Analysis of S‐allele genetic diversity in Sicilian almond germplasm comparing different molecular methods

Italian almond germplasm is characterized by a wide diversity in several growing areas among which Sicily is one of the most important. Analysis with consensus and specific primers and DNA sequencing was performed to investigate S‐RNase genetic diversity and to elucidate the homology rate within a genetic pool of 27 Italian accessions. Interestingly, some of the self‐compatible cultivars did not show the presence of S_f allele. Amplicons from consensus and allele‐specific PCR primers revealed a high level of variability. Sequencing of all the S‐RNase amplicons derived from consensus primers allowed the identification of two new S‐RNase alleles (S₅₁ and S₅₂). Surprisingly, despite the AA replacement mutation, S₅₁ did not exhibit any change of its S‐RNase function. Additionally, several mutations, with no effect on amino acid composition, were detected in the intron and/or in the ORF of four known alleles (S_g, S₁₀, S₃₁ and S₃₅). Genetic variation, regarding point mutations and only detected by sequencing, was revealed among 11 of 27 tested cultivars. The new sources of variability might have an interest for product traceability.     

Identification and characterization of new S‐alleles associated with self‐incompatibility in almond

Almond is a highly heterozygous species with a high number of S‐alleles controlling its gametophytic self‐incompatibility system (GSI). In this work, we have analysed 14 Spanish local almond cultivars for S‐RNase allele diversity. Five new S‐RNase alleles were identified by cloning and sequencing, S₃₁ (804 bp) in ‘Pou de Felanitx’ and ‘Totsol’, S₃₂ (855 bp) in ‘Taiatona’, S₃₃ (1165 bp) in ‘Pou d’Establiments’ and ‘Muel’, S₃₄ (1663 bp) in ‘Pané‐Barquets’ and S₃₅ (1658 bp) in ‘Planeta de les Garrigues’. Additionally, seven already known almond alleles could be recognized in the local cultivars studied. The high number of new alleles identified reveals the wide diversity of almond germplasm still existing and requiring characterization, and points to the possibility of new findings by a wider study focusing on other provenances. The almond S‐RNases have been compared to those of other Prunus species, showing a high identity and confirming that the S‐RNase gene in this genus presents a probable common ancestor.     

SFB‐based S‐haplotyping of apricot (Prunus armeniaca) with DHPLC

Most cultivars that belong to the Rosaceae are self‐incompatible and depend on cross‐pollination. The pollen donor and pollen recipient have to flower synchronously and must be genetically compatible. Genetic compatibility is governed by the S‐locus, which holds the S‐RNase and S‐haplotype‐specific F‐Box (SFB) genes. Thus, the S‐genotype of cultivars is an important feature and is characterized molecularly by the S‐RNase and SFB alleles which are distinctive for each S‐haplotype. Here, we report the usage of DNA chromatography (denaturing high‐performance liquid chromatography – DHPLC) for identifying the S‐genotypes of European apricots on the basis of their SFB alleles. DHPLC is amenable to high‐throughput automation, and therefore is valuable for breeding and for high‐quality plant typing in the nursery.     

Development of a molecular marker for self‐compatible S4′ haplotype in sweet cherry (Prunus avium L.) using high‐resolution melting

Sweet cherry (Prunus avium L.) has stylar gametophytic self‐incompatibility, which is controlled by the multi‐allelic S‐locus and encompasses the highly polymorphic genes for the S‐ribonuclease (S‐RNase) and S‐haplotype‐specific F‐box (SFB), which are female and male determinants, respectively. The self‐compatible mutant SFB4′ corresponds to an allele variant of SFB4 and presents a frameshift mutation. Even though male‐determinant molecular markers can discriminate between SFB4 and SFB4′ alleles, the methods required are laborious, time‐consuming and expensive, and not suitable for massive analysis and integration into breeding programmes. Our aim was to develop molecular markers for the evaluation of self‐compatibility alleles in sweet cherry, that could be used as a high‐throughput screening strategy to identify SFB4 and SFB4′ alleles, based on a marker for male determinacy. Our results were consistent using primers flanking the mutation responsible for the SFB4′ allele. We designed a specific molecular marker and confirmed it in sweet cherry commercial varieties. This new molecular marker is feasible for self‐compatibility alleles in the male determinant in sweet cherry‐assisted breeding programs.     

Genetic diversity,population structure,pollen morphology and cross‐compatibility among Chinese Cymbidiums

A total of 85 Chinese Cymbidiums and one Zygopetalum were collected and analysed using 29 simple sequence repeats (SSRs) and 12 intersimple sequence repeats (ISSRs). These Chinese Cymbidiums are some of the most popular cultivars in market. The pairwise genetic distance between these accessions averaged 0.897, ranging from 0.259 to 1.000. A model‐based clustering analysis revealed five genetic groups with serious admixture ancestry, as indicated by a fixation index of 0.156 and only 10.67% genetic variation among groups. By analysing the cross‐compatibility and pollen morphology, Chinese Cymbidiums are revealed to have high compatibility for intra‐ and interspecific crosses. Notably, Zygopetalum mackayi had high compatibility with Chinese Cymbidiums and shared similar morphologies in terms of pollinia and pollen grain. This study is the first to report a successful cross between Zygopetalum and Chinese Cymbidiums. The high compatibility offers the potential for introducing Zygopetalum mackayi's superior traits into Chinese Cymbidiums. The similar pollen morphology as well as the low genetic differentiation might be related to the high cross‐compatibility among these accessions.     

Inheritance of resistance to the peach root‐knot nematode (Meloidogyne floridensis) in interspecific crosses between peach (Prunus persica) and its wild relative (Prunus kansuensis)

The peach root‐knot nematode, Meloidogyne floridensis (MF), infects majority of available nematode‐resistant peach rootstocks which are mostly derived from peach (Prunus persica) and Chinese wild peach (P. davidiana). Interspecific hybridization of peach with its wild relative, Kansu peach (P. kansuensis), offers potential for broadening the resistance spectrum in standard peach rootstocks. We investigated the inheritance of resistance to MF in segregating populations of peach (‘Okinawa’ or ‘Flordaguard’) × P. kansuensis. A total of 379 individuals from 13 F₂ and BC₁F₁ families were challenged with a pathogenic MF isolate “MFGnv14” and were classified as resistant (R) or susceptible (S) based on root galling intensity. Segregation analyses in F₂ progeny revealed the involvement of a major locus with a dominant or recessive allele determining resistance in progeny segregating 3R:1S and 1R:3S, respectively. Testcrosses with a homozygous‐susceptible peach genotype (‘Flordaguard’ or ‘UFSharp’) confirmed P. kansuensis as a source of new resistance and the heterozygous allelic status of P. kansuensis at the locus conferring resistance to MF. We propose a single‐locus dominant/recessive model for the inheritance of resistance.     

Identification and classification of S haplotypes in radish (Raphanus sativus)

Radish (Raphanus sativus L.) is a typical cross‐pollinated crop that exhibits obvious heterosis. Self‐incompatibility is an important character for F₁ hybrid breeding of radish. Knowledge of the S haplotypes of breeding lines is very important for breeders to avoid cross‐incompatibility of the parental lines. In the present study, the S haplotypes of 63 radish inbred lines, which were independently cultivated by our research group, were identified by PCR amplification, sequencing and BLAST analyses of the SRK and SLG genes. Finally, fifty‐four inbred lines were classified into 15 class I S haplotypes, including three new types, RsS‐38, RsS‐39 and RsS‐40. Additionally, three class II S haplotypes were identified in nine radish inbred lines. Partial SRK or SLG sequences were completed, such as RsS‐11 Lim (SRK‐S), RsS‐26 (SRK‐K), RsS‐5 Lim (SRK‐K and SRK‐S) and RsS‐9 (SRK‐K). The identified S haplotypes were verified with a cross‐pollination test, and RsS‐9 has weaker self‐incompatibility than other S haplotypes. These information will not only contribute to the production of hybrid seeds but also to the development of new self‐compatible inbred lines, which were advantageous of the production of maintain line and male line in CMS breeding system.     

Use of non‐adapted quantitative trait loci for increasing Fusarium head blight resistance for breeding semi‐dwarf wheat

Semi‐dwarf wheat is an important prerequisite for releasing a successful commercial cultivar in high‐yielding environments. In Northern Europe, this aim is achieved by using one of the dwarfing genes Rht‐B1 (formerly known as Rht‐1) or Rht‐D1 (Rht‐2). Both genes, however, result in a higher susceptibility to Fusarium head blight (FHB). We analysed the possibility to use the two non‐adapted FHB resistance quantitative trait loci Fhb1 and Fhb5 (syn. QFhs.ifa‐5A) to counterbalance the negative effect of the dwarfing allele Rht‐D1b in a winter wheat population of 585 doubled‐haploid (DH) lines segregating for the three loci. All lines were inoculated with Fusarium culmorum at four locations and analysed for FHB severity, plant height, and heading date. The DH population showed a significant (p < 0.001) genotypic variation for FHB severity ranging from 3.6% to 65.9% with a very high entry‐mean heritability of 0.95. The dwarfing allele Rht‐D1b reduced plant height by 24 cm, but nearly doubled the FHB susceptibility (24.74% vs. 12.74%). The resistance alleles of Fhb1 and Fhb5 reduced FHB susceptibility by 6.5 and 11.3 percentage points, respectively. Taken all three loci together, Fhb5 alone was already able to reduce FHB susceptibility to the same extent as Rht‐D1b increased it. This opens new avenues for selecting semi‐dwarf wheat by marker‐assisted introgression of Fhb5 without the enhancement of FHB susceptibility.     

Reproductive compatibility studies between wild and cultivated strawberries (Fragaria × ananassa) to obtain “bridge species” for breeding programmes

The Fragaria genus has become relevant due to worldwide economic importance of the octoploid hybrid F. × ananassa. Interspecific hybridizations are widely employed in breeding programmes to introduce useful characteristics from wild species to cultivated varieties, although many reproductive barriers prevent gene flow between species. Hybrid genotypes that can act as a bridge between related wild species and cultivated strawberry could be useful to overcome those barriers. The aim of this work was to obtain interspecific hybrids of Fragaria and to analyse possible reproductive barriers. In the Banco de Germoplasma de Frutilla‐UNT, crossings were carried out among wild species of Fragaria with different ploidy levels and cultivated strawberry. To confirm hybrid condition of genotypes obtained, histological techniques and microsatellite markers were used. When wild species were crossed with cultivated strawberry in both directions, there was low production of achenes, while in crosses between wild species of Fragaria genus, achene production was very high. The percentage of germination of achenes was high when crossed species had the same ploidy level, and very low or null when they presented different ploidy levels. Although pre‐zygotic incompatibility associated to the degree of domestication and postzygotic barriers related to different ploidy levels of the progenitors were detected, new hybrid genotypes of Fragaria were obtained. These new hybrids could be used as “bridge species” in breeding programmes, since preliminary results showed no incompatibility barriers when they were crossed with Duchesnea indica.     

Marker‐assisted breeding of Pi‐1 and Piz‐5 genes imparting resistance to rice blast in PRR78, restorer line of Pusa RH‐10 Basmati rice hybrid

Rice blast, caused by fungus Magnaporthe grisea, is a serious disease causing considerable economic damage worldwide. Best way to overcome disease is to breed for disease‐resistant cultivars/parental lines of hybrids. Pusa RH10, first aromatic, fine‐grain rice hybrid released and cultivated extensively in India. Hybrid and its parental lines, Pusa 6A and PRR78, are highly susceptible to blast. CO39 pyramid carrying two dominant, broad‐spectrum blast‐resistance genes, viz. Pi‐1 and Piz‐5, used as a donor parent to introgress these genes into PRR78 using marker‐assisted backcrossing (MABC). Microsatellite markers RM5926 and AP5659‐5 tightly linked to Pi‐1 and Piz‐5 genes, respectively, were used for foreground selection to derive introgression lines. Further, these lines were evaluated for agronomic performance, disease reaction and cooking quality traits along with PRR78. Most of the improved lines were on par with PRR78 for all traits evaluated except gelatinization temperature. Recurrent parent genome percentage (RPG) study also revealed similarity of these lines with PRR78. Hybrids derived using improved PRR78 lines were superior over Pusa RH10 in terms of yield.     

Survey and new insights in the application of PCR‐based molecular markers for identification of HMW‐GS at the Glu‐B1 locus in durum and bread wheat

Wheat, among all cereal grains, possesses unique characteristics conferred by gluten; in particular, high molecular weight glutenin subunits (HMW‐GS) are of considerable interest as they strictly relate to bread‐making quality and contribute to strengthening and stabilizing dough. Thus, the identification of allelic composition, in particular at the Glu‐B1 locus, is very important to wheat quality improvement. Several PCR‐based molecular markers to tag‐specific HMW glutenin genes encoding Bx and By subunits have been developed in recent years. This study provides a survey of the molecular markers developed for the HMW‐GS at the Glu‐B1 locus. In addition, a selection of molecular markers was tested on 31 durum and bread wheat cultivars containing the By8, By16, By9, Bx17, Bx6, Bx14 and Bx17 Glu‐B1 alleles, and a new assignation was defined for the ZSBy9_aF1/R3 molecular marker that was specific for the By20 allele. We believe the results constitute a practical guide for results that might be achieved by these molecular markers on populations and cultivars with high variability at the Glu‐B1 locus.     

QTL analysis for cooking traits of super rice with a high‐density SNP genetic map and fine mapping of a novel boiled grain length locus

Hybrid rice has contributed substantially to the improvement of grain production worldwide, yet its poor cooking and tasting characteristics have long been recognized. In this study, 132 recombinant inbred lines derived from LYPJ were used to identify quantitative trait loci (QTLs) for 12 cooking traits with the high‐density SNP linkage map recently developed by our team. We identified 17 QTLs on chromosomes 1, 2, 4, 5, 6, 7, 8, 9 and 11, which accounted for 7.50% to 23.50% of the phenotypic variations. A novel major QTL qBGL7 for boiled grain length was further fine‐mapped to an interval of 440 Kb between the two markers RM21906 and gl3 using a BC₃F₂ population. Two near‐isogenic lines with extreme boiled grain length, GX5‐176 and GX5‐101, could be directly used in improving cooking quality. We also identified a QTL for soaked grain width expansion rate, qSGWE6, in the Wx gene region on chromosome 6. The Wx differential regulation coincided with sequential variation between the two parents. Our work offered a theoretical basis for molecular breeding of high‐quality hybrid rice.     

Marker‐assisted selection for rice blast resistance genes Pi2 and Pi9 through high‐resolution melting of a gene‐targeted amplicon

The use of host resistance (R) genes is considered the most cost‐effective option to control the rice blast disease. The two allelic R genes Pi2 and Pi9 confer very broad‐spectrum resistance against blast isolates collected worldwide. However, the two genes have not yet been widely deployed in rice breeding programmes. Availability of specific markers for them would facilitate incorporating the two R genes into new rice lines through marker‐assisted selection. Herein, we report the development and utilization of a robust and specific marker for the Pi2 and Pi9. This marker was derived from polymorphisms within the target gene, and achieved simultaneously distinguish Pi2 and Pi9 from other alleles through high‐resolution melting of a small amplicon. With the marker, we were able to transfer the Pi2 into an elite restorer line through marker‐assisted backcrossing, successfully obtained effective resistance to blast disease, and we were also able to, respectively, incorporate the Pi2 and Pi9 with two other R genes. As the additive effect, blast resistance in these stacking lines harbouring three R genes were significantly improved.     

Validation of a male‐linked gene locus (OGI) for sex identification in persimmon (Diospyros kaki Thunb.) and its application in F1 progeny

It is crucial to develop a rapid technique for identifying sexuality in the seedling stage of persimmon (Diospyros kaki Thunb.), and the elimination of male progeny has been regarded as an important strategy for enhancing breeding efficiency. In this study, phenotype characterization and genotyping of the male‐linked OGI marker were carried out using 205 accessions, including persimmon cultivars, F₁ progeny and nine related Diospyros species. All persimmon cultivars displayed consistent results regarding OGI amplification and sex phenotype. A total of 143 F₁ progeny were derived from 11 crosses, among which 95 individuals had flowered. In the flowering full‐sib families, the amplification of the OGI marker in agreement with the sex phenotype was obtained in 85 plants (89.5%). The segregation of OGI in ‘Huashi 1’ × ‘Luotian Tianshi’ and ‘Huashi 1’ × Male 3 F₁ populations fit a 1 : 1 ratio. Furthermore, high OGI transferability was observed in nine related species. Overall, the results indicated that the OGI locus could be used to distinguish male from female persimmon plants at an early stage.     

Development and characterization of a new set of 164 polymorphic EST‐SSR markers for diversity and breeding studies in rubber tree (Hevea brasiliensis Müll. Arg.)

Despite its economic importance and recent genome release, the need for molecular tools for Hevea brasiliensis is high. In the frame of a disease resistance study, EST sequences were retrieved from public database or generated by sequencing SSH libraries. Sequences were trimmed and microsatellite motifs searched using an ad hoc bioinformatic pipeline, and pairs of primers for the amplification of candidate markers were generated. We found a total of 10 499 unigenes from both sources of sequences, and 673 microsatellites motifs were detected using the default parameters of the pipeline. Two hundred sixty‐four primer pairs were tested and 226 (85.6%) successfully amplified. Out of the amplified candidate markers, 164 exhibited polymorphism. Relationships based on dendrograms using simple matching index and diversity statistics based on EST‐SSRs were compared with Genomic SSRs, showing the potentialities of EST‐derived microsatellites for resistance studies but also for population genetics approaches.     

Development of gene‐based markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa

Turnip mosaic virus (TuMV) is responsible for a serious disease that affects the production of Chinese cabbage. Previous studies have cloned a series of TuMV resistance genes and developed molecular markers. In this study, a derived cleaved amplified polymorphism sequence (dCAPS) marker and a Kompetitive Allele Specific PCR (KASP) marker were developed based on a single recessive gene, retr02, which confers broad‐spectrum TuMV resistance in Chinese cabbage by means of an additional G at the junction of exon 1 and intron 1. The two markers were able to detect the retr02 allele in Chinese cabbage accessions used in breeding programmes. Compared with the dCAPS marker, the KASP marker was flexible, cost‐effective and quick to process, which is likely to be beneficial in establishing high‐throughput assays for marker‐assisted selection.     

Development of AS‐PCR marker based on a key mutation confirmed by resequencing of Wx‐mp in Milky Princess and its application in japonica soft rice (Oryza sativa L.) breeding

Soft rice with low amylose content (AC) ranging by 5–15% is a unique type with special eating and appearance quality and has become popular in the rice market. We resequenced the Wx‐mp, a key allele from Milky Princess, a Japanese low AC variety, and found that the +473 mutation in exon 4 is the key mutation in both Wx‐mp and its ancestor allele, Wx‐mq from Milky Queen. Based on this functional mutation, an allele‐specific PCR (AS‐PCR) marker was developed and proven in a breeding population derived from a cross between a Chinese late variety Nan Keng 46 (Wx‐mp/Wx‐mp) and an early line Ning 63121(Wx‐b/Wx‐b). Based on the marker‐aided selection by our newly developed AS‐PCR marker for Wx‐mp and the known ST10 marker for Stvb‐i, a total of 12 Wx‐mp homozygotes were selected from 198 F₂ progenies, and four of them were immune to rice stripe virus (RSV) with averagely 11.3 days earlier heading than Nan Keng 46 without significant change in grain yield.