Additionally, an in situ large-scale comparison between the seed-based sampling strategy and the current routine leaf-based sampling protocol was conducted by sampling single seeds and leaf punches from 1466 F6 plants from the head rows in the line stage testing (LST) breeding class of IRRI’s irrigated lowland rice breeding program that are routinely prioritized for MAS (Additional file 1: Table S1). Lines in the LST class were grown in 2-row plots with 6 plants per row spaced 40 cm from each other at IRRI’s ZES field facilities in Los Baños, Philippines. During the implementation of each sampling activity, the number of workers, time spent on each step, and operational costs were recorded to compare the relative difference in cost-effectiveness between the two protocols.
When the cost of genotyping is higher than those associated with generating a fixed line, MAS activities to determine homozygous genotypes for high priority traits in fixed lines are more cost-effective than in segregating generations. In this study we demonstrated that a single whole seed in rice can be effectively used for MAS, avoiding the complexity of seed chipping an asymmetrical rice seeds, and ensuring a high probability that the genotypic profile of the sampled seed matches the remnant seed from the same plant, as demonstrated by the high genotypic concordance rates reported in this study.
To be effective a high-throughput MAS system requires simple and rapid DNA extraction methods  to be combined with simple and effective sampling strategies that permit high quality data return and tracking of information. In this study a high-throughput single seed-based sampling method was compared to a conventional leaf-based sampling method using DNA extraction protocols developed at Intertek-AgriTech (http://www.intertek.com/agriculture/agritech/) for routine MAS applications in rice. The reported single seed-based sampling strategy for forward breeding applications of MAS should also be compatible with different DNA extraction protocols developed for rice in different studies [16, 24, 26, 31]. The quality and quantity of DNA obtained from these methods though should be validated as outlined in this study in order to ensure suitability for MAS deployment.
DNA isolation, quantification and genotyping
Single seed-based sampling for MAS at scale. a Call rates from each SNP assay estimated using 1466 single seed and leaf samples. b Distributions of genotypic concordance for all 1466 accessions between single seed and leaf-based sampling strategies. c Call rate distribution across single seed and leaf samples for 1466 rice lines. Solid and dashed lines show the average call rates of 99.24% and 97.5% estimated on single seed and leaf-based samples respectively
Comparison on CT values between accessions with different physical and chemical grain properties was done to evaluate the potential impact these properties have on DNA concentration as judged by CT values (Fig. 1b–f, Additional file 1: Table S4). Grains with different pericarp color, size, width, amylose content and alkali digestibility were compared. An analysis of variance showed no significant differences between CT values obtained from grains with different physical or chemical properties (Fig. 1b–f, Additional file 1: Table S4).
For this protocol a single seed from each sample was collected from a barcoded seed envelope containing F6 seeds derived from a single F5 plant. Each seed envelope was scanned using a handheld Zebra Scanner (model DS3678, http://www.zebra.com) linked to the mobile application “Coordinate” (http://www.phenoapps.org) which simultaneously creates a 96-well plate layout and stores the line information in real time during sampling. A single seed was then collected from the envelope and placed in a 96-deep-well plate on the coordinates defined by the ‘Coordinate’ application. These steps are repeated for each sample until the whole plate is completed leaving the last two wells empty for negative/positive controls. The full plate was covered with a silicon cap mat or sticky paper and was shipped immediately to Intertek-AgriTech (http://www.intertek.com/agriculture/agritech/) for DNA extraction, marker assay and scoring. A detailed version of this protocol can be found in Additional file 2.
To test if a single F6 seed yielded sufficient DNA to run at least ten independent SNP KASP assays, CT values from 96 seed samples from 24 rice accessions were generated and analyzed. The average CT value observed among the single seed samples was 24.3 (with standard deviation (SD) of 0.76) and ranged from 23 to 27 (Fig. 1a). The average CT value obtained from 12 independent leaf tissue samples was 23.9, which is equivalent to 7–8 ng/μL (Fig. 1a, Additional file 1: Table S3). No significant differences were observed between the CT values from DNA extracted from leaf and mature seed samples (Fig. 1a, Additional file 1: Table S3).
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DeltaRow crops can be easily harvested with current silage and grain maize harvesters. Trials conducted over several years have confirmed that this technology allows higher yields to be achieved. LEMKEN will launch its DeltaRow seeding technology on the market with its new Azurit single-seed drill.
Single-seed drilling with DeltaRow
DeltaRow technology involves a singling unit depositing seeds in an extended furrow comprising twin rows spaced 12.5 cm apart. Seed singling is performed by two synchronised perforated discs offset against each other by half a grain spacing. As each of the perforated discs only singles every other seed, their diameter can be kept narrow and their circumferential speed be minimised. The greater time interval available for singling results in outstanding quality of placement at high speeds of travel. A fertiliser band is placed centrally underneath the DeltaRow. Placement in a triangular seeding pattern ensures that seeds are positioned optimally in relation to fertiliser and obtain maximum benefit from it.
In its Azurit single-seed drill, LEMKEN has opted for the novel DeltaRow technology, which provides each and every plant with 70% more surface area for growth than conventional placement in single rows. This in turn ensures plants have better access to light and are better supplied with water and nutrients.
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Their goals were to develop a system that:
DrFrancis Ogbannaya, Project Manager, GRDC
Keeping a finger on the pulse
Finally, the material is sent to the breeding program who can use the lines for molecular mapping for gene discovery, running field trials to select lines with valuable traits, or as parents in crossing programs.
There has been keen interest in their LED techniques from key breeding companies in Australia and at international conferences.
This technology has now become a critical component of the breeding programs as it allows us to fast-track potential variety releases, providing better varieties to growers in a shorter timeframe.