Identification of Accession-Specific Variants and Development of KASP Markers for Assessing the Genetic Makeup of Crop 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 testing for purity was done with a grow-out test (GOT) in the field, but these tests are time consuming and costly. Seed testing with molecular markers was introduced as a replacement for GOT early in the last decade. Recently, Kompetitive allele specific PCR (KASP) markers are promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and could be used with only a small number of accessions or varieties due to the limited genetic information and reference genomes available. Results Here, we identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. Furthermore, the total 2,925 SNPs were selected as accession-specific SNPs, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by 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 distinguish individuals from the mixed population including 50 target accessions from B. rapa core collection and outgroup. 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).

A long-amplicon viability-qPCR test for quantifying living pathogens that cause bacterial spot in tomato seed

Bacterial spot is one of the most serious diseases of tomato. It is caused by four species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated and/or infected seed can serve as a major source of inoculum for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. Current seed testing protocols rely mainly on plating the seed extract and conventional PCR, however, the plating method cannot detect viable but non-culturable cells and the conventional PCR assay has limited capability to differentiate DNA extracted from viable versus dead bacterial cells. To improve the sensitivity and specificity of the tomato seed testing method for the bacterial spot pathogens, a long-amplicon qPCR assay coupled with propidium monoazide (PMA-qPCR) was developed to quantify selectively the four pathogenic Xanthomonas species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed extracts, and seed extracts of inoculated and naturally-infected seed. A crude DNA extraction protocol also was developed and PMA-qPCR with crude bacterial DNA extracts resulted in accurate quantification of 104-108 CFU/ml of viable bacteria when mixed with dead cells at concentrations as high as 107 CFU/ml in the seed extracts. With DNA purified from concentrated seed extracts, the PMA-qPCR assay was able to detect DNA of the target pathogens in seed samples spiked with ≥75 CFU/ml (~0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally-infected seed samples showed that the PMA-qPCR assay had greater sensitivity than plating the seed extracts on the semi-selective MTMB and CKTM media for all four target species. Being much faster and more sensitive than dilution plating, the PMA-qPCR assay has a promising potential to serve as a standalone tool or used in combination with the plating method to improve tomato seed testing and advance the production of clean seed.

Weed seed contamination in imported seed lots entering New Zealand

Imports of seeds for sowing are a major pathway for the introduction of contaminant seeds, and many agricultural weeds globally naturalised originally have entered through this pathway. Effective management of this pathway is a significant means of reducing future plant introductions and helps minimise agricultural losses. Using a national border inspection database, we examined the frequency, origin and identity of contaminant seeds within seed for sowing shipments entering New Zealand between 2014–2018. Our analysis looked at 41,610 seed lots across 1,420 crop seed species from over 90 countries. Overall, contamination was rare, occurring in 1.9% of all seed lots. Among the different crop types, the arable category had the lowest percentage of seed lots contaminated (0.5%) and the forage category had the highest (12.6%). Crop seeds Capsicum, Phaseolus and Solanum had the lowest contamination rates (0.0%). Forage crops Medicago (27.3%) and Trifolium (19.8%) had the highest contamination rates. Out of 191 genera recorded as contaminants, Chenopodium was the most common. Regulated quarantine weeds were the rarest contaminant type, only occurring in 0.06% of seed lots. Sorghum halepense was the most common quarantine species and was only found in vegetable seed lots. Vegetable crop seed lots accounted for approximately half of all quarantine species detections, Raphanus sativus being the most contaminated vegetable crop. Larger seed lots were significantly more contaminated and more likely to contain a quarantine species than smaller seed lots. These findings support International Seed Testing Association rules on maximum seed lot weights. Low contamination rates suggest industry practices are effective in minimising contaminant seeds. Considering New Zealand inspects every imported seed lot, utilises a working sample size 5 times larger than International Seed Testing Association rules require, trades crop seed with approximately half of the world’s countries and imports thousands of crop seed species, our study provides a unique overview of contaminant seeds that move throughout the seed for sowing system.

Detection of Xanthomonas translucens pv. undulosa from wheat by quantitative PCR

A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen Xanthomonas translucens pv. undulosa (Xtu). The protocol can also detect X. translucens pv. translucens (Xtt), but cannot differentiate the two pathovars. When tested on DNA from plant, non-target bacteria and culture of microorganisms from wheat seeds, the qPCR showed a high specificity. On purified Xtu DNA, the qPCR was more sensitive than a loop-mediated isothermal amplification (LAMP) assay. When DNA samples from a set of serial dilutions of Xtu cells were tested, the qPCR method could repeatedly generate quantification cycle (Cq) values from the dilutions containing 1,000 cells. Since 2 uL of the total of 50 uL DNA was used in one reaction, one qPCR reaction could detect the presence of the bacteria in samples containing as few as 40 bacterial cells. The qPCR could detect the bacteria from both infected seed and leaf tissues. For seed testing, a protocol for template preparation was standardized, which allowed one qPCR reaction to test DNA from the surface of one seed. Thus, the qPCR system could theoretically detect Xtu and/or Xtt in samples where the bacteria had an average concentration at or larger than 40 cells per seed.

The Use of Multispectral Imaging and Single Seed and Bulk Near-Infrared Spectroscopy to Characterize Seed Covering Structures: Methods and Applications in Seed Testing and Research

The objective of seed testing is to provide high-quality seeds in terms of high varietal identity and purity, germination capacity, and seed health. Across the seed industry, it is widely acknowledged that quality assessment needs an upgrade and improvement by inclusion of faster and more cost-effective techniques. Consequently, there is a need to develop and apply new techniques alongside the classical testing methods, to increase efficiency, reduce analysis time, and meet the needs of stakeholders in seed testing. Multispectral imaging (MSI) and near-infrared spectroscopy (NIRS) are both quick and non-destructive methods that attract attention in seed research and in the seed industry. This review addresses the potential benefits and challenges of using MSI and NIRS for seed testing with a comprehensive focus on applications in physical and physiological seed quality as well as seed health.

Effects of pre-sowing seed treatments for improving germination and the growth of pepper and tomato seedlings

The aim of this study was to establish the effect of different seed treatments on germination, and the growth of the embryonic stem and the radicle of tomato and pepper varieties. Four treatments were used in the study: MIX (Coveron + zinc (Zn 0.5%) + boron (B 0.025%)); Coveron; zinc (ZnSO4, Zn 0.5%) and boron (B 0.025%). The treatments were applied on seeds of following four pepper varieties: Šorokšari, Somborka, Kraljica, and Mirtima and three tomato varieties: Rio Grande, Saint Pierre, and Tomato apple of Novi Sad (Novosadski jabučar). Germination and the growth increase of both the embryonic stem (cm) and the radicle (cm) were observed in the germination cabinets and pots containing soil in two laboratories – locations. After the treatment applied to the pepper seeds and testing in the laboratory germination cabinet the following was established: i) the maximum increase in germination of 90% was when the MIX and Zn treatment was applied to seeds, ii) the growth increase of embryonic stems of 2.7cm was recorded when the MIX treatment was applied, iii) the growth increase of radicles of 1.7cm was gained when the Coveron and MIX treatment was applied. Tests performed in pots showed that Coveron was the most efficient treatment. Treatments on tomato seeds during the seed testing in the germination cabinet provided: vi) the germination increase of 13% with the MIX treatment, vii) the growth increase of the embryonic stem of 2.6 cm with the same treatment, vii) the growth increase of 1.7 cm of radicles. Coveron was the most efficient treatment in tests in pots.

Effects of Different Growth Regulators on Seed Germination and Vigor of Pot Marigold (Calendula Officinalis. L)

Flowers are valued to mankind from the dawn of civilization. Flowers are used for various purposes in our day to day life like worshipping, religious and social functions, wedding, interior decoration, natural colours, dyes, scents and self adornment. Calendula officinalis is used as sudorific, blood refiner, blood sugar reducer and also use as anti-inflammatory skin. Seed testing is an essential step for evaluation of planting value of seeds, to minimize the risk of failure in planting low quality seeds. The importance of seed testing in agricultural crops has long been realized. Seed testing is also necessary to determine the need for drying and processing, to determine the quality standards under seed certification and seed law enforcement program, to identify seed quality problems and their probable causes and to provide basis for price fixation and consumer discrimination of seed lots etc.the 200ppm concentration of GA3 shows the significant and positive effect on the various germination and seedling parameters under study shows that GA3 is the best priming substance for the healthy and better germination in pot marigold. SAARC J. Agri., 18(2): 227-231 (2020)

Assessment of the effectiveness of storage structures for maintaining the quality of maize seed stored at different moisture levels

One of the main causes of food insecurity is the timely unavailability of quality seeds for smallholder farmers in developing countries. Improved storage technologies are effective in reducing storage losses. Thus, the objective of this research is to assess the traditional and emerging seed storage structures/materials for the maintenance of seed qualities. The effect of different storage conditions (moisture content of seed at the time of storage, i.e. 12±0.15% and 13.5±0.18%; storage containers such as metal bin, earthen pot, Purdue Improved Crop Storage (PICS) bag, and jute bag) on seed qualities of maize were assessed. The seed was collected from Chitwan, Nepal and a laboratory experiment was conducted at the central seed testing laboratory, seed quality control center (SQQC), Hariharbhawan, Lalitpur. The experiment was done using a two-factor complete randomized design in a four replicates design. Data regarding the seed qualities (seed moisture percentage, germination percentage, root and shoot length, and vigor index) just before store and also at 45, 90, 135, 180, and 240 days after storage (DAS) was performed as per the standard followed by International Seed Testing Association (ISTA). The germination percentage, root, and shoot length decreased with increasing storage duration. The seed stored in the higher moisture level had significantly higher seed moisture throughout the storage duration. Seed stored in the traditional structures (jute bag and earthen pot) had lower seed moisture at 120 and 180 DAS. The seed stored at lower moisture resulted in a higher germination percentage, long root, and shoot length. Up to six months of storage germination of maize seeds stored in the earthen pots, PICS bag, and Jute bags were statistically similar. The traditional storage structures are equally effective for the maintenance of seed quality of maize.