggest that upregulation of a suite of flowering genes including MdFT, and possibly MdBFT, in the vasculature is part of the underlying mechanism of apple dwarfing rootstocks. Genes involved with response to biotic and abiotic stress and disease were also upregulated in the M.9 rootstock, suggesting that stress, possibly mediated by JA and ABA signalling, also plays a role in the M.9-induced phenotype.     

Evaluation of Genetic Diversity in Chinese Wild Apple Species Along with Apple Cultivars Using SSR Markers

China, one of the primary centers of genetic diversity for the genus Malus, is very rich in wild apple germplasm. In this study, genetic diversity in 29 Malus accessions, including 12 accessions from 7 Chinese Malus species, 4 Chinese landraces, and 13 introduced apple cultivars, was assessed using a set of 19 single-locus simple sequence repeat (SSR) markers distributed across all 17 linkage groups of the apple genome. The number of alleles detected at each locus ranged from 2 to 11, with an average of 5.3 per SSR marker. In some accessions, 16 unique alleles were identified. Ten out of these 16 unique alleles (62.5%) were detected exclusively in wild species, indicating that these Chinese wild apple species have considerable genetic diversity and can be used in breeding programs to increase the genetic diversity of apple cultivars. Using 19 SSRs, an unweighted pair-group method with arithmetic average cluster analysis was conducted, and the resulting dendrogram revealed that all cultivars, except for E??peMeBckoe, were clustered together in the same group. The Russian cultivar E??peMeBckoe was closely related to the Chinese crabapple Baihaitang (M. prunifolia), with a high similarity coefficient value of 0.94. Of the two M. sieversii accessions used, one accession showed a close relationship to apple cultivars, while the other accession was closely related to wild apple species, suggesting the presence of a wider genetic diversity in Chinese M. sieversii species. The influence of SSR marker selection on genetic diversity analysis in this Malus collection was also discussed.     

Genetic mapping of the crown gall resistance gene of the wild apple <Emphasis Type="Italic">Malus sieboldii</Emphasis>

Crown gall, caused by Agrobacterium tumefaciens, causes severe damage to apple saplings resulting in weak growth and loss of commercial value. Developing molecular markers linked to crown gall resistance genes, and establishing a marker-assisted selection (MAS) for such a trait would be an effective way to improve rootstock breeding for crown gall resistance. The wild apple Malus sieboldii Sanashi 63 carries the crown gall resistance gene Cg effective against the A. tumefaciens strain Peach CG8331 (biovar 2). Applying the genome scanning approach on the mapping population JM7 (cgcg) × Malus sieboldii Sanashi 63 (Cgcg), Cg was mapped on the linkage group (LG) 2. The constructed linkage map of LG 2 of Sanashi 63 spans 59.8 cM and has an average marker density of 3.5 cM per marker. The 191 bp allele of the simple sequence repeat (SSR) NZmsEB119405 co-segregated perfectly with Cg in a segregating population of 119 individuals. Quantitative trait loci, accounting for 75.3% to 84.3% of phenotypic variation were detected in the same position. Testing eight additional rootstocks with the NZmsEB119405 SSR marker revealed that the 191 bp allele is also present in crown gall-susceptible rootstock accessions. Only the markers CH03b01 and NZmsPal92 mapping at 0.9 and 4.3 cM from Cg, respectively, showed “private” alleles associated to Cg.     

Construction of a dense genetic linkage map for apple rootstocks using SSRs developed from Malus ESTs and Pyrus genomic sequences

Marker-assisted selection (MAS) offers quick and reliable prediction of the phenotypes of seedlings in large populations and thus opens new approaches for selection to breeders of apple (Malus x domestica Borkh.). The development of framework maps enables the discovery of genetic markers linked to desired traits. Although genetic maps have been reported for apple scion cultivars, none has previously been constructed for apple rootstocks. We report the construction of framework genetic maps in a cross between ‘M.9’ (‘Malling 9’) and ‘R.5’ (‘Robusta 5’) apple rootstocks. The maps comprise 224 simple sequence repeat (SSR) markers, 18 sequence-characterised amplified regions, 14 single nucleotide polymorphisms and 42 random amplified polymorphic DNAs. A new set of 47 polymorphic SSRs was developed from apple EST sequences and used for construction of this rootstock map. All 17 linkage groups have been identified and aligned to existing apple genetic maps. The maps span 1,175.7 cM (‘M.9’) and 1,086.7 cM (‘R.5’). To improve the efficiency of mapping markers to this framework map, we developed a bin mapping set. Applications of these new genetic maps include the elucidation of the genetic basis of the dwarfing effect of the apple rootstock ‘M.9’ and the analysis of disease and insect resistance traits such as fire blight (Erwinia amylovora), apple scab (Venturia inaequalis) and woolly apple aphid (Eriosoma lanigerum). Markers for traits mapped in this population will be of direct use to apple breeders for MAS and for identification of causative genes by map-based cloning.     

High throughput analysis of grape genetic diversity as a tool for germplasm collection management

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI?=?0.839 and 0.865, respectively) than V. vinifera cultivars (GDI?=?0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI?=?0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.     

A genetic linkage map of an apple rootstock progeny anchored to the Malus genome sequence

An apple rootstock progeny raised from the cross between the very dwarfing ??M.27?? and the more vigorous ??M.116?? (??M.M.106???×???M.27??) was used for the construction of a linkage map comprising a total of 324 loci: 252 previously mapped SSRs, 71 newly characterised or previously unmapped SSR loci (including 36 amplified by 33 out of the 35 novel markers reported here), and the self-incompatibility locus. The map spanned the 17 linkage groups (LG) expected for apple covering a genetic distance of 1,229.5?cM, an estimated 91% of the Malus genome. Linkage groups were well populated and, although marker density ranged from 2.3 to 6.2?cM/SSR, just 15 gaps of more than 15?cM were observed. Moreover, only 17.5% of markers displayed segregation distortion and, unsurprisingly in a semi-compatible backcross, distortion was particularly pronounced surrounding the self-incompatibility locus (S) at the bottom of LG17. DNA sequences of 273 SSR markers and the S locus, representing a total of 314 loci in this investigation, were used to anchor to the ??Golden Delicious?? genome sequence. More than 260 of these loci were located on the expected pseudo-chromosome on the ??Golden Delicious?? genome or on its homeologous pseudo-chromosome. In total, 282.4?Mbp of sequence from 142 genome sequence scaffolds of the Malus genome were anchored to the ??M.27???×???M.116?? map, providing an interface between the marker data and the underlying genome sequence. This will be exploited for the identification of genes responsible for traits of agronomic importance such as dwarfing and water use efficiency.     

Genetic diversity,population structure,and linkage disequilibrium of elite and local apple accessions from Belgium using the IRSC array

The identification of molecular markers associated with economic and quality traits will help improve breeding for new apple (Malus × domestica Borkh.) cultivars. Tools such as the 8K apple SNP array developed by the RosBREED consortium allow for high-throughput genotyping of SNP polymorphisms within collections. However, genetic characterization and the identification of population stratification and kinship within germplasm collections is a fundamental prerequisite for identifying robust marker–trait associations. In this study, a collection of apple germplasm originally developed for plant architectural studies and consisting of both non-commercial/local and elite accessions was genotyped using the 8K apple SNP array to identify cryptic relationships between accessions, to analyze population structure and to calculate the linkage disequilibrium (LD). A total of nine pairs of synonyms and several triploids accessions were identified within the 130 accessions genotyped. In addition, most of the known parent-child relations were confirmed, and several putative, previously unknown parent-child relations were identified among the local accessions. No clear subgroups could be identified although some separation between local and elite accessions was evident. The study of LD showed a rapid decay in our collection, indicating that a larger number of SNPs is necessary to perform whole genome association mapping. Finally, an association mapping effort for architectural traits was carried out on a small number of accessions to estimate the feasibility of this approach.     

Drought tolerance of apple rootstocks: Production and partitioning of dry matter

The drought tolerance of the commercial apple ( Malus domestica Borkh.) rootstocks M9, M26, M27 and MM111, and some new selections from the rootstock breeding programme at HRI-East Malling (AR69-7, AR295-6, AR360-19, AR486-1 and AR628-2), was assessed using potted, glasshouse-grown, unworked rootstocks. After an initial period of growth under well-watered conditions the amount of irrigation was gradually reduced, for some treatments, to simulate natural drying in the soil. At the end of a six-month growth period, the rootstocks were harvested and the production of dry matter and its partitioning to various plant parts determined. The rootstocks exhibited large differences in shoot and root dry matter, and root length but not all the rootstocks showed declines in root mass or length in response to the droughting treatment. The dwarfing rootstocks tended to have smaller amounts of both coarse (>2 mm diameter) and fine roots (<2 mm diameter), than the more vigorous rootstocks. Irrespective of rootstock or irrigation treatment there was a close linear relationship between coarse and fine root. There was also no change in the length/weight relationship for fine roots irrespective of rootstock or irrigation treatment, i.e. 42 m of fine root weighed 1 g dry weight. In some cases the amount of root produced could be directly correlated with the rootstock known potential to control scion vigour, but this was not true for all the rootstocks examined. The absence of this relationship was particularly evident in some of the new selections of rootstock. The possible causes for these differences, compared with commercially used rootstocks, is discussed in relation to the origin and parentage of the rootstock selections. Despite this lack of a root length/vigour relationship, the amount of dry matter partitioned to shoot growth reflected the rootstocks' known vigour. The different responses of these rootstocks to drought are discussed along with their implications for understanding the mechanisms by which rootstocks are thought to dwarf scion shoots.     

SSR and RAPID Profile Based Grouping of Selected Jute Germplasm with Respect to Fibre Fineness Trait

With an aim to develop mapping population on fibre fineness trait, grouping of 16 selected jute accessions, eight each from Corchorus olitorius and Corchorus capsularis which showed promising agronomic characteristics, was carried out using fibre fineness data and DNA fingerprinting using SSR and RAPID primers. Based on fibre fineness trait two subgroups depicting the fine and coarse fibre yielding accessions were obtained in each species. A total of 26 RAPID primers and 22 pairs of SSR primers yielded 277 and 41 scorable bands, respectively. High level of polymorphism was detected between fine and coarse fibre yielding jute accessions. Dendrogram showed that all the accessions formed two main clusters between C. olitorius and C. capsularis and each main cluster subdivided in two clusters containing fine and coarse fibre jute accessions. RAPID and SSR marker data-sets showed high levels of positive correlation (Mantel test, r = 0.97). Grouping of jute accessions based on morphological and molecular data was highly correlated. This study will be useful in future jute breeding programs.     

Rootstock genotype succession influences apple replant disease and root-zone microbial community composition in an orchard soil

Apple replant disease (ARD) is a soil-borne disease complex that affects young apple trees in replanted orchards, resulting in stunted growth and reduced yields. Newly developed rootstock genotypes with tolerance to ARD may help to control this disease. We determined the effects of rootstock genotype rotations during orchard renovation, by investigating root-zone soil microbial consortia and the relative severity of ARD on seven rootstock genotypes (M.9, M.26, G.30, G.41, G.65, G.935, and CG.6210) planted in soil where trees on four of those same rootstocks (M.9, M.26, G.30 and CG.6210) had grown for the previous 15 years. Rootstock genotyping indicated that genetic distances among rootstocks were loosely correlated with their differential responses to ARD. Root-zone fungal and bacterial community composition, assessed by DNA fingerprinting (T-RFLP), differed between M.26 and CG.6210. Soil bacterial communities were influenced most by which rootstock had grown in the soil previously, while fungal communities were influenced more by the current replanted rootstock. In a clone library of bacteria from M.26 and CG.6210 root-zone soil, β-Proteobacteria was the most abundant phylum (25% of sequences). Sequences representing the Burkholderia cepacia complex were obtained only from CG.6210 soil. Rootstock genotypes that were grown in the orchard soil previously affected subsequent ARD severity, but replanting with the same or closely related rootstocks did not necessarily exacerbate this disease problem. Our results suggest that genotype-specific interactions with soil microbial consortia are linked with apple rootstock tolerance or susceptibility to ARD.     

<Emphasis Type="Italic">MdPIN1b</Emphasis> encodes a putative auxin efflux carrier and has different expression patterns in BC and M9 apple rootstocks

Key message

Lower promoter activity is closely associated with lower MdPIN1b expression in the M9 interstem, which might contribute to the dwarfing effect in apple trees.

Abstract

Apple trees grafted onto dwarfing rootstock Malling 9 (M9) produce dwarfing tree architecture with high yield and widely applying in production. Previously, we have reported that in Malus ‘Red Fuji’ (RF) trees growing on M9 interstem and Baleng Crab (BC) rootstock, IAA content was relatively higher in bark tissue of M9 interstem than that in scion or rootstock. As IAA polar transportation largely depends on the PIN-FORMED (PIN) auxin efflux carrier. Herein, we identify two putative auxin efflux carrier genes in Malus genus, MdPIN1a and MdPIN1b, which were closely related to the AtPIN1. We found that MdPIN1b was expressed preferentially in BC and M9, and the expression of MdPIN1b was significantly lower in the phloem of M9 interstem than that in the scion and rootstock. The distinct expression of MdPIN1b and IAA content were concentrated in the cambium and adjacent xylem or phloem, and MdPIN1b protein was localized on cell plasma membrane in onion epidermal cells transiently expressing 35S:MdPIN1b-GFP fusion protein. Interestingly, an MdPIN1b mutant allele in the promoter region upstream of M9 exhibited decreased MdPIN1b expression compared to BC. MdPIN1b over-expressing interstem in tobacco exhibited increased polar auxin transport. It is proposed that natural allelic differences decreased promoter activity is closely associated with lower MdPIN1b expression in the M9 interstem, which might limit the basipetal transport of auxin, and in turn might contribute to the dwarfing effect. Taken together, these results reveal allelic variation underlying an important apple rootstock trait, and specifically a novel molecular genetic mechanism underlying dwarfing mechanism.

     

Genetic Diversity and Characterization of a Core Collection of Malus Germplasm Using Simple Sequence Repeats (SSRs)

Simple sequence repeats (SSRs) were used to assess genetic diversity and study genetic relatedness in a large collection of Malus germplasm. A total of 164 accessions from the Malus core collection, maintained at the University of Illinois, were genotyped using apple SSR markers. Each of the accessions was genotyped using a single robust SSR marker from each of the 17 different linkage groups in Malus. Data were subjected to principal component analysis, and a dendrogram was constructed to establish genetic relatedness. As expected, this diverse core collection showed high allelic diversity; moreover, this allelic diversity was higher than that previously reported. Cluster analysis revealed the presence of four distinct clusters of accessions in this collection.     

Heritability of adventitious rooting of grapevine dormant canes

One of the most important viticultural characteristics of a grapevine rootstock is the ability to form roots on dormant lignified canes (rootstrike). North American species of Vitis are the primary source of germplasm for grapevine rootstocks and vary widely in their rate of rootstrike. Breeders have hybridized grape species in order to introgress traits to produce commercial rootstocks. A combination of 26 parents consisting of improved and wild accessions of Vitis spp. was used to generate 27 families. The percentage of rootstrike of dormant canes was observed over several years for 552 individuals. A logistic generalized linear mixed model (GLMM) method was used to estimate the narrow sense heritability (h ²) of rootstrike. Heritability was found to be moderate (h ²?=?0.307?±?0.050). The model also estimated breeding values of all parents and progeny. A GLMM method can be used to estimate breeding values of germplasm to identify individuals with commercially acceptable rates of rootstrike with a defined probability of transmitting this trait to progeny. This is useful for the introgression of traits into potentially new commercial rootstocks. The pattern of normal distribution of rooting indicates that it is possible to identify individuals with good rootstrike from Vitis species that are generally considered to have low rootstrike. Selection of individuals with a higher breeding value will increase the efficiency of rootstock breeding.     

Rootstock genotype and orchard replant position rather than soil fumigation or compost amendment determine tree growth and rhizosphere bacterial community composition in an apple replant soil

Apple replant disease (ARD) is a complex soilborne disease syndrome that often causes problems when renovating old orchard sites. Soil fumigants sometimes control ARD, but biological and cultural alternatives are needed. In this study the growth of two widely used clonal apple (Malus domestica) rootstocks (M7 and M26) were compared to three new rootstocks from the Cornell-Geneva series (CG16, CG30 and CG210 (a.k.a. CG6210)) in an orchard site with a history of ARD, in Ithaca, NY. Trees were planted in two distinguishable positions – the previous tree rows versus the old inter-row grass lanes. Additionally, we compared the effects of compost amendment and fumigation with dichloropropene plus chloropicrin on tree growth on each replant rootstock. Rhizosphere bacteria and actinobacteria were assessed using PCR-DGGE for the rootstocks M7, M26, CG30 and CG210. Tree growth on the rootstocks M7, M26 and CG16 was suppressed in the old tree rows relative to grass lanes, while trees on CG30 and CG210 rootstocks grew equally well in both positions. The species composition of rhizosphere bacteria and actinobacteria differed significantly between the planting positions and between the rootstocks M7 and M26 compared to CG30 and CG210. In contrast, the preplant compost or fumigation soil treatments had no effect on tree growth and little impact on rhizosphere bacterial community composition. Abbreviations: ARD – apple replant disease; DGGE – denaturing gradient gel electrophoresis; PCR – polymerase chain reaction.     

A functional genetic marker for apple red skin coloration across different environments

The red skin color desired by most apple consumers is not easy to achieve in warm climates, as the expression of MYB10, which regulates red pigmentation in apple, is influenced negatively by high temperatures. We describe the development and validation of a genetic marker for red skin coloration that effectively predicts color in a warm summer environment in Spain, as well as more temperate climates in New Zealand and Italy. Following the determination of a major-effect quantitative trait locus (QTL) controlling red skin coloration on linkage group (LG)9, using four segregating populations grown in New Zealand, and screened using the IRSC apple 8-K single-nucleotide polymorphism (SNP) array, the most significant SNP marker (ss475879531) was transformed into a marker suitable for use in a real-time PCR assay. This marker was validated using five apple seedling populations growing in a warm summer environment in Spain, demonstrating that the marker system efficiently predicts red skin coloration and can be used for marker assisted selection, even under conditions considered adverse for skin color development.     

Linkage Mapping and Molecular Diversity at the Flower Sex Locus in Wild and Cultivated Grapevine Reveal a Prominent SSR Haplotype in Hermaphrodite Plants

Cultivars used for wine and table grape have self-fertile hermaphrodite flowers whereas wild European vines and American and Asian species are dioecious, having either male or female flowers. Consistent with previous studies, the flower sex trait was mapped as a single major locus on chromosome 2 based on a pure Vitis vinifera population segregating for hermaphrodite and female progeny, and a hybrid population producing all three flower sex types. The sex locus was placed between the same SSR and SNP markers on both genetic maps, although abnormal segregation hampered to fine map the genomic region. From a total of 55 possible haplotypes inferred for three SSR markers around the sex locus, in a population of 132 V. sylvestris accessions and 171 V. vinifera cultivars, one of them accounted for 66 % of the hermaphrodite individuals and may be the result of domestication. Specific size variants of the VVIB23 microsatellite sequence within the 3′-UTR of a putative YABBY1 gene were found to be statistically significantly associated with the sex alleles M, H and f; these markers can provide assistance in defining the status of wild grapevine germplasm.     

Aligned genetic linkage maps of apple rootstock cultivar ‘JM7’ and Malus sieboldii ‘Sanashi 63’ constructed with novel EST-SSRs

Identification of markers associated with genes of interest and quantitative trait loci (QTLs), combined with high-density genetic linkage maps, can help reduce labor and costs by enabling marker-assisted selection (MAS). In this study, a dwarfing apple rootstock cultivar ??JM7?? (Malus prunifolia × Malus pumila ??Malling 9??) and wild apple Malus sieboldii ??Sanashi 63?? (section Sorbomalus) were used for constructing genetic linkage maps. Here, a species from section Sorbomalus was used for the first time as a target species in a genome-wide mapping study. We also developed and mapped 137 novel-expressed sequence tag-simple sequence repeat (EST-SSR) markers. The genetic linkage maps of ??JM7?? and ??Sanashi 63?? consisted of 415 and 310 loci and spanned 998.0 and 981.8?cM, respectively, comparable to the reference map of Malus × domestica ??Discovery??. A BLASTN search revealed that all of the EST-SSR sequences used in this study exhibited very high homology to one or more previously characterized apple genome contigs. Although the most homologous contigs of 89 EST-SSRs were located within the same linkage groups (LGs) identified by mapping analysis, the other 48 EST-SSRs were aligned into contigs positioned in different LGs than those identified by mapping. When search criteria were expanded to include the five most homologous contigs of each EST-SSR, at least one of the top five contigs for 15 of these 48 EST-SSRs corresponded to the LG obtained by mapping. The maps of ??JM7?? and ??Sanashi 63?? may be useful for analyzing important rootstock characteristics and identifying markers for MAS.     

Ιn vitro plant regeneration from leaf explants of the cherry rootstocks CAB-6P,Gisela 6, and MxM 14 using sodium nitroprusside

Conventional multiplication of cherry (Prunus cerasus L.) rootstocks utilizes division, cuttings, and propagation through seed, which are relatively slow and labor intensive and result in genetic variability. Tissue culture, on the other hand, ensures rapid, large-scale, and low-cost production of genetically identical, physiologically uniform, and pathogen-free plants. In the cherry rootstocks CAB-6P, Gisela 6, and MxM 14, sodium nitroprusside (SNP) promoted callus induction, in vitro shoot proliferation, and rooting from leaf explants in a medium containing 17.6 μM benzyladenine and 2.68 μM α-naphthaleneacetic acid. CAB-6P explants treated with 10 μM SNP gave the maximum shoot number (5), whereas 30 μM SNP gave the longest shoots and the greatest shoot induction rate (26.67%). Best rooting was obtained with 50 μM SNP. In Gisela 6 rootstock, the shoot number (10) and shoot length (20.5 mm) were maximal in the control group without plant growth regulators. The shoot induction rate was enhanced (40%) with 40 μM SNP. SNP at 40 μM resulted in root formation, while 30 μM produced the largest callus size, and 10 μM SNP resulted in the maximum callus fresh weight. MxM 14 leaves treated with 30 μM SNP gave the maximum shoot number (3), root number (7.56), and shoot induction rate (40%), whereas 40 μM SNP gave the longest shoots (12 mm) and roots (20 mm). Best results for callus size, callus fresh weight, and callus induction rate (100%) in the CAB-6P and MxM 14 rootstocks were observed with 30 and 40 μM SNP, respectively. Rooted explants with shoots were gradually acclimatized to the external environment with a high survival percentage (85%). An efficient protocol of indirect organogenesis was established for the three cherry rootstocks using SNP.     

A versatile PCR marker for pungency in Capsicum spp.

Pungency in Capsicum spp. is an important quality trait for pepper breeding. The perception of pungency in pepper is due to the presence of a group of compounds named capsaicinoids, only found within the Capsicum genus. How pungency is controlled at genetic and molecular levels has not been completely elucidated. The use of molecular markers to assess pungency trait is required for molecular breeding, despite the difficulty of development of universal markers for this trait. In this work, a DNA sequence possibly related to pungency with a high similarity to Pun1 locus was studied, and sequence analysis of this homolog revealed a 15?bp deletion in non-pungent pepper accessions. An allele-specific pair of primers was designed and specific fragments of 479?bp from non-pungent and 494?bp from pungent accessions were obtained. Polymorphism of this marker, named MAP1, was tested in a wide range of accessions, belonging to several Capsicum species, including pungent and non-pungent accessions of C. annuum L., and pungent accessions of C. chinense, C. baccatum, C. frutescens, C. pubescens, C. galapagoense, C. eximium, C. tovarii, C. cardenasii, and C. chacoense. All these Capsicum accessions were correctly discriminated. The marker suitability to assess pungency in domesticated and wild Capsicum species was demonstrated, and therefore it will be very useful in marker assisted selection (MAS). Moreover, MAP1 was located in a saturated pepper linkage map and its possible relationship with the Pun1 locus has been discussed. Among the available markers for this complex quality trait, the marker developed in this study is the most universal so far.