Self-pollinated Progeny Research Articles

Diploid aposporous sunflower forms triploid BIII progeny displaying increased apospory levels and non-random genetic mutations

Apomixis (asexual reproduction via seeds) has the potential to revolutionize sunflower breeding. In previous studies, we identified a diploid sunflower line (Rf975) that naturally exhibits extra gametophytes resembling aposporous apomictic embryo sacs (AES). Here, we investigated the nature (reduced vs. unreduced) and viability of these AES-like gametophytes by examining the formation of triploid (3x) BIII hybrids (2n + n) in the progeny of Rf975. Flow cytometry analysis of immature seeds revealed that, on average, 42.8% of self-pollinated Rf975 progeny were triploids, although only 36.6% of them reached maturity. Cytoembryological analysis showed that 100% of triploids exhibited some degree of apospory, with an average expressivity of 61.9%. Abnormal pollen grains and limited viable seeds were also noted. A segregant F2 progeny, comprising diploid and triploid individuals, was generated by crossing Rf975 with HA89, a genetically divergent sexual diploid. SNP-based progeny tests discarded that diploid Rf975 forms clonal matroclinal progeny at levels greater than 18%. Furthermore, specific non-random genetic and DNA methylation changes were detected in the F2 triploids compared to F2 diploids and parental plants, highlighting recurrent (epi)genetic alterations occurring during triploidization. This research could contribute to the future implementation of apomixis-based strategies in sunflower breeding.

Heritable Phenotypic Variations in the Self-Pollinated Progeny from Grafted Cucumber with Pumpkin as Rootstock

This study investigated whether grafting cucumber (Cucumis sativus L.) onto pumpkin (Cucurbita moschata Duch.) rootstocks can induce phenotypic variations in the self-bred cucumber progeny. The phenotypic traits of the self-pollinated progeny of grafted ‘Zhenghuang 1409’ cucumber were significantly different from those of the naturally rooted ‘Zhenghuang 1409’ parent line. Compared with the ‘Zhenghuang 1409’ cucumber inbred line, the first self-pollinated and naturally rooted generation of grafted cucumber displayed reduced length and width of the leaf at the node of the second female flower, a tapered petiole and stem, increased internode length, shorter plants, decreased fruit diameter, longer fruit stalk, thicker pulp, decreased seed length and width, and a lower 100-grain weight. Similar changes in most of these phenotypic traits were observed in the second generation of self-pollinated plants from grafted cucumber. By observing plant phenotypic traits, two leaf phenotypic variations were observed in the first generation of selfed grafted cucumber, and these two phenotypes were inherited in the second selfed generation.

The Application of Fluorescence In Situ Hybridization in the Prescreening of Veronica Hybrids.

Fluorescence in situ hybridization (FISH), a molecular cytogenetic technique that enables the visualization and identification of specific DNA sequences within chromosomes, has emerged as a pivotal tool in plant breeding programs, particularly in the case of Veronica species. Veronica, a genus with a complex reproductive system, often poses challenges in accurately identifying hybrids because of its tendency to hybridize, which leads to intricate genetic variation. This study focused on the use of FISH as a prescreening method to identify true hybrids in Veronica breeding programs. FISH analysis was first performed on the parents to identify their 45S and 5S rDNA signals, along with their respective chromosome numbers. The signals were then compared with those of the twenty progenies with reference to their supposed parents. Five true hybrids, seven self-pollinated progenies, and eight false hybrids were identified through FISH. The findings highlight the significance of FISH as a screening method that contributes significantly to the efficiency of Veronica breeding programs by ensuring the preservation of desired genetic traits and minimizing the inadvertent inclusion of misidentified hybrids. To conclude, this study underscores the vital role of FISH in enhancing the precision and success of breeding programs and opens new avenues for improved breeding strategies and crop development.

Efficient induction and rapid identification of haploid grains in tetraploid wheat by editing genes TtMTL and pyramiding anthocyanin markers.

Doubled haploid (DH) technology provides an effective way to generate homozygous genetic and breeding materials over a short period of time. We produced three types of homozygous TtMTL gene-edited mutants (mtl-a, mtl-b, and mtl-ab) by CRISPR/Cas9 in durum wheat. PCR restriction enzymes and sequencing confirmed that the editing efficiency was up to 53.5%. The seed-setting rates of the three types of mutants ranged from 20% to 60%. Abnormal grain phenotypes of kernel, embryo, and both embryo and endosperm abortions were observed in the progenies of the mutants. The average frequency of embryo-less grains was 25.3%. Chromosome counting, guard cell length, and flow cytometry confirmed that the haploid induction rate was in the range of 3%-21% in the cross- and self-pollinated progenies of the mtl mutants (mtl-a and mtl-ab). Furthermore, we co-transformed two vectors, pCRISPR/Cas9-MTL and pBD68-(ZmR + ZmC1), into durum wheat, to pyramide Ttmtl-edited mutations and embryo-specifically expressed anthocyanin markers, and developed a homozygous durum haploid inducer with purple embryo (DHIPE). Using DHIPE as the male parent to be crossed with the wild-type Kronos, the grains with white embryos were identified as haploid, while the grains with purple embryos were diploid. These findings will promote the breeding of new tetraploid wheat varieties.

Natural Albino Mutant of Daylily (Hemerocallis spp.) Reveals a Link between Drought Sensitivity and Photosynthetic Pigments Metabolism.

Mutant analysis remains one of the main genetic tools for characterising unclarified gene functions in plants, especially in non-model plants. Daylily (Hemerocallis spp.) is a popular perennial ornamental plant grown worldwide. Analysis of daylily mutants can enhance understanding of genes regulating the albino phenotype and improve the cultivar quality of daylily. The natural albino mutant (Alb-⁣/-) was isolated by screening a self-pollinated progeny of daylily cultivar 'black-eyed stella'. Transmission electron microscopy was used in analysing the structure of plastids between mutant and wild-type seedlings. The content of chlorophyll, carotenoids and chlorophyll precursors in plants was measured by ultraviolet spectrophotometry. RNA sequencing and physiological measurements were performed to explore the association between drought tolerance and mutation. All the seedlings of the daylily albino mutants died spontaneously within fifteen days after germination when grown in soil. The carotenoid and chlorophyll content in the leaves of the mutant plants significantly decreased compared with those of the wild-type control. The mutant plants displayed stunted growth, and their leaves were white or light yellow in color. Abnormal plastids such as those showing endomembrane vesiculation and lacking stacking were discovered in the leaves of mutant plants. Furthermore, genetic analysis revealed that a single recessive nuclear gene mutation led to the albino trait, RNA sequencing and real-time quantitative PCR validation showed extensive differences in gene expression between the mutant plants and the wild-type control, and most of the genes related to chlorophyll metabolism were down-regulated, with foldchange ranging from 0.20-0.49. Additionally, the surviving homozygous plants (Alb+⁣/+), which do not contain this mutation, were also isolated by analysing the phenotype of their self-pollinated progeny. The net photosynthesis rate and light saturation point of Alb+⁣/+ were higher than those of heterozygous (Alb+⁣/-) plants. Additionally, the Alb+⁣/+ plants were more tolerant to drought conditions than the Alb+⁣/- plants, suggesting that a heterozygous Alb- mutation is sufficient to negatively affect photosynthetic efficiency and drought tolerance. The albino mutation negatively affects photosynthetic efficiency and drought tolerance, and homozygous mutation is required for the characteristic albino phenotype. This work highlights the link between albino mutation, photosynthetic pigment metabolism and drought sensitivity in daylily.

Genome composition in Brassica interspecific hybrids affects chromosome inheritance and viability of progeny

Interspecific hybridization is widespread in nature and can result in the formation of new hybrid species as well as the transfer of traits between species. However, the fate of newly formed hybrid lineages is relatively understudied. We undertook pairwise crossing between multiple genotypes of three Brassica allotetraploid species Brassica juncea (2n = AABB), Brassica carinata (2n = BBCC), and Brassica napus (2n = AACC) to generate AABC, BBAC, and CCAB interspecific hybrids and investigated chromosome inheritance and fertility in these hybrids and their self-pollinated progeny. Surprisingly, despite the presence of a complete diploid genome in all hybrids, hybrid fertility was very low. AABC and BBAC first generation (F1) hybrids both averaged ~16% pollen viability compared to 3.5% in CCAB hybrids: most CCAB hybrid flowers were male-sterile. AABC and CCAB F1 hybrid plants averaged 5.5 and 0.5 seeds per plant, respectively, and BBAC F1 hybrids ~56 seeds/plant. In the second generation (S1), all confirmed self-pollinated progeny resulting from CCAB hybrids were sterile, producing no self-pollinated seeds. Three AABC S1 hybrids putatively resulting from unreduced gametes produced 3, 14, and 182 seeds each, while other AABC S1 hybrids averaged 1.5 seeds/plant (0–8). BBAC S1 hybrids averaged 44 seeds/plant (range 0–403). We also observed strong bias towards retention rather than loss of the haploid genomes, suggesting that the subgenomes in the Brassica allotetraploids are already highly interdependent, such that loss of one subgenome is detrimental to fertility and viability. Our results suggest that relationships between subgenomes determine hybridization outcomes in these species.

A Genome-Wide Association Study and Genomic Prediction for Fiber and Sucrose Contents in a Mapping Population of LCP 85-384 Sugarcane.

Sugarcane (Saccharum spp. hybrids) is an economically important crop for both sugar and biofuel industries. Fiber and sucrose contents are the two most critical quantitative traits in sugarcane breeding that require multiple-year and multiple-location evaluations. Marker-assisted selection (MAS) could significantly reduce the time and cost of developing new sugarcane varieties. The objectives of this study were to conduct a genome-wide association study (GWAS) to identify DNA markers associated with fiber and sucrose contents and to perform genomic prediction (GP) for the two traits. Fiber and sucrose data were collected from 237 self-pollinated progenies of LCP 85-384, the most popular Louisiana sugarcane cultivar from 1999 to 2007. The GWAS was performed using 1310 polymorphic DNA marker alleles with three models of TASSEL 5, single marker regression (SMR), general linear model (GLM) and mixed linear model (MLM), and the fixed and random model circulating probability unification (FarmCPU) of R package. The results showed that 13 and 9 markers were associated with fiber and sucrose contents, respectively. The GP was performed by cross-prediction with five models, ridge regression best linear unbiased prediction (rrBLUP), Bayesian ridge regression (BRR), Bayesian A (BA), Bayesian B (BB) and Bayesian least absolute shrinkage and selection operator (BL). The accuracy of GP varied from 55.8% to 58.9% for fiber content and 54.6% to 57.2% for sucrose content. Upon validation, these markers can be applied in MAS and genomic selection (GS) to select superior sugarcane with good fiber and high sucrose contents.

Synthetic apomixis with normal hybrid rice seed production

Synthetic apomixis with normal hybrid rice seed production

Effects of TaMTL-Edited Mutations on Grain Phenotype and Storage Component Composition in Wheat

Wheat nutrition and processing-quality are primarily based on the endosperm ingredients. However, the effect of embryos on grain traits and components remains unclear. In this study, we found that in the cross-pollinated and self-pollinated progenies of the four wheat mtl mutants (mtl-A, mtl-AD, mtl-BD, and mtl-ABD) the haploid induction rates were 0–15.6% and 0–14.1%, and the embryo abortion rates were 0–27.4% and 0–24.1%, respectively, in which mtl-A had no effect on haploid induction and embryo development. The embryoless grains (ELG) were comparable to the normal grains (NG) from mtl-AD, mtl-BD, and mtl-ABD in grain length, grain width and thousand-kernel weight, but the grain traits were significantly less than those in NG from mutant mtl-A. During grain filling period, mtl-ABD had similar ELG ratio and amount of starch granule (SG) and protein body (PB) in ELG and NG. At maturity stage, the morphological features of A-type and B-type SG in ELG were similar to those in NG in mtl mutants; however, amylose, gliadin, and glutenin contents were higher in ELG, and total starch, albumin and globulin contents were higher in NG. Our results clarified the effect of the wheat mtl mutants on haploid induction and grain traits and nutrition composition in this crop, and provided new clues for studying the development of embryo and endosperm and their interaction in plants.

Quantitative Trait Loci Analysis Based on High-Density Mapping of Single-Nucleotide Polymorphisms by Genotyping-by-Sequencing Against Pine Wilt Disease in Japanese Black Pine (Pinus thunbergii).

Identifying genes/loci for resistance to pine wilt disease (PWD) caused by the pine wood nematode (PWN) is beneficial for improving resistance breeding in Pinus thunbergii, but to date, genetic information using molecular markers has been limited. Here, we constructed a high-density linkage map using genotyping-by-sequencing (GBS) and conducted quantitative trait loci (QTL) analysis for PWD resistance for the self-pollinated progeny of “Namikata 73,” which is the most resistant variety among resistant varieties of P. thunbergii, following inoculation tests with PWN. An S1 mapping population consisting of the 116 progenies derived from self-pollination of the resistant variety, “Namikata 73” (resistance rank 5 to PWN), was inoculated with PWN isolate Ka-4 and evaluated for disease symptoms. To construct a high-density linkage map, we used single-nucleotide polymorphisms (SNPs) identified by GBS based on next-generation sequencing technology and some anchor DNA markers, expressed sequence tag (EST)-derived SNP markers and EST-derived simple sequence repeat (SSR) markers, and genomic SSR markers. The linkage map had 13 linkage groups (LGs) consisting of 2,365 markers including 2,243 GBS-SNP markers over a total map distance of 1968.4 centimorgans (cM). Results from QTL analysis using phenotype data and the linkage map indicated that PWD resistance is controlled by a single locus located on LG-3, as identified in a previous study. This locus showed overdominant genetic action in the present study. With the confirmation of PWD1 in two different mapping populations (present study and a previous study), the locus associated with this region is thought to be a good target for marker-assisted selection in P. thunbergii breeding programs in order to obtain high levels of resistance to PWD caused by PWN.

A Heterozygous Genotype-Dependent Branched-Spike Wheat and the Potential Genetic Mechanism Revealed by Transcriptome Sequencing.

Simple SummaryThis paper reported a novel type of branched spike wheat from a natural mutation event. The branched spike was controlled by a heterozygous genotype. The genetic patterns showed that gametophytic male sterility probably contributed to the heterozygous genotype responsible for the branched spike trait. Expressional levels and Wheat FRIZZY PANICLE (WFZP) sequencing between the mutant with the branched spike and the wild-type with the normal spike showed that WFZP was not the causal gene for the branched spike. Data from transcriptome sequencing indicated that carbohydrate metabolism might be involved in the formation of the branched spike trait.Wheat (Triticum aestivum L.) spike architecture is an important trait associated with spike development and grain yield. Here, we report a naturally occurring wheat mutant with branched spikelets (BSL) from its wild-type YD-16, which has a normal spike trait and confers a moderate level of resistance to wheat Fusarium head blight (FHB). The lateral meristems positioned at the basal parts of the rachis node of the BSL mutant develop into ramified spikelets characterized as multiple spikelets. The BSL mutant shows three to four-day longer growth period but less 1000-grain weight than the wild type, and it becomes highly susceptible to FHB infection, indicating that the locus controlling the BSL trait may have undergone an intensively artificial and/or natural selection in modern breeding process. The self-pollinated descendants of the lines with the BSL trait consistently segregated with an equal ratio of branched and normal spikelets (NSL) wheat, and homozygotes with the BSL trait could not be achieved even after nine cycles of self-pollination. Distinct segregation patterns both from the self-pollinated progenies of the BSL plants and from the reciprocal crosses between the BSL plants with their sister NSL plants suggested that gametophytic male sterility was probably associated with the heterozygosity for the BSL trait. Transcriptome sequencing of the RNA bulks contrasting in the two types of spike trait at the heading stage indicated that the genes on chromosome 2DS may be critical for the BSL trait formation since 329 out of 2540 differentially expressed genes (DEGs) were located on that chromosome, and most of them were down-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that carbohydrate metabolism may be involved in the BSL trait expression. This work provides valuable clues into understanding development and domestication of wheat spike as well as the association of the BSL trait with FHB susceptibility.

Investigation and screening of chrysanthemum resources to identify self-compatible mutants

• There are wide variations in self-compatibility among different chrysanthemum cultivars. • The pollen germination rates are not significantly related to self-compatibility. • A self-compatible chrysanthemum mutant 'Hongguan' is identifed. • One SRK , two SCRs , two Exo70A1s and one THL1 may be involved in self-compatibility of C. × morifolium . Chrysanthemum ( Chrysanthemum × morifolium ) is an important horticultural crop with great ornamental and economic value. Most chrysanthemum cultivars are self-incompatible, which limits the creation of pure chrysanthemum lines which are the material basis for breeding and theoretical research. Here, artificial self-pollination and natural cross-pollination were used to analyze the self-compatibility (SC) and cross-compatibility (CC) of 85 chrysanthemums cultivated by our research group. By conducting a phenotypic study, observing of pollen germination in vitro and in vivo and gene expression analyses, we explored the reasons for the self-compatibility of chrysanthemum and identified self-compatible mutants. It was found that there were wide variations in self-compatibility among different chrysanthemum cultivars and the SC index ranged from 0 to 5.64, which could classify the chrysanthemum cultivars into three types: self-compatibility (SC index ≥ 1), self-incompatibility (SI, 1 > SC index > 0) and high self-incompatibility (SC index = 0). Seven chrysanthemum cultivars with self-pollinated progenies (‘Hongguan’, K183−8, A22, B12, D87, D76 and S8) were selected, and the pollen germination rates in vitro and in vivo were not significantly related to SC. The pollen tube of ‘Hongguan’ could pass through its own stigma and grow toward the embryo sac along the style, and there was no obvious inbreeding depression in self-pollinated progenies of ‘Hongguan’. In addition, we found that one S -locus receptor kinase ( SRK ), two S -locus cysteine-rich protein ( SCR ), two exocyst complex component EXo70A1 ( Exo70A1 ) and one thioredoxin h-like 1 ( THL1 ) were important for regulating self-compatibility of C. × morifolium . Our results not only laid a material foundation for creating pure lines through continuous self-pollination and analyzing the regulation mechanism underlying SC variation but also provided the possibility for theoretical research on the important ornamental traits of chrysanthemums in the future.

Genome-Wide Duplication of Allotetraploid Brassica napus Produces Novel Characteristics and Extensive Ploidy Variation in Self-Pollinated Progeny.

Whole genome duplications (WGDs) have played a major role in angiosperm species evolution. Polyploid plants have undergone multiple cycles of ancient WGD events during their evolutionary history. However, little attention has been paid to the additional WGD of the existing allopolyploids. In this study, we explored the influences of additional WGD on the allopolyploid Brassica napus. Compared to tetraploid B. napus, octoploid B. napus (AAAACCCC, 2n = 8x =76) showed significant differences in phenotype, reproductive ability and the ploidy of self-pollinated progeny. Genome duplication also altered a key reproductive organ feature in B. napus, that is, increased the number of pollen apertures. Unlike autopolyploids produced from the diploid Brassica species, the octoploid B. napus produced from allotetraploid B. napus had a relatively stable meiotic process, high pollen viability and moderate fertility under self-pollination conditions, indicating that sub-genomic interactions may be important for the successful establishment of higher-order polyploids. Doubling the genome of B. napus provided us with an opportunity to gain insight into the flexibility of the Brassica genomes. The genome size of self-pollinated progeny of octoploid B. napus varied greatly, and was accompanied by extensive genomic instability, such as aneuploidy, mixed-ploidy and mitotic abnormality. The octoploid B. napus could go through any of genome reduction, equilibrium or expansion in the short-term, thus providing a novel karyotype library for the Brassica genus. Our results reveal the short-term evolutionary consequences of recurrent polyploidization events, and help to deepen our understanding of polyploid plant evolution.

Plant breeding evaluation of the red currant cultivar ‘Podarok Pobeditelyam’ and its inbred progeny

Background. Inbreeding, as a way to produce progenies from organisms in different degrees of kinship, is an important genetic and breeding technique used to improve various crops, including fruit and berry plants. Inbreeding has not been extensively used with red currant, so a study of the possibility to employ closely related crosses in breeding practice with this crop remains relevant. The purpose of this study was to assess the breeding potential of the red currant cultivar ‘Podarok Pobeditelyam’ by disclosing economic and biological characteristics in its inbred progeny.Methods and materials. The research was carried out at the red currant breeding site of the Russian Research Institute of Fruit Crop Breeding (VNIISPK, Orel) in 2016–2018. The seedlings obtained from the self-pollination of a new red currant cultivar, ‘Podarok Pobeditelyam’, were the objects of the study. Records and observations were carried out according to the program and methodology for breeding and variety studies of fruit, berry and nut crops.Results and conclusion. The percentage of long-cluster seedlings (cluster length > 10 cm) in the inbred progeny of cv. ‘Podarok Pobeditelyam’ was 21.8%; segregation of large-fruited seedlings (berry weight > 0.65 g) was observed at the level of 17%; the maximum degree of transgression (Tsmax) was 42%; and the incidence of transgressive seedlings was 37%. The analysis of the progeny showed that cv. ‘Podarok Pobeditelyam’ is a homozygote for the dominant Rc gene that controls the red color of the berries, and displays donor properties for the trait “dry detachment of berries”. The study of the cultivar’s self-pollinated progeny did not reveal any inbreeding depression in F1 seedlings from closely related crosses as far as plant condition or productivity is concerned. The selected seedlings are of interest for further variety research and use in breeding practice as new source material.

Characterization and map-based cloning of miniature2-m1, a gene controlling kernel size in maize

Kernel development plays an important role in determining kernel size in maize. Here we present the cloning and characterization of a maize gene, nitrate transporter1.5 (NRT1.5), which controls small kernel phenotype by playing an important role in kernel development. A novel recessive small kernel mutant miniature2-m1 (mn2-m1) was isolated from self-pollinated progenies of breeding materials. The mutant spontaneously showed small kernel character arresting both embryo and endosperm development at an early stage after pollination. Utilizing 21 polymorphic SSR markers, the mn2-m1 locus was limited to a 209.9-kb interval using 9 176 recessive individuals of a BC1 segregating population from mn2-m1/B73. Only one annotated gene was located in this 209.9 kb region, Zm00001d019294, which was predicted to encode nitrate transporter1.5 (NRT1.5). Allelism tests confirmed that mn2-m1 was allelic to miniature2-m2 (mn2-m2) and miniature2-710B (mn2-710B). The mn2-m1 and mn2-m2 alleles both had nucleotide deletions in the coding region resulting in premature termination, and the mn2-710B allele had some missence mutations. Subcellular localization showed that Miniature 2 (MN2) is localized in the plasma membrane. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of MN2 and some genes involved in the basal endosperm transfer layer (BETL) and embryo surrounding region (ESR) development were affected in mn2-m1 seeds. These results suggested that MN2 plays an important role in maize seed development.

Genetic engineering of eggplant accumulating β-carotene in fruit.

Genetic engineering of eggplant using fruit-specific EEF48 promoter-driven bacterial PSY gene, crtB, confers β-carotene accumulation in fruit. Eggplant (Solanum melongena L.) is globally cultivated especially in Asia and is an important source of nutrients in the diets of low-income consumers in developing countries. Since fruits of eggplant have low provitamin A carotenoid content, it is expected to develop eggplant with high carotenoid content for combatting vitamin A deficiency. To achieve this, the present study implemented a metabolic engineering strategy to modify the carotenoid biosynthetic pathway in eggplant. Expression analysis of carotenogenic genes in eggplant tissues showed that the expression of the endogenous phytoene synthase (PSY) was low in fruit and callus. Orange-colored calluses were generated from ectopic expression of crtB gene, which encodes bacterial PSY, in eggplant cells. The orange calluses accumulated > 20μgg-1FW of β-carotene, which was approximately 150-fold higher than that of the untransformed calluses. These observations suggest that the PSY expression is the rate-limiting step for β-carotene production in callus and fruit. Since the orange calluses did not regenerate plants, we chose eggplant EEF48 gene, which is presumably expressed in fruit. We amplified its promoter region by TAIL-PCR and showed that the EEF48 promoter is indeed active in eggplant fruit. Subsequently, transgenic eggplant lines having EEF48 promoter-driven crtB were produced. Among the transgenic lines produced, one line set fruit containing 1.50μgg-1FW of β-carotene, which was 30-fold higher than that of the untransformed fruits (0.05μgg-1FW). The self-pollinated progenies showed a 3:1 segregation ratio for the presence and absence of the transgene, which was linked to the β-carotene accumulation in fruit. These results provide a strategy for improvement of carotenoid content in eggplant fruit.

Development of chromosome-specific markers for a study on introgressive hybridization of potato with the wild Mexican allotetraploid species Solanum stoloniferum Schltdl

In order to involve valuable germplasm of the wild Mexican allotetraploid potato species Solanum stoloniferum Schltdl. (genomic composition ААВВ) into breeding, pentaploid interspecific hybrids (ААAAВ) with cultivated potato S. tuberosum L. (АААА) and their backcross progenies are usually used. Homologous synapsis in meiosis of such hybrids is expected only between chromosomes of the A subgenome, therefore a question arose about a possibility of introgressing genetic material of the subgenome B into the A genome of cultivated potato. In this connection, development of various schemes for the B subgenome introgression into the genome of cultivated potato is considered as a topical issue. The previous research has yielded four schemes of S. stoloniferum involvement into breeding, which imply backcrossing with cultivated potato of the following interspecific hybrids: (1) hexaploids (genomic composition ААААВВ, the conventional introgression scheme), (2) tetraploids (putatively, АААВ), (3) self-pollination progeny of a 4x hybrid and (4) pentaploid hybrids with a putative genome composition of АААВВ. The present paper presents the first results of the development of chromosome-specific DNA markers for the identification of S. stoloniferum chromosomes in interspecific hybrids. An S. stoloniferum accession PI 205522 with a high degree of resistance to late blight and PVY had been found to possess several DNA-markers of the R-genes conferring resistance to these pathogens and was used in hybridization as a promising parent. A set of 23 SSR- and CAPS markers with the known chromosome location in S. tuberosum was generated. These markers detect polymorphism between parent genotypes, i.e., the diploid clone IGC 10/1.21 of cultivated potatoes S. tuberosum, and accession PI 205522 of S. stoloniferum. All the markers specific for the wild species were found in triploid (ААВ) and pentaploid (АААВВ) hybrids of S. stoloniferum × S. tuberosum. This set of markers will be used for efficiency assessment of different schemes for S. stoloniferum genetic material introgression into the obtained BC2-BC3 generations after crossing the interspecific hybrids with cultivated potato.

Maximising recombination across macadamia populations to generate linkage maps for genome anchoring

The Proteaceae genus Macadamia has a recent history of domestication as a commercial nut crop. We aimed to establish the first sequence-based haploid-correlated reference genetic linkage maps for this primarily outcrossing perennial tree crop, with marker density suitable for genome anchoring. Four first generation populations were used to maximise the segregation patterns available within full-sib, biparental and self-pollinated progeny. This allowed us to combine segregation data from overlapping subsets of >4,000 informative sequence-tagged markers to increase the effective coverage of the karyotype represented by the recombinant crossover events detected. All maps had 14 linkage groups, corresponding to the Macadamia haploid chromosome number, and enabled the anchoring and orientation of sequence scaffolds to construct a pseudo-chromosomal genome assembly for macadamia. Comparison of individual maps indicated a high level of congruence, with minor discrepancies satisfactorily resolved within the integrated maps. The combined set of maps significantly improved marker density and the proportion (70%) of the genome sequence assembly anchored. Overall, increasing our understanding of the genetic landscape and genome for this nut crop represents a substantial advance in macadamia genetics and genomics. The set of maps, large number of sequence-based markers and the reconstructed genome provide a toolkit to underpin future breeding that should help to extend the macadamia industry as well as provide resources for the long term conservation of natural populations in eastern Australia of this unique genus.

Genetic dissection of thousand-seed weight and fine mapping of cqSW.A03-2 via linkage and association analysis in rapeseed (Brassica napus L.).

KEY MESSAGE: cqSW.A03-2, one of the six identified quantitative trait loci associated with thousand-seed weight in rapeseed, is mapped to a 61.6-kb region on chromosome A03 and corresponds to the candidate gene BnaA03G37960D. Seed weight is an important factor that determines the seed yield of oilseed rape (Brassica napus L.). To elucidate the genetic mechanism of thousand-seed weight (TSW), quantitative trait locus (QTL) mapping was conducted using a double haploid population derived from the cross between an elite line ZY50 and a pol cytoplasmic male sterility restorer line 7-5. The genetic basis of TSW was dissected into six major QTLs. One major QTL denoted as cqSW.A03-2, which explained 8.46-13.70% of the phenotypic variation, was detected across multiple environments. To uncover the genetic basis of cqSW.A03-2, a set of near-isogenic lines were developed. Based on the test of self-pollinated progenies, cqSW.A03-2 was identified as a single Mendelian factor and the ZY50 allele at cqSW.A03-2 showed a positive effect on TSW. Fine mapping delimited the cqSW.A03-2 locus into a 61.6-kb region, and 18 genes within this region were predicted. Candidate gene association analysis and expression analysis indicated that a histidine kinase gene (BnaA03G37960D) is likely to be the candidate gene for the cqSW.A03-2 locus. Our results may contribute to a better understanding of the molecular mechanism of seed weight regulation and promote the breeding program for yield improvement in rapeseed.

Bastard Cabbage (Rapistrum rugosum L.) Resistance to Tribenuron-Methyl and Iodosulfuron-Methyl-Sodium in Spain and Alternative Herbicides for Its Control

Complaints about the lack of control of Rapistrum rugosum with tribenuron-methyl and iodosulfuron-methyl-sodium in winter cereals in Northeastern Spain motivated this study. During 2015–2018, greenhouse trials were conducted to test the responses of two possibly resistant (R1 and R2) and two susceptible populations to both active ingredients to determine the response of these populations to alternative herbicides. In the first trial that was repeated twice, populations were treated with both active ingredients (three rates, six replicates), and the lack of control confirmed resistance both times. The second trial was conducted on the self-pollinated progeny of the initial populations (13 rates, 6 replicates) to confirm the heritable character of resistance and to determine the resistance factors related to survival and biomass. Resistance factors based on biomass were 188 and 253 for tribenuron-methyl and 42 and 26 for iodosulfuron-methyl-sodium for R1 and R2, respectively, confirming the strong resistance of the progeny. In the third trial, nine active ingredients (a.i.) registered for broadleaved weed control in winter cereals were tested on the four populations (two rates, four replicates). All the alternative herbicides, except florasulam, results in important phytotoxicity to all tested populations, with 100% efficacy for several a.i. This work is the first report of R. rugosum that is resistant to iodosulfuron-methyl-sodium and the first report in Europe of R. rugosum that is resistant to tribenuron-methyl.