Marker-assisted introgression to improve the oleic acid content in the TMV 7 groundnut (Arachis hypogaea L.) variety suitable for the oil industry.

BACKGROUND: Improving the quality and shelf life of groundnut oil is one of the foremost objectives of groundnut breeding programmes. This can be achieved by marker-assisted introgression, a technique that efficiently and precisely enables breeders to develop plants with enhanced qualities. This study focused on improving the oleic acid content of an elite groundnut variety, TMV 7, by introgressing a recessive mutation responsible for the increase in oleic acid from ICG 15419. Hybridization was performed between the donor and recurrent parents to develop the F1, BC1F1, BC2F1 and BC2F2 populations. Introgressed lines with increased oleic acid in the genetic background of TMV 7 were identified using allele-specific marker, F435-F, F435SUB-R and a set of SSR markers were employed to recover the genome of the recurrent parent. RESULTS: With two backcrosses, a total of ten homozygous plants in the BC2F2 population were identified with oleic acid content ranging from 54.23 to 57.72% causing an increase of 36% over the recurrent parent. Among the ten lines, the line IL-23 exhibited the highest level of recurrent parent genome recovery of 91.12%. CONCLUSIONS: The phenotypic evaluation of 10 homozygous introgressed lines indicated fewer differences for all other traits under study compared to the recurrent parent, except for oleic acid and linoleic acid content confirming the genetic background of the recurrent parent. The identified lines will be subjected to multilocation trials before their commercial release.

Comparison of phenotypic and genotypic frequency of phosphine resistance in select field populations of Tribolium castaneum from India.

BACKGROUND: Tribolium castaneum causes substantial damage to stored grains, leading to economic losses. The present study evaluates phosphine resistance in adult and larval stages of T. castaneum from north and northeast India, where continuous and long-term phosphine use in large-scale storage conditions intensifies resistance, posing risks to grain quality, safety, and industry profitability. METHODS AND RESULTS: This study utilized T. castaneum bioassays and CAPS markers restriction digestion methodology to assess resistance. The phenotypic results indicated a lower LC50 value in larvae compared to adults, while the resistance ratio remained consistent across both stages. Similarly, the genotypic analysis revealed comparable resistance levels regardless of the developmental stage. We categorized the freshly collected populations based on resistance ratios, with Shillong showing weak resistance, Delhi and Sonipat displaying moderate resistance, and Karnal, Hapur, Moga, and Patiala exhibiting strong resistance to phosphine. Further validation by accessing findings and exploring the relationship between phenotypic and genotypic variations using Principal Component Analysis (PCA). This comprehensive study enhances our understanding of T. castaneum resistance levels, providing valuable insights for the development of targeted pest management strategies. CONCLUSION: This study provides insights into the current phenotypic and genotypic resistance levels of T. castaneum in North and North East India. Understanding this is crucial for developing effective pest management strategies and future research on biological and physiological aspects of phosphine resistance in insects, enabling the formulation of effective management practices. Addressing phosphine resistance is vital for sustainable pest management and the long-term viability of the agricultural and food industries.

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the XfmrpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Loss of chromatin remodeler DDM1 causes segregation distortion in Arabidopsis thaliana.

MAIN CONCLUSION: In ddm1 mutants, the DNA methylation is primarily affected in the heterochromatic region of the chromosomes, which is associated with the segregation distortion of SNPs in the F2 progenies. Segregation distortion (SD) is common in most genetic mapping experiments and a valuable resource to determine how gene loci induce deviation. Meiotic DNA crossing over and SD are under the control of several types of epigenetic modifications. DNA methylation is an important regulatory epigenetic modification that is inherited across generations. In the present study, we investigated the relationship between SD and DNA methylation. The ecotypes Col-0/C24 and chromatin remodeler mutants ddm1-10/Col and ddm1-15/C24 were reciprocally crossed to obtain F2 generations. A total of 300 plants for each reciprocally crossed plant in the F2 generations were subjected to next-generation sequencing to detect the single-nucleotide polymorphisms (SNPs) as DNA markers. All SNPs were analyzed using the Chi-square test method to determine their segregation ratio in F2 generations. Through the segregation ratio, whole-genome SNPs were classified into 16 classes. In class 10, the SNPs in the reciprocal crosses of wild type showed the expected Mendelian ratio of 1:2:1, while those in the reciprocal crosses of ddm1 mutants showed distortion. In contrast, all SNPs in class 16 displayed a normal 1:2:1 ratio, and class 1 showed SD, regardless of wild type or mutants, as assessed using CAPS (cleaved amplified polymorphic sequences) marker analysis to confirm the next-generation sequencing. In ddm1 mutants, the DNA methylation is highly reduced throughout the whole genome and more significantly in the heterochromatic regions of chromosomes. Our results showed that the ddm1 mutants exhibit low levels of DNA methylation, which facilitates the SD of SNPs primarily located in the heterochromatic region of chromosomes by reducing the heterozygous ratio. The present study will provide a strong base for future research focusing on the impact of DNA methylation on trait segregation and plant evolution.

Genetic Mapping of ms1s, a Recessive Gene for Male Sterility in Common Wheat.

The utilization of heterosis is an important way to improve wheat yield, and the production of wheat hybrid seeds mainly relies on male-sterile lines. Male sterility in line 15 Fan 03 derived from a cross of 72,180 and Xiaoyan 6 is controlled by a single recessive gene. The gene was mapped to the distal region of chromosome 4BS in a genetic interval of 1.4 cM and physical distance of 6.57 Mb between SSR markers Ms4BS42 and Ms4BS199 using an F2 population with 1205 individuals. Sterile individuals had a deletion of 4.57 Mb in the region presumed to carry the Ms1 locus. The allele for sterility was therefore named ms1s. Three CAPS markers were developed and verified from the region upstream of the deleted fragment and can be used for ms1s marker-assisted selection in wheat hybrid breeding. This work will enrich the utilization of male sterility genetic resources.

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene.

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low ("00") cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new "00" varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5'-C↓TAG-3' sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

A simple molecular identification method of the Thrips tabaci (Thysanoptera: Thripidae) cryptic species complex.

The onion thrips (Thrips tabaci Lindeman, 1889) is a key pest of a wide range of crops because of its ecological attributes such as polyphagy, high reproduction rate, ability to transmit tospoviruses and resistance to insecticides. Recent studies revealed that T. tabaci is a cryptic species complex and it has three lineages (leek-associated arrhenotokous L1-biotype, leek-associated thelytokous L2-biotype and tobacco-associated arrhenotokous T-biotype), however, the adults remain indistinguishable. T. tabaci individuals were collected from different locations of Hungary to create laboratory colonies from each biotypes. Mitochondrial COI (mtCOI) region was sequenced from morphologically identified individuals. After sequence analysis SNPs were identified and used for CAPS marker development, which were suitable for distinguishing the three T. tabaci lineages. Genetic analysis of the T. tabaci species complex based on mtCOI gene confirmed the three well-known biotypes (L1, L2, T) and a new biotype because the new molecular evidence presented in this study suggests T-biotype of T. tabaci forming two distinct (sub)clades (T1 and T2). This genetic finding indicates that the genetic variability of T. tabaci populations is still not fully mapped. We validated our developed marker on thrips individuals from our thrips colonies. The results demonstrated that the new marker effectively identifies the different T. tabaci biotypes. We believe that our reliable genotyping method will be useful in further studies focusing on T. tabaci biotypes and in pest management by scanning the composition of sympatric T. tabaci populations.

Mikan Genome Database (MiGD): integrated database of genome annotation, genomic diversity, and CAPS marker information for mandarin molecular breeding.

Citrus species are some of the most valuable and widely consumed fruits globally. The genome sequences of representative citrus (e.g., Citrus clementina, C. sinensis, C. grandis) species have been released but the research base for mandarin molecular breeding is still poor. We assembled the genomes of Citrus unshiu and Poncirus trifoliata, two important species for citrus industry in Japan, using hybrid de novo assembly of Illumina and PacBio sequence data, and developed the Mikan Genome Database (MiGD). The assembled genome sizes of C. unshiu and P. trifoliata are 346 and 292 Mb, respectively, similar to those of citrus species in public databases; they are predicted to possess 41,489 and 34,333 protein-coding genes in their draft genome sequences, with 9,642 and 8,377 specific genes when compared to C. clementina, respectively. MiGD is an integrated database of genome annotation, genetic diversity, and Cleaved Amplified Polymorphic Sequence (CAPS) marker information, with these contents being mutually linked by genes. MiGD facilitates access to genome sequences of interest from previously reported linkage maps through CAPS markers and obtains polymorphism information through the multiple genome browser TASUKE. The genomic resources in MiGD (https://mikan.dna.affrc.go.jp) could provide valuable information for mandarin molecular breeding in Japan.

Development of new SNP derived cleaved amplified polymorphic sequence marker set and its successful utilization in the genetic analysis of seed color variation in barley.

The aim of the present study was to develop a new cost effective PCR based CAPS marker set using advantages of high-throughput SNP genotyping. Initially, SNP survey was made using 20 diverse barley genotypes via 9k iSelect array genotyping that resulted in 6334 polymorphic SNP markers. Principle component analysis using this marker data showed fine differentiation of barley diverse gene pool. Till this end, we developed 200 SNP derived CAPS markers distributed across the genome covering around 991cM with an average marker density of 5.09cM. Further, we genotyped 68 CAPS markers in an F2 population (Cheri×ICB181160) segregating for seed color variation in barley. Genetic mapping of seed color revealed putative linkage of single nuclear gene on chromosome 1H. These findings showed the proof of concept for the development and utility of a newer cost effective genomic tool kit to analyze broader genetic resources of barley worldwide.

Development of genome-wide PCR-based markers from insertion, deletion and single nucleotide polymorphisms for closely related Japanese rice cultivars and identification of QTLs for the appearance of cooked rice and polished rice.

Appearance of rice grain is an important property, affecting its acceptance by consumers. Moreover, appearance is a complex characteristic involving many components, including glossiness and whiteness. The genetic bases for the glossiness of cooked rice and the whiteness of polished rice (WPR) were determined using 133 recombinant inbred lines (RILs) derived from a cross between two closely related cultivars from Hokkaido, Joiku462, with high glossiness and whiteness, and Yukihikari, an ancestor of Joiku462 with low glossiness and whiteness. Analyses identified 167 genome-wide InDel markers, five cleaved amplified polymorphic sequences (CAPS) and eight derived CAPS markers differentiating the parental lines. The glossiness area (GLA) and glossiness strength (GLS) of cooked rice and WPR were determined for RILs in two locations, Pippu and Sapporo, Hokkaido. Four QTLs were detected. qGLA10 and qGLS9 were detected on chromosomes 10 and 9, respectively, with both being significant at both geographic locations. qWPR1 on chromosome 1 was significant at Pippu, and qWPR4 on chromosome 4 was significant at Sapporo. The Joiku462 alleles at all QTLs increased each trait. The PCR-based markers flanking these four QTLs may be useful for improvement of GLA, GLS and WPR.

Allelic expression of AhNSP2-B07 due to parent of origin affects peanut nodulation.

Legumes are well-known for establishing a symbiotic relationship with rhizobia in root nodules to fix nitrogen from the atmosphere. The nodulation signaling pathway 2 (NSP2) gene plays a critical role in the symbiotic signaling pathway. In cultivated peanut, an allotetraploid (2n = 4x = 40, AABB) legume crop, natural polymorphisms in a pair of NSP2 homoeologs (Na and Nb) located on chromosomes A08 and B07, respectively, can cause loss of nodulation. Interestingly, some heterozygous (NBnb) progeny produced nodules, while some others do not, suggesting non-Mendelian inheritance in the segregating population at the Nb locus. In this study, we investigated the non-Mendelian inheritance at the NB locus. Selfing populations were developed to validate the genotypical and phenotypical segregating ratios. Allelic expression was detected in roots, ovaries, and pollens of heterozygous plants. Bisulfite PCR and sequencing of the Nb gene in gametic tissue were performed to detect the DNA methylation variations of this gene in different gametic tissues. The results showed that only one allele at the Nb locus expressed in peanut roots during symbiosis. In the heterozygous (Nbnb) plants, if dominant allele expressed, the plants produced nodules, if recessive allele expressed, then no nodules were produced. qRT-PCR experiments revealed that the expression of Nb gene in the ovary was extremely low, about seven times lower than that in pollen, regardless of genotypes or phenotypes of the plants at this locus. The results indicated that Nb gene expression in peanut depends on the parent of origin and is imprinted in female gametes. However, no significant differences of DNA methylation level were detected between these two gametic tissues by bisulfite PCR and sequencing. The results suggested that the remarkable low expression of Nb in female gametes may not be caused by DNA methylation. This study provided a unique genetic basis of a key gene involved in peanut symbiosis, which could facilitate understanding the regulation of gene expression in symbiosis in polyploid legumes.

A rapid method for detection of the root-knot nematode resistance gene, Mi-1.2, in tomato cultivars.

Molecular markers have been widely used in plant breeding to improve the accuracy and efficiency of trait selection. In particular, molecular markers are powerful in facilitating the introgression of resistance genes by circumventing costly and time-consuming infection assays. To achieve their practical use, it is important to ensure the tight linkage between the markers and the traits. Here we report a new cleaved amplified polymorphic sequence (CAPS) marker, Mi1713, for the root-knot nematode resistance gene, Mi-1.2, in cultivated tomato. The Mi1713 marker is designed in the conserved region of Mi-1.2 and its homologs in tomato and other nightshade species. Combined with a single-step procedure for preparing PCR templates, the Mi1713 marker enables rapid and reliable screening for the presence of Mi-1.2. The approach described in this study is applicable in designing CAPS markers for various genes or alleles of interest in tomato and other crops.

Intergeneric hybridization of marguerite (Argyranthemum frutescens (L.) Sch. Bip.) and Roman chamomile (Chamaemelum nobile (L.) All.) using ovule culture and confirmation of hybridity.

To introduce useful characteristics such as fragrance into Argyranthemum frutescens (L.) and to expand the variation, we conducted crosses using A. frutescens as the seed parent and Chamaemelum nobile (L.) All. as the pollen parent. All the tested cross combinations between the three strains of A. frutescens and one strain of C. nobile produced embryos, and healthy plants were obtained by ovule culture. The obtained plantlets had a white ray floret, and the leaf shape was intermediate to those of the parents. The individuals obtained from this cross were subjected to two methods to determine hybridity: flow cytometry analyses and cleaved amplified polymorphic sequence (CAPS) markers. For the CAPS marker, we selected the internal transcribed spacer (ITS) region, which is highly variable among the genera, as the region to be amplified. We selected restriction enzymes BmgT120 I and Afl II, which selectively cut common sequences in the genus Argyranthemum, based on the sequence analysis of one parent strain each of A. frutescens and C. nobile and alignment with known sequences of related species. Flow cytometry analyses and CAPS markers revealed that the individuals obtained from the cross between A. frutescens and C. nobile are intergeneric hybrids. In addition, these established methods were capable of quickly and reliably identifying hybrids between A. frutescens and C. nobile. This report shows for the first time that crossbreeding between A. frutescens (seed parent) and C. nobile (pollen parent) is possible, and further development of Argyranthemum breeding, such as the expansion of variation by intergeneric crosses, is expected.

Experimental Outcrossing in Agaricus bisporus Revealed a Major and Unexpected Involvement of Airborne Mycelium Fragments.

Agaricus bisporus var. bisporus, the button mushroom, has a predominantly pseudohomothallic life cycle. Most of its spores are heterokaryotic and give rise to fertile heterokaryons. However, previous studies have suggested that outcrossing should not be rare in wild populations. In order to discover how outcrossing occurs, we experimentally favored it between aerial propagules of a fruiting donor mycelium and a delayed receiver mycelium that only invaded culture trays. Two donor/receiver pairs were studied, and potentially hybrid basidiomata collected on the receiver trays were analyzed with a mitochondrial marker, two unlinked nuclear CAPS markers, then haplotype markers based on DNA sequences obtained after PCR cloning of the rDNA ITS region and the fruk gene. For one of the two pairs, most basidiomata were hybrids between the donor and the receiver. Genotyping of the hybrids revealed only two genotypes consistent with outcrossing involving airborne mycelium fragments rather than basidiospores. The resident receiver heterokaryon that provided its mitochondria to the hybrid basidiomata is suspected to have had a trophic contribution to their growth and successful fruiting. The high level of heterozygosity and the cultivar introgression previously revealed in wild populations of this pseudohomothallic species may result from outcrossing involving airborne pieces of mycelium.

Identification of the gene coding for seed cotyledon albumin SCA in the pea (Pisum L.) genome.

Albumins SCA and SAA are short, highly hydrophilic proteins accumulated in large quantities in the cotyledons and seed axes, respectively, of a dry pea (Pisum sativum L.) seed. SCA was earlier shown to have two allelic variants differing in mobility in polyacrylamide gel electrophoresis in acid medium. Using them, the corresponding gene SCA was mapped on Linkage Group V. This protein was used as a useful genetic and phylogeographical marker, which still required electrophoretic analysis of the protein while the DNA sequence of the corresponding SCA gene remained unknown. Based on the length, the positive charge under acidic conditions and the number of lysine residues of SCA and SAA albumins, estimated earlier electrophoretically, the data available in public databases were searched for candidates for the SCA gene among coding sequences residing in the region of the pea genome which, taking into account the synteny of the pea and Medicago truncatula genomes, corresponds to the map position of SCA. Then we sequenced them in a number of pea accessions. Concordance of the earlier electrophoretic data and sequence variation indicated the sequence Psat0s797g0160 of the reference pea genome to be the SCA gene. The sequence Psat0s797g0240 could encode a minor related albumin SA-a2, while a candidate gene for albumin SAA is still missing (as well as electrophoretic variation of both latter albumins). DNA amplif ication using original primers SCA1_3f and SCA1_3r from genomic DNA and restriction by endonuclease HindII made it possible to distinguish the SCA alleles coding for protein products with different charges without sequencing the gene. Thus, the gene encoding the highly hydrophilic albumin SCA accumulated in pea seeds, the alleles of which are useful for classif ication of pea wild relatives, has now been identif ied in the pea genome and a convenient CAPS marker has been developed on its basis.

Genome-wide analysis of the serine carboxypeptidase-like (SCPL) proteins in Brassica napus L.

The serine carboxypeptidase-like protein (SCPL) family plays a key part in plant growth, development and stress responses. However, the serine carboxypeptidase-like (SCPL) proteins in Brassica napus L. (B. napus) have not been reported yet. Here, we identified a total of 117 putative SCPL genes in B. napus, which were unevenly distributed on all 19 chromosomes and were divided into three groups (carboxypeptidase Ⅰ to Ⅲ) according to their phylogenetic relationships. Synteny and duplication analysis revealed that the SCPL gene family of B. napus was amplified during allopolyploidization, in which the whole genome triplication and dispersed duplication played critical roles. After the separation of Brassica and Arabidopsis lineages, orthologous gene analysis showed that many SCPL genes were lost during the evolutionary process in B. rapa, B. oleracea and B. napus. Subsequently, the analyses of the gene structure, conserved motifs, cis-element and expression patterns showed that the members in the same group were highly conserved. Furthermore, candidate gene based association study suggested the role of BnSCPL52 in controlling seed number per silique, seed weight and silique length and a CAPS marker was developed to distinguish different haplotypes. Our results provide an overview of rapeseed SCPL genes that enable us for further functional research and benefit the marker-assisted breeding in Brassica napus.

Development of CAPS Markers for Evaluation of Genetic Diversity and Population Structure in the Germplasm of Button Mushroom (Agaricus bisporus).

Agaricus bisporus is a globally cultivated mushroom with high economic value. Despite its widespread cultivation, commercial button mushroom strains have little genetic diversity and discrimination of strains for identification and breeding purposes is challenging. Molecular markers suitable for diversity analyses of germplasms with similar genotypes and discrimination between accessions are needed to support the development of new varieties. To develop cleaved amplified polymorphic sequences (CAPs) markers, single nucleotide polymorphism (SNP) mining was performed based on the A. bisporus genome and resequencing data. A total of 70 sets of CAPs markers were developed and applied to 41 A. bisporus accessions for diversity, multivariate, and population structure analyses. Of the 70 SNPs, 62.85% (44/70) were transitions (G/A or C/T) and 37.15% (26/70) were transversions (A/C, A/T, C/G, or G/T). The number of alleles per locus was 1 or 2 (average = 1.9), and expected heterozygosity and gene diversity were 0.0-0.499 (mean = 0.265) and 0.0-0.9367 (mean = 0.3599), respectively. Multivariate and cluster analyses of accessions produced similar groups, with F-statistic values of 0.134 and 0.153 for distance-based and model-based groups, respectively. A minimum set of 10 markers optimized for accession identification were selected based on high index of genetic diversity (GD, range 0.299-0.499) and major allele frequency (MAF, range 0.524-0.817). The CAPS markers can be used to evaluate genetic diversity and population structure and will facilitate the management of emerging genetic resources.

Gametophytic self-incompatibility in Andean capuli (Prunus serotina subsp. capuli): allelic diversity at the S-RNase locus influences normal pollen-tube formation during fertilization.

Capuli (Prunus serotina subsp. capuli) is a tree species that is widely distributed in the northern Andes. In Prunus, fruit set and productivity appears to be limited by gametophytic self-incompatibility (GSI) which is controlled by the S-Locus. For the first time, this research reveals the molecular structure of the capuli S-RNase (a proxy for S-Locus diversity) and documents how S-Locus diversity influences GSI in the species. To this end, the capuli S-RNase gene was amplified and sequenced in order to design a CAPS (Cleaved Amplified Polymorphic Sequence) marker system that could unequivocally detect S-alleles by targeting the highly polymorphic C2-C3 S-RNase intra-genic region. The devised system proved highly effective. When used to assess S-Locus diversity in 15 P. serotina accessions, it could identify 18 S-alleles; 7 more than when using standard methodologies for the identification of S-alleles in Prunus species. CAPS marker information was subsequently used to formulate experimental crosses between compatible and incompatible individuals (as defined by their S-allelic identity). Crosses between heterozygote individuals with contrasting S-alleles resulted in normal pollen tube formation and growth. In crosses between individuals with exactly similar S-allele identities, pollen tubes often showed morphological alterations and arrested development, but for some (suspected) incompatible crosses, pollen tubes could reach the ovary. The latter indicates the possibility of a genotype-specific breakdown of GSI in the species. Overall, this supports the notion that S-Locus diversity influences the reproductive patterns of Andean capuli and that it should be considered in the design of orchards and the production of basic propagation materials.

Mitochondrial Genome of Fagus sylvatica L. as a Source for Taxonomic Marker Development in the Fagales.

European beech, Fagus sylvatica L., is one of the most important and widespread deciduous tree species in Central Europe and is widely managed for its hard wood. The complete DNA sequence of the mitochondrial genome of Fagus sylvatica L. was assembled and annotated based on Illumina MiSeq reads and validated using long reads from nanopore MinION sequencing. The genome assembled into a single DNA sequence of 504,715 bp in length containing 58 genes with predicted function, including 35 protein-coding, 20 tRNA and three rRNA genes. Additionally, 23 putative protein-coding genes were predicted supported by RNA-Seq data. Aiming at the development of taxon-specific mitochondrial genetic markers, the tool SNPtax was developed and applied to select genic SNPs potentially specific for different taxa within the Fagales. Further validation of a small SNP set resulted in the development of four CAPS markers specific for Fagus, Fagaceae, or Fagales, respectively, when considering over 100 individuals from a total of 69 species of deciduous trees and conifers from up to 15 families included in the marker validation. The CAPS marker set is suitable to identify the genus Fagus in DNA samples from tree tissues or wood products, including wood composite products.

Analysis of genetic diversity and population structure using SSR markers and validation of a Cleavage Amplified Polymorphic Sequences (CAPS) marker involving the sodium transporter OsHKT1;5 in saline tolerant rice (Oryza sativa L.) landraces.

Naturally evolved saline tolerant rice landraces found along the coastline of India are a valuable genomic resource to explore the complex, polygenic nature of salinity tolerance. In the present study, a set of 28 genome wide SSR markers, 11 salt responsive genic SSR markers and 8 Saltol QTL linked SSR markers were used to estimate genetic relatedness and population structure within a collection of 47 rice landraces (including a tolerant and 2 sensitive checks) originating from geographically divergent coastal regions of India. All three marker types identified substantial genetic variation among the landraces, as evident from their higher PIC values (0.53 for genomic SSRs, 0.43 for Genic SSRs and 0.59 for Saltol SSRs). The markers RM431, RM484 (Genomic SSRs), OsCAX (D), OsCAX (T) (Genic SSRs) and RM562 (Saltol SSR) were identified as good candidates to be used in breeding programs for improving salinity tolerance in rice. STRUCTURE analysis divided the landraces into five distinct populations, with classification correlating with their geographical locations. Principal coordinate and hierarchical cluster analyses (UPGMA and neighbor joining) are in close agreement with STRUCTURE results. AMOVA analysis indicated a higher magnitude of genetic differentiation within individuals of groups (58%), than among groups (42%). We also report the development and validation of a new Cleavage Amplified Polymorphic Sequence (CAPS) marker (OsHKT1;5V395) that targets a codon in the sodium transporter gene OsHKT1;5 (Saltol/SKC1 locus) that is associated with sodium transport rates in the above rice landraces. The CAPS marker was found to be present in all landraces except in IR29, Kamini, Gheus, Matla 1 and Matla 2. Significant molecular genetic diversity established among the analyzed salt tolerant rice landraces will aid in future association mapping; the CAPS marker, OsHKT1;5V395 can be used to map rice landraces for the presence of the SNP (Single Nucleotide Polymorphism) associated with increased sodium transport rates and concomitant salinity tolerance in rice.