Identification of accession-specific variants and development of KASP markers for assessing the genetic makeup of Brassica rapa seeds.

BACKGROUND: Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed purity has been assessed with a grow-out test (GOT) in the field, a time consuming and costly venture. Early in the last decade, seed testing with molecular markers was introduced as a replacement for GOT, and Kompetitive allele specific PCR (KASP) markers were recognized as promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and applicable to only a small number of accessions or varieties due to the limited genetic information and reference genomes available. RESULTS: We identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. From these, we identified 2,925 SNPs as accession-specific, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by the Next Generation Sequencing (NGS) analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguised individuals from the mixed population including 50 target accessions from B. rapa core collection and the outgroup. Additionally, the marker set we developed here discriminated F1 hybrids and their parental lines with distinct clusters. CONCLUSIONS: This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).

Rice Seed Purity Identification Technology Using Hyperspectral Image with LASSO Logistic Regression Model.

Hyperspectral technology is used to obtain spectral and spatial information of samples simultaneously and demonstrates significant potential for use in seed purity identification. However, it has certain limitations, such as high acquisition cost and massive redundant information. This study integrates the advantages of the sparse feature of the least absolute shrinkage and selection operator (LASSO) algorithm and the classification feature of the logistic regression model (LRM). We propose a hyperspectral rice seed purity identification method based on the LASSO logistic regression model (LLRM). The feasibility of using LLRM for the selection of feature wavelength bands and seed purity identification are discussed using four types of rice seeds as research objects. The results of 13 different adulteration cases revealed that the value of the regularisation parameter was different in each case. The recognition accuracy of LLRM and average recognition accuracy were 91.67-100% and 98.47%, respectively. Furthermore, the recognition accuracy of full-band LRM was 71.60-100%. However, the average recognition accuracy was merely 89.63%. These results indicate that LLRM can select the feature wavelength bands stably and improve the recognition accuracy of rice seeds, demonstrating the feasibility of developing a hyperspectral technology with LLRM for seed purity identification.

Ex-post assessment of genetically modified, low level presence in Canadian flax.

Canada is the world's largest producer and exporter of flaxseed. In 2009, DNA from deregistered genetically modified (GM) CDC Triffid was detected in a shipment of Canadian flaxseed exported to Europe, causing a large decrease in the amount of flax planted in Canada and a major shift in export markets. The flax industry in Canada undertook major changes to ensure the removal of transgenic flax from the supply chain. To demonstrate compliance, Canada adopted a protocol involving testing grain samples (post-harvest) using an RT-PCR test for the construct found in CDC Triffid. Efforts to remove the presence of GM flax from the value chain included reconstituting major flax varieties from GM-free plants. The reconstituted varieties represented the majority of planting seed in 2014. This study re-evaluates GM flax presence in Canadian grain stocks for an updated dataset (2009-2015) using a previously described simulation model to estimate low-level GM presence. Additionally, losses to the Canadian economy resulting from the reduction in flax production and export opportunities, costs associated with reconstituting major flax varieties, and testing for the presence of GM flax along the flax value chain are estimated.

A novel method for identifying rice seed purity using hybrid machine learning algorithms.

In the grain industry, identifying seed purity is a crucial task because it is an important factor in evaluating seed quality. For rice seeds, this attribute enables the minimization of unexpected influences of other varieties on rice yield, nutrient composition, and price. However, in practice, they are often mixed with seeds from other varieties. This study proposes a novel method for automatically identifying the purity of a specific rice variety using hybrid machine learning algorithms. The core concept involves leveraging deep learning architectures to extract pertinent features from raw data, followed by the application of machine learning algorithms for classification. Several experiments are conducted to evaluate the performance of the proposed model through practical implementation. The results demonstrate that the novel method substantially outperformed the existing methods, demonstrating the potential for effective rice seed purity identification systems.

A Set of Molecular Markers to Accelerate Breeding and Determine Seed Purity of CMS Three-Line Hybrids in Brassica napus.

Cytoplasmic male sterility (CMS) is the main mechanism employed to utilize the heterosis of Brassica napus. CMS three-line rapeseed hybrids have dramatically enhanced yield and brought about the global revolution of hybrid varieties, replacing conventional crop varieties. Over the last half century, China has led the development of hybrid Brassica napus varieties. Two sterile lines, polima (pol) and shaan 2A, were of particular importance for the establishment of three-line hybrid systems in rapeseed, which has opened up a new era of heterosis utilization. However, in current breeding practices, it takes up to three years to identify the restorer or maintainer relationship and the cytoplasmic type of any inbred material. This greatly affects the breeding speed of new varieties and inhibits the rapid development of the rapeseed industry. To address this problem, we developed a set of molecular markers for the identification of fertile cytoplasmic gene N and sterile cytoplasmic gene S, as well as for the fertile nucleus gene R and sterile nucleus gene r, based on differences in the gene sequences between the CMS line, maintainer line and restorer line of Brassica napus. Combining these markers can accurately identify the CMS line, maintainer and restorer of both the pol and shaan systems, as well as their hybrids. These markers can not only be used to identify of the maintainer and restorer relationship of inbred materials; they can also be used as general molecular markers to identify the CMS-type hybrid purity of pol and shaan systems.

Simple, fast and inexpensive hot sodium hydroxide and tris DNA extraction method for genotyping tomato and melon seeds.

Seed commerce is a highly profitable global market. Most commercialized seeds are hybrid seeds originating from a controlled cross between two selected parental lines. The market value of hybrid seeds depends on their hybrid genetic purity. DNA molecular markers are a reliable and widespread tool to genotype plant materials; however, DNA extraction from seeds is challenging, often laborious and expensive. With the ultimate goal of creating a tomato and melon hybrid seeds purity test, various challenges arise. To overcome these problems and with the purpose of crude DNA extraction, a simple, fast, inexpensive and easily scalable adaptation of the hot sodium hydroxide and tris method coupled to a competitive allele-specific PCR genotyping method is proposed.

Detection of seed purity of hybrid wheat using reflectance and transmittance hyperspectral imaging technology.

Chemical hybridization and genic male sterility systems are two main methods of hybrid wheat production; however, complete sterility of female wheat plants cannot be guaranteed owing to the influence of the growth stage and weather. Consequently, hybrid wheat seeds are inevitably mixed with few parent seeds, especially female seeds. Therefore, seed purity is a key factor in the popularization of hybrid wheat. However, traditional seed purity detection and variety identification methods are time-consuming, laborious, and destructive. Therefore, to establish a non-destructive classification method for hybrid and female parent seeds, three hybrid wheat varieties (Jingmai 9, Jingmai 11, and Jingmai 183) and their parent seeds were sampled. The transmittance and reflectance spectra of all seeds were collected via hyperspectral imaging technology, and a classification model was established using partial least squares-discriminant analysis (PLS-DA) combined with various preprocessing methods. The transmittance spectrum significantly improved the classification of hybrids and female parents compared to that obtained using reflectance spectrum. Specifically, using transmittance spectrum combined with a characteristic wavelength-screening algorithm, the Detrend-CARS-PLS-DA model was established, and the accuracy rates in the testing sets of Jingmai 9, Jingmai 11, and Jingmai 183 were 95.69%, 98.25%, and 97.25%, respectively. In conclusion, transmittance hyperspectral imaging combined with a machine learning algorithm can effectively distinguish female parent seeds from hybrid seeds. These results provide a reference for rapid seed purity detection in the hybrid production process. Owing to the non-destructive and rapid nature of hyperspectral imaging, the detection of hybrid wheat seed purity can be improved by online sorting in the future.

The xantha Marker Trait Is Associated with Altered Tetrapyrrole Biosynthesis and Deregulated Transcription of PhANGs in Rice.

The xantha marker trait, which is controlled by a down-regulating epi-mutation of OsGUN4, has been applied to the production of hybrid rice. However, the molecular basis for the ability of xantha mutants to attain high photosynthetic capacity even with decreased chlorophyll contents has not been characterized. In the present study, we observed that the total chlorophyll content of the xantha mutant was only 27.2% of that of the wild-type (WT) plants. However, the xantha mutant still accumulated 59.9% of the WT δ-aminolevulinic acid content, 72.8% of the WT Mg-protoporphyrin IX content, and 63.0% of the WT protochlorophyllide a content. Additionally, the protoporphyrin IX and heme contents in the mutant increased to 155.0 and 160.0%, respectively, of the WT levels. A search for homologs resulted in the identification of 124 rice genes involved in tetrapyrrole biosynthesis and photosynthesis. With the exception of OsGUN4, OsHO-1, and OsHO-2, the expression levels of the genes involved in tetrapyrrole biosynthesis were significantly higher in the xantha mutant than in the WT plants, as were all 72 photosynthesis-associated nuclear genes. In contrast, there were no differences between the xantha mutant and WT plants regarding the expression of all 22 photosynthesis-associated chloroplast genes. Furthermore, the abundance of 1O2 and the expression levels of 1O2-related genes were lower in the xantha mutant than in the WT plants, indicating 1O2-mediated retrograde signaling was repressed in the mutant plants. These results suggested that the abundance of protoporphyrin IX used for chlorophyll synthesis decreased in the mutant, which ultimately decreased the amount of chlorophyll in the xantha mutant. Additionally, the up-regulated expression of photosynthesis-associated nuclear genes enabled the mutant to attain a high photosynthetic capacity. Our findings confirm that OsGUN4 plays an important role in tetrapyrrole biosynthesis and photosynthesis in rice. GUN4, chlorophyll synthesis pathways, and photosynthetic activities are highly conserved in plants and hence, novel traits (e.g., xantha marker trait) may be generated in other cereal crops by modifying the GUN4 gene.

Putative Microsatellite DNA Marker-Based Wheat Genomic Resource for Varietal Improvement and Management.

Wheat fulfills 20% of global caloric requirement. World needs 60% more wheat for 9 billion population by 2050 but climate change with increasing temperature is projected to affect wheat productivity adversely. Trait improvement and management of wheat germplasm requires genomic resource. Simple Sequence Repeats (SSRs) being highly polymorphic and ubiquitously distributed in the genome, can be a marker of choice but there is no structured marker database with options to generate primer pairs for genotyping on desired chromosome/physical location. Previously associated markers with different wheat trait are also not available in any database. Limitations of in vitro SSR discovery can be overcome by genome-wide in silico mining of SSR. Triticum aestivum SSR database (TaSSRDb) is an integrated online database with three-tier architecture, developed using PHP and MySQL and accessible at http://webtom.cabgrid.res.in/wheatssr/. For genotyping, Primer3 standalone code computes primers on user request. Chromosome-wise SSR calling for all the three sub genomes along with choice of motif types is provided in addition to the primer generation for desired marker. We report here a database of highest number of SSRs (476,169) from complex, hexaploid wheat genome (~17 GB) along with previously reported 268 SSR markers associated with 11 traits. Highest (116.93 SSRs/Mb) and lowest (74.57 SSRs/Mb) SSR densities were found on 2D and 3A chromosome, respectively. To obtain homozygous locus, e-PCR was done. Such 30 loci were randomly selected for PCR validation in panel of 18 wheat Advance Varietal Trial (AVT) lines. TaSSRDb can be a valuable genomic resource tool for linkage mapping, gene/QTL (Quantitative trait locus) discovery, diversity analysis, traceability and variety identification. Varietal specific profiling and differentiation can supplement DUS (Distinctiveness, Uniformity, and Stability) testing, EDV (Essentially Derived Variety)/IV (Initial Variety) disputes, seed purity and hybrid wheat testing. All these are required in germplasm management as well as also in the endeavor of wheat productivity.

Single-seed PCR-RFLP analysis for the identification of S haplotypes in commercial F1 hybrid cultivars of broccoli and cabbage.

Several simple methods of DNA preparation from plant tissues were evaluated for PCR-RFLP analyses of SLG and SRK alleles, which can be used for the identification of S haplotypes of breeding lines in broccoli and cabbage (Brassica oleracea L.) and in purity tests of F1 hybrid seeds. On the five methods tested, the NaI method was found to be the most suitable for the amplification of the SLG and SRK alleles. This method enables the use of a single seed as testing material. Using this method, we identified S haplotypes of 31 broccoli and 31 cabbage cultivars. Ninety-four percent of the cultivars of broccoli and 97% of those of cabbage were-single cross F1 hybrids. Nine and 15 S haplotypes were found in broccoli and cabbage, respectively. The small number of S haplotypes in broccoli suggests the importance of incorporating new S haplotypes in the breeding program.

Ospapst1, a useful mutant for identifying seed purity and authenticity in hybrid rice.

The stability and completeness of male sterility is still a challenge in some male sterile rice lines, especially those of photoperiod/thermo-sensitive genic male sterility (P/TGMS). Leaf color marker is a widely practiced approach to reduce the impact of self-pollinated seeds of male sterile lines. The papst1 is a leaf color mutant. The newly emerged leaves of papst1 are chlorosis and have an impaired photosynthesis. But the other agronomic traits, such as germination rate, duration of maturation and seed weight, are not changed. The papst1/PAPST1 F1 showed the wild-type leaf phenotype. The papst1/PAPST1 F2 progenies displayed an approximately 3:1 segregation ratio of WT phenotype:mutant phenotype (72: 28, χ(2) = 0.48, p > 0.05), suggesting that papst1 mutant phenotype is caused by a single repressive gene. Map-based cloning and sequencing analysis revealed that a point mutation was occurred in Os01 g16040 (OsPAPST1). Given these results, the Ospapst1 mutant is a useful mutant for identifying seed purity and authenticity in hybrid rice.

SSR genetic diversity assessment of popular pigeonpea varieties in Malawi reveals unique fingerprints

Background: Pigeonpea (Cajanus cajan (L.) Millsp.) is a drought tolerant legume of the Fabaceae family and the only cultivated species in the genus Cajanus. It is mainly cultivated in the semi-arid tropics of Asia and Oceania, Africa and America. In Malawi, it is grown as a source of food and income and for soil improvement in intercropping systems. However, varietal contamination due to natural outcrossing causes significant quality reduction and yield losses. In this study, 48 polymorphic SSR markers were used to assess the diversity among all pigeonpea varieties cultivated in Malawi to determine if a genetic fingerprint could be identified to distinguish the popular varieties. Results: A total of 212 alleles were observed with an average of 5.58 alleles per marker and a maximum of 14 alleles produced by CCttc019 (Marker 40). Polymorphic information content (PIC), ranged from 0.03 to 0.89 with an average of 0.30. A neighbor-joining tree produced 4 clusters. The most commonly cultivated varieties, which include released varieties and cultivated land races, were well-spread across all the clusters observed, indicating that they generally represented the genetic diversity available in Malawi, although substantial variation was evident that can still be exploited through further breeding. Conclusion: Screening of the allelic data associated with the five most popular cultivated varieties, revealed 6 markers - CCB1, CCB7, Ccac035, CCttc003, Ccac026 and CCttc019 - which displayed unique allelic profiles for each of the five varieties. This genetic fingerprint can potentially be applied for seed certification to confirm the genetic purity of seeds that are delivered to Malawi farmers.