Patent ID: 12196648

Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is in no way intended to limit the present teachings, application, or uses. Throughout this specification, like reference numerals will be used to refer to like elements.

FIG.1illustrates an automated seed sampler system10, in accordance with various embodiments of the present disclosure. Generally, the seed sampler system10includes a seed loading station100, a seed orientation system200, a seed transport subsystem300, a milling station400, a sampling station500, a sample collection and transport subsystem600, a liquid delivery subsystem700, a sample deposit subsystem800, a seed treatment station900and a seed deposit subsystem1000.

The seed sampler system10is structured and operable to isolate a seed from a seed bin104of the seed loading station100, orient the seed at the seed orientation station200and transfer the seed to the milling station400, via the transport subsystem300. The seed sampler system10is further structured and operable to remove a portion of the seed coat material at the milling station400, transfer the seed to the sampling station500, via the seed transport subsystem300, where sample material is extracted from the seed at the point where the seed coat material has been removed. The seed sampler system10is still further structured and operable to convey the extracted sample to the sample deposit subsystem800, via the sample transport subsystem700, and deposit the extracted sample into a sample tray14located on the sample deposit subsystem800. In various embodiments, the sample material is collected in a disposable sample tube and delivered to the sample tray14using liquid, as described further below. Further yet, the seed sampler system10is structured and operable to treat, e.g., apply a protective coating to, the exposed portion of the seed at the seed treatment station900and convey the seed to the seed deposit subsystem1000, where the seed is deposited into a seed tray18located on a platform of the seed deposit subsystem1000.

It should be understood that the seed sampler system10, as shown and described herein, includes various stationary braces, beams, platforms, pedestals, stands, etc. to which various components, devices, mechanisms, systems, subsystems, assemblies and sub-assemblies described herein are coupled, connected and/or mounted. Although such braces, beams, platforms, pedestals, stands, etc. are necessary to the construction of the seed sampler system10, description of their placement, orientation and interconnections are not necessary for one skilled in the art to easily and fully comprehend the structure, function and operation of the seed sampler system10. Particularly, such braces, beams, platforms, pedestals, stands, etc. are clearly illustrated throughout the figures and, as such, their placement, orientation and interconnections are easily understood by one skilled in the art. Therefore, for simplicity, such braces, beams, platforms, pedestals, stands, etc. will be referred to herein merely as system support structures, absent further description of their placement, orientation and interconnections.

Referring now toFIGS.2and3, in various embodiments, the seed loading station includes the seed bin104and a separating wheel108. The separating wheel108is mounted for rotation in a vertical plane such that a portion of the separating wheel108extends into an interior reservoir of the seed bin104. Another portion of the separating wheel108extends outside of the seed bin104such that a face120of the separating wheel108is positioned adjacent a seed collector124. The seed separating wheel108includes a plurality of spaced apart recessed ports128that extend through the face120and are communicatively coupled to a vacuum system (not shown) such that a vacuum can be provided at each of the recessed ports128.

To initiate operation of the seed sampler system10, seeds to be sampled and tested are placed in the seed bin104interior reservoir and a vacuum is provided to at least some of the recessed ports128, e.g., the recessed ports128in the face120of the portion of the separating wheel108extending into the interior reservoir of the seed bin104. The seed separating wheel108is then incrementally rotated, via an indexing motor132, such that recessed ports128sequentially rotate through the interior reservoir of the seed bin104, out of the seed bin104, and past seed collector124before re-entering the interior reservoir of the seed bin104. As the separating wheel incrementally rotates and the recessed ports128incrementally pass through the seed bin104interior reservoir, individual seeds are picked up and held at each recessed port128by the vacuum provided at the respective recessed ports128. As the separating wheel108incrementally rotates, the seeds are carried out of the seed bin104to the seed collector124where each seed is removed from the face120of the separating wheel108. After each seed is removed from the separating wheel108, the seed is funneled to a loading station transfer tube136. The seed is then passed through the loading station transfer tube136, via gravity, vacuum or forced air, into a seed imaging fixture204of the seed orientation system200. The loading station transfer tube136is sized to have an inside diameter that will only allow the seed to pass through the loading station transfer tube136in a longitudinal orientation. That is, the seed can only pass through the loading station transfer tube136in either a tip-up or tip-down orientation and the inside diameter will not allow the seed to tumble or flip as it passes through the loading station transfer tube136.

In various embodiments, the seed collector124includes a wiper (not shown) that physically dislodges each seed from the respective recessed port128as the separating wheel108incrementally rotates past the seed collector124. Thereafter, the dislodged seed passes through the loading station transfer tube136to the imaging fixture204. Alternatively, in various other embodiments, each seed can be released from respective recessed port128by temporarily terminating the vacuum at each individual recessed port128as the individual recessed port128is positioned adjacent the seed collector124. Thereafter, the dislodged seed is transferred to the imaging fixture204, via the loading station transfer tube136. In still other embodiments, each seed can be blown from the respective recessed port128by temporarily providing forced air at each individual recessed port128as the individual recessed port128is positioned adjacent the seed collector124. Thereafter, the dislodged seed is transferred to the imaging fixture204, via the loading station transfer tube136.

Additionally, in various embodiments the seed loading station100can include a bulk seed hopper140having a shaped surface and a vibrating feeder mechanism144. Large amounts of seed can be placed in the hopper140where the seed is funneled onto the vibrating feed mechanism144. The vibrating feeder mechanism144can be controlled to meter seeds into the seed bin104where the seeds are separated and transferred to the imaging fixture204of the seed orienting system200, as described above.

Referring now toFIGS.3and4, the seed orientation system200comprises the seed imaging fixture204, an imaging device208, and a seed orienting device212mounted to a stationary center platform214of the seed sampler system10. The seed imaging fixture204includes a window216and an internal seed orientation area that is visible through the window216. The orienting device212includes a flipper actuator220operable to rotate the seed while the seed is suspended in the seed orientation area. The imaging fixture204is connected to an end of the loading station transfer tube136and the imaging device208is mounted to a system support structure adjacent the imaging fixture such that the imaging device208is positioned to view a seed suspended in the seed orientation area through the window216.

When a seed is transferred to the imaging fixture204, via the loading station transfer tube136, the seed is suspended within the seed orientation area, adjacent the window216, and viewed by the imaging device208through the window216. In various other embodiments, the seed is levitated within the seed orientation area using air provided through an orientation system transfer tube224connected to the bottom of the imaging fixture204, opposite the loading station transfer tube136. Or, in various embodiments, the seed can be physically held within the seed orientation area using any suitable mechanical holding means.

As the seed is suspended adjacent the window216, an image of the seed within the imaging fixture204is collected by the imaging device208. The imaging device208can be any imaging device suitable for collecting images through the window216of the seeds suspended within the seed orientation area. For example, in various embodiments, the imaging device208comprises a high speed, high resolution digital camera, such as a disruptive visual technology (DVT) machine vision camera. The image is communicated to a computer based system controller (not shown), where an orientation of the seed, i.e., tip-up or tip-down, is determined. In a various embodiments, the seed imaging device208additionally locates a centroid of the seed and identifies the farthest point from the centroid as the tip.

If the seed is determined to be tip-down, the seed is conveyed in the tip-down orientation, via the orientation system transfer tube224, to one of a plurality of seed holders304. If the seed is determined to be tip-up, the flipper actuator220is commanded by the system controller to rotate the seed 180° to place the seed in the tip-down orientation. For example, the flipper actuator220can be air-operated such that air is used to rotate the seed until the tip-down orientation is detected by the imaging device208. Or, the flipper actuator can be a mechanical actuator that rotates the seed held by a suitable mechanical holding device to place the seed in the tip-down orientation. Once in the tip-down orientation, the seed is conveyed in the tip-down orientation, via the orientation system transfer tube224, to one of the seed holders304. Orienting the seeds in the tip-down position minimizes the impact to the seed's viability when a sample is removed from the seed, as described below. In various embodiments, the seeds are conveyed via the orientation system transfer tube224utilizing gravity, i.e., the seeds fall from the imaging fixture204, through the transfer tube224and into one of the seed holders304. Additionally, each seed is maintained in the proper orientation, i.e., tip-down, during conveyance to the respective seed holder304by providing the orientation system transfer tube224with an inside diameter sized such that the seeds cannot rotate to the tip-up position.

As used herein, the system controller can be a single computer based system, or a plurality of subsystems networked together to coordinate the simultaneous operations of the seed sample system10, described herein. For example, the system controller can include a plurality of controller subsystems, e.g., a controller subsystem for each station described herein. Each controller subsystem could include one or more processors or microprocessors that communicate with various seed sampler system sensors, devices, mechanisms, motors, tools, etc., and are networked together with a main computer system to cooperatively operate all the stations, systems and subsystems of the seed sampler system10. Or alternatively, the system controller could comprise a single computer communicatively connected to all the various sensors, devices, mechanisms, motors, tools, etc., to cooperatively operate all the stations, systems and subsystems of the seed sampler system10.

The seed holders304are mounted to, and equally spaced around a perimeter area of, a motorized turntable308of the seed transport subsystem300. The orientation system transfer tube224is connected at a first end to the seed imaging fixture204such that a second end of the orientation system transfer tube224is positioned a specific distance above a perimeter portion of the turntable308. More particularly, the second end of the orientation system transfer tube224is positioned above the turntable308a distance sufficient to allow the seed holders304to pass under the orientation system transfer tube second end. However, the second end of the orientation system transfer tube224is also positioned above the turntable308such that there is only a small amount of clearance between the second end and the holders304. Therefore, each seed will remain in the tip-down orientation as it transitions from the orientation system transfer tube224to one of the seed holders304.

Referring now toFIGS.5,6and7, each seed holder304is structured and used to rigidly retain a respective seed in the tip-down orientation. Each seed holder304includes a pair of opposing clamp heads312slidingly positioned within opposing clamp pockets316. The opposing clamp pockets316are separated by a seed channel318laterally formed along a centerline C of the seed holder304. Each clamp head312is connected to a respective clamp piston320via a respective clamp shaft324. Each clamp piston320is slidingly housed within a respective longitudinal internal piston cylinder328of seed holder304. A compression spring332is positioned within each piston cylinder328between a base of the respective piston and a bottom of the respective piston cylinder328. Accordingly, each clamp head312is biased toward the centerline C of the seed holder304. When a seed holder304is in an idle state, that is, when the respective seed holder is not holding a seed or being manipulated to hold a seed, the opposing clamp heads312will be biased by the springs332to a fully extended, or deployed, position. When the clamp heads312are in the deployed position, a top of each respective piston320will extend into a respective fork passageway336extending laterally through the seed holder304on opposing sides of the seed channel318.

Each clamp head312is fabricated from a slightly soft, resilient material, such as neoprene, such that a seed held between the opposing clamp heads312, as described below, will not be damaged.

As described above, the seed holders304are mounted to, and equally spaced around a perimeter area of, the turntable308. Prior to, subsequent to, or substantially simultaneously with the seed orientation process described above, the turntable308is rotated to place an empty, i.e., absent a seed, seed holder308under the orientation system transfer tube224. More specifically, the seed channel318is positioned under the orientation system transfer tube224. When a seed holder304is positioned under the orientation system transfer tube224an automated clamp head spreader340is activated to spread the clamp heads312such that a seed can be received between the clamp heads312. The clamp head spreader340is mounted to system support structure adjacent the seed orienting device212and includes a pair of fork tangs344coupled to a fork base348. The clamp head spreader340is operable to extend the fork base348and tangs344toward the seed holder304. For example, the clamp head spreader340can be a pneumatic device operable to extend and retract the fork base348. Each fork tang344has a chamfered distal end portion and is sized to fit within the fork passageways336.

Upon activation of the clamp head spreader340, the fork base348is extended toward the seed holder304such that the tangs344are inserted into the fork passageways336. As each tang344slides into the respective fork passageway336the chamfered distal end portions slide between the top of each respective piston320and an inner wall of the fork passageway336. As the tangs344are extended further into each fork passageway336, the chamfer of each tang forces the respective piston320outward and away from the centerline C of the seed holder. Accordingly, as the pistons320are moved outward and away from the centerline C, the clamp heads312are also moved outward and away from each other and the centerline C. Thus, the clamp heads312are moved to a retracted position where a seed can be placed between them.

Once the clamp heads312have been retracted, a properly oriented seed can be conveyed through the orientation system transfer tube224and positioned in the tip-down orientation between the clamp heads312. In various embodiments, the seed sampler system10additionally includes a seed height positioning subsystem360for positioning the seed at a specific height within the respective seed holder304. The seed height positioning subsystem includes a vertical positioner364mounted to system support structure below the perimeter area of the turntable308, directly opposite the orientation system transfer tube224, and a datum plate actuator368mounted to the center platform214directly opposite the clamp head spreader340. The vertical positioner364includes a spring loaded plunger372mounted to a positioner head376and the datum plate actuator368includes a datum plate380mounted to a datum plate actuator head384. The vertical positioner364is operable to extend the positioner head376and plunger372toward a bottom of the turntable308directly opposite the seed holder centerline C. For example, the vertical positioner364can be a pneumatic device operable to extend and retract the plunger372. Similarly, the datum plate actuator368is operable to extend the actuator head384and datum plate380over the top of the seed holder seed channel318. For example, the datum plate actuator368can be a pneumatic device operable to extend and retract the datum plate380.

Once the seed has been positioned between the retracted clamp heads312, the positioner head376is extended upward to insert a plunger shaft388through a hole (not shown) in the bottom of the turntable308and a coaxially aligned hole (not shown) in the bottom of the seed holder seed channel318. Substantially simultaneously, the datum plate actuator368extends the actuator head384to position the datum plate380a specified distance above the seed holder304, directly above the hole in the bottom of the seed holder seed channel318. More specifically, as positioner head376is moved upward, the plunger shaft388is extended into the coaxially aligned holes and contacts the tip of the seed. The seed is then pushed upward between the clamp heads312until the crown of the seed contacts the datum plate380. The spring loaded structure of the plunger372allows the shaft388to retract within the plunger372when the seed crown contacts the datum plate380so that the seed is held in place without damaging the seed. Accordingly, the crown of the seed is located at a specific height relative to the top of the turntable308.

With the seed crown held against the datum plate380by the spring loaded plunger372, the clamp head spreader340is operated to retract the fork base348and withdraw the tangs344from the respective passageways336. Upon withdrawal of the tangs344, the springs332bias the clamp heads312toward the deployed position and firmly clamp the seed between the clamp heads312. The datum plate380and plunger shaft388are subsequently retracted leaving the seed properly positioned, or ‘loaded’, in the respective seed holder304. The system controller then rotates the turntable308to position the ‘loaded’ seed holder304beneath the milling station400and the next empty seed holder304beneath the seed orienting device212.

Referring now toFIG.8, as described above, the seed sampler system10includes the seed transport subsystem300for conveying the seeds between individual stations of the sampler system, e.g., the seed loading station100, milling station400, sampling station500, etc. Generally, the seed transport subsystem300can be any suitable conveyance mechanism such as, for example, a belt conveyor, roller conveyor, and the like. In various embodiments, however, the transport subsystem300comprises the round turntable308that is pivotally mounted at its center for rotation. The turntable308is virtually divided into a plurality of sectors, with each sector containing a seed holder304. The number of sectors available on the turntable308may be even or odd with a number chosen which depends in large part on the diameter of the turntable308, the size of the seed holders304and the needs of the transport application.

The circular turntable308is pivotally mounted at its center to a shaft and bearing system390. In various embodiments, a shaft (not shown) of the shaft and bearing system390can be directly coupled to an actuating motor392. Alternatively, the shaft may be separate from the actuating motor392and driven for rotation by a suitable chain drive, pulley drive or gear drive. In various implementations, the actuating motor392can be a high torque stepper motor.

In operation, the actuating motor392for the turntable308is actuated to step forward (which can be either clockwise or counter clockwise, depending on configuration) to rotationally move the turntable308from station to station of the sampler system10. Therefore, the seed holders304are aligned with auxiliary devices, such as the loading station100, milling station400, sampling station500, etc. In this configuration, an auxiliary device can be positioned about the turntable308at stations which are in alignment with each position and thus have precise access to the seeds and seed holders304. To the extent necessary, the peripheral edges of the turntable308may be supported with rollers, guides, slides, or the like, to assist with smooth rotation of the turntable conveyor.

Referring toFIG.8further, as described above, once each seed holder304is ‘loaded’ with a seed, the system controller rotates the turntable308to position the ‘loaded’ seed holder304beneath the milling station400. The milling station400includes at least one milling tool404mounted to system support structure above the perimeter area of the turntable308. The one or more milling tools404are used to remove a portion of the seed coat from each seed when the respective seed holder304is positioned beneath the milling station400. Each milling tool404includes a Z-axis actuator408operable to lower and raise at least a portion of the respective milling tool404along the Z-axis. Each milling tool404is controlled by the system controller and can be electrically, pneumatically or hydraulically operated.

The milling tool(s)404can be any suitable mechanism for removing a portion of seed coat material from each seed. For example, in various embodiments, each milling tool404is a rotary device including the Z-axis actuator408and a rotary drive412operationally coupled to a bit chuck416. Each Z-axis actuator408is operable to lower and raise the respective bit chuck416and a milling tool bit420held within the bit chuck416along the Z-axis. The milling tool bit420can be any instrument suitable for removing the seed coat material, such as a mill bit, drill bit, a router bit, a broach, or a scraping tool. For example, in various embodiments, the milling tool bit420comprises an end mill bit. Each Z-axis actuator408is controlled by the system controller to lower the respective Z-axis actuator408a specific predetermined distance. The rotary drive412of each rotary milling tool404functions to rotate, or spin, the respective bit chuck416and any milling tool bit420held within the bit chuck416.

In operation, when a seed holder304is positioned below a rotary milling tool404, the rotary drive412is activated to begin spinning the bit chuck416and milling tool bit420. The Z-axis actuator408is then commanded to lower the respective bit chuck416and milling tool bit420a specific predetermined distance. As the spinning milling tool bit420is lowered, it contacts the crown of the seed and removes the seed coat from at least a portion of the crown. This exposes a portion of the inner seed material that can be extracted and utilized to test and analyze the various traits of the respective seed, as described below.

In various embodiments, the milling station400comprises at least two milling tools404mounted to a milling station horizontal movement stage424that is mounted to system support structure. The milling station horizontal movement stage424is controlled by the system controller to position a selected one of the milling tools404above a seed holder304positioned below the milling station400. The selected milling tool404is then operated as described above to remove the seed coat from at least a portion of the respective seed crown. Subsequently, the system controller can position a second one of the milling tools404above a subsequent seed holder304positioned below the milling station400. The second selected milling tool404is then operated as described above to remove the seed coat from at least a portion of the respective seed crown. In such embodiments, the milling station400can additionally include at least one milling bit cleaning assembly428for cleaning the bit416of the idle, i.e., not in use, milling tool404. That is, while one milling tool404is operable to remove the seed coat from a respective seed, the bit420of an idle second milling tool404can be cleaned by a cleaning assembly428in preparation for the next milling operation. In various embodiments, the milling bit cleaning assemblies428utilize air pressure and or vacuum pressure to remove and/or collect any seed coat residue that may collect on the bits420of the milling tools404.

Referring now toFIG.9, once the seed coat has been removed from a seed, the system controller rotates the turntable308to position the respective seed holder304beneath the sampling station500. The sampling station500includes at least one sampling tool504mounted to system support structure anchored to the center platform214above the turntable308. The one or more sampling tools504are used to remove a portion, i.e., a sample, of the exposed inner seed material when the respective seed holder304is positioned beneath the sampling station500. Each sampling tool504includes a Z-axis actuator508operable to lower and raise at least a portion of the respective sampling tool504along the Z-axis. Each sampling tool504is controlled by the system controller and can be electrically, pneumatically or hydraulically operated.

The sampling tool(s)504can be any suitable mechanism for removing a sample of the exposed inner seed material from each seed. For example, in various embodiments, each sampling tool504is a rotary device including the Z-axis actuator508and a rotary drive512operationally coupled to a bit chuck516. Each Z-axis actuator508is operable to lower and raise the respective bit chuck516and a sampling tool bit520held within the bit chuck516along the Z-axis. The sampling tool bit520can be any instrument having an outer diameter smaller than the circumference of the area of exposed inner seed material, and suitable for removing a sample from the exposed inner seed material, such as a drill bit, a router bit, a broach, or a coring tube. It is important that the sampling tool bit520be of a smaller diameter than the milling tool bit420to ensure that sample material is obtained from an area where the seed coat material has been removed, thereby substantially eliminating any seed coat material from contaminating the sample material collected.

For example, in various embodiments, the sampling tool bit520comprises a spade tip drill bit having an outer diameter that is smaller than an outer diameter of the milling tool bit420. Each Z-axis actuator508is controlled by the system controller to lower the respective Z-axis actuator508a specific predetermined distance. The rotary drive512of each rotary sampling tool454functions to rotate, or spin, the respective bit chuck516and any sampling tool bit520held within the bit chuck516.

In operation, when a seed holder304is positioned below a rotary sampling tool504, the rotary drive512is activated to begin spinning the bit chuck516and sampling tool bit520. The Z-axis actuator508is then commanded to lower the respective bit chuck516and sampling tool bit520a specific predetermined distance. As the spinning sampling tool bit520is lowered, it contacts the exposed inner material of the seed and cuts away a sample of the inner material. The sample is then removed, or extracted, to be tested and analyzed for various traits and/or characteristics of the respective seed, as described below.

In various embodiments, the sampling station500comprises at least two sampling tools504mounted to a sampling station horizontal movement stage524that is mounted to system support structure. The sampling station horizontal movement stage524is controlled by the system controller to position a selected one of the sampling tools504above a seed holder304positioned below the sampling station500. The selected sampling tool504is then operated as described above to remove the sample from the exposed inner material of the respective seed. Subsequently, the system controller can position a second one of the sampling tools504above a subsequent seed holder304positioned below the sampling station500. The second selected sampling tool504is then operated as described above to remove the sample from the exposed inner material of the respective seed. In such embodiments, the sampling station500can additionally include at least one sampling bit cleaning assembly528for cleaning the sampling bit520of the idle, i.e., not in use, sampling tool504. That is, while one sampling tool504is operable to remove the sample from a respective seed, the sampling bit520of an idle second sampling tool504can be cleaned by a sampling bit cleaning assembly528in preparation for the next sampling operation. In various embodiments, the sampling bit cleaning assemblies528utilize air pressure and or vacuum pressure to remove and/or collect any inner seed material residue that may collect on the sampling bits520of the sampling tools504.

Referring now toFIGS.9and10, the sample collection and transport (SCT) subsystem600is controlled by the system controller to operate in synchronized coordination with the sampling station500to collect each sample as it is removed from each seed. The SCT subsystem600includes a motorized rotating platform604driven by an actuating motor (not shown) similar to the turntable308actuating motor392(shown inFIG.8). The SCT subsystem additionally includes a plurality of collection tube placement (CTP) devices608equally spaced around, and mounted to a perimeter area of the rotating platform604. Each CTP device608includes a pivot bar612having a hollow tube mount616mounted through a transverse bore (not shown) in the pivot bar612. The tube mount616includes a distal end618structured to accept a base620of a collection tube624and a proximal end628adapted to receive pneumatic tubing (not shown).

Each CTP device608further includes a pivot bar actuator632controllable by the system controller to rotate the pivot bar612to various positions about a longitudinal axis of the pivot bar612. In various embodiments, the pivot bar actuator632is operable to pivot the tube mount616between a flushing position, as illustrated inFIG.11, a collection position, as illustrated inFIG.10, and a load and deposit position, as illustrated inFIGS.13and17. The CTP device608additionally includes a stop arm636connected to the pivot bar612and an adjustable stop640, e.g., a set screw, adjustably engaged with the stop arm636. The stop arm636and adjustable stop640pivot with the pivot bar612and function to accurately stop rotation of the pivot bar612so that the tube mount616is in the collection position.

Simultaneously with the operation of the seed loading station100, the milling station400and the sampling station500, the SCT subsystem600operates to load the collection tube624on the tube mounts616of each CTP device608, collect the samples in the collection tubes624as each sample is being removed, and deposit the collected samples in the sample trays14. Loading the collection tubes624on the tube mounts616and depositing the collected sample in the sample trays14, will be described further below with reference toFIG.17, andFIGS.12and13, respectively. The collection tubes624can be any container or device suitable for mounting on the tube mounts616and collecting the samples as described below. For example, in various embodiments, the collection tubes624are disposable such that each sample is collected in a clean collection tube624. An example of such a disposable collection tube624is a filtered pipette.

As described above, the SCT subsystem600is controlled by the system controller to operate in synchronized coordination with the sampling station500to collect each sample as it is removed from each seed. More specifically, prior to removing the sample from the seed, the system controller rotates the platform604to position a CTP device608adjacent the sampling station500. Particularly, a CTP device608is positioned adjacent the sampling station500such that the respective tube mount616is aligned with the seed held within an adjacent seed holder304that has been positioned below a sampling device504, via the controlled rotation of the turntable308. Prior to positioning the CTP device608adjacent the seed holder304positioned at the sampling station500, the SCT system600has loaded a collection tube624on the respective tube mount distal end618and the respective pivot bar actuator632has raised the collection tube624to a position above the collection position, e.g., the flushing position. Once the CTP device608is positioned adjacent the respective seed holder304, the pivot bar actuator632lowers the loaded collection tube624until the adjustable stop640contacts a stop plate648mounted to system support structure between the turntable308and the platform604adjacent the sampling station500. The adjustable stop640is preset, i.e., pre-adjusted, such that the rotation of the pivot bar612is stopped to precisely locate a tip672of the collection tube624in very close proximity to, or in contact with, the crown of the seed held in the adjacent seed holder304.

The sampling bit620of a sampling tool504is then lowered to begin removing the sample, as described above. As sampling bit620is lowered, a vacuum is provided at the collection tube tip672. The vacuum is provided via vacuum tube (not shown) connected to the proximal end628of the tube mount616. The vacuum tube is also connected to a vacuum source (not shown) such that the vacuum is through the vacuum tube, the hollow tube mount616and the collection tube624. Accordingly, as the sampling bit620removes the sample material, the sample is drawn into the collection tube624, where the sample is collected. In various embodiments, the sampling station500can include a positive pressure device (not shown) to assist the vacuum provided at the respective seed to collect substantially all the removed sample in the respective collection tube624.

Each collection tube includes a filter676that prevents the sample from being drawn into the tube mount616and vacuum tube. Once the sample has been collected, the pivot bar actuator632raises the collection tube624to the flush position and the respective CTP device608is advanced to a position adjacent the liquid delivery subsystem700. Consequently, another CTP device608and empty collection tube624are positioned adjacent a subsequent seed holder304and un-sampled seed that have been moved to the sampling station.

Referring now toFIGS.11and12, the liquid delivery subsystem700includes a liquid injection device704mounted to a linear actuator708operable to extend and retract the liquid injection device704along a linear axis M. More specifically, the linear actuator708is operable to insert and withdraw an injection needle712, fastened to the liquid injection device704, into and out of the tip672of the respective collection tube624. When a collection tube624with a collected sample has been raised to the flush position and advanced to be positioned adjacent the liquid delivery subsystem700, the linear actuator708and injection needle712are in the retracted position, as illustrated inFIG.11. The pivot bar actuator632and rotating platform604are controlled by the system controller such that when the CTP device608is adjacent the liquid delivery subsystem700and the collection tube624is raised to the flush position, a linear axis of the collection tube624is substantially coaxial with the linear axis M of the liquid injection device704, as shown inFIG.11.

Once the linear axis of the collection tube624is positioned to be coaxial with the M axis, the linear actuator708extends to insert the injection needle712into the tip672of the collection tube624. The liquid injection device704is connected to an extraction fluid supply source (not shown) via a fluid port716coupled to a metering valve720of the liquid injection device704. Therefore, once the injection needle712is inserted into the collection tube tip672, the fluid injection device704injects a metered amount of extraction fluid into the collection tube624. The injected extraction fluid flushes, or washes, the interior sides of the collection tube624and creates an aqueous solution with the respective sample, herein referred to as an aqueous sample. Thus, any of the collected sample that may have gathered on the interior walls of the collection tube624is flushed off so that substantially all the collected sample is suspended in the resulting aqueous solution. The extraction liquid can be any liquid suitable for delivering substantially all the sample material collected within each respective collection tube624, without interfering with the desired analysis, e.g., chemical and genetic analysis, of the sample material. For example, in various embodiments, the extraction liquid may comprise distilled water or any suitable solvent compatible with the desired sample analysis.

Once the collected sample has been mixed with the extraction liquid, the linear actuator708retracts to withdraw the injection needle712from collection tube tip672. The system controller then advances the rotating platform604to position the CTP device608above the sample deposit subsystem800. The system controller additionally commands the respective pivot bar actuator632to position the collection tube in the load and deposit position. The load and deposit position points the tube mount616and mounted collection tube624downward to a substantially vertical orientation.

Referring now toFIG.13, the sample deposit subsystem800includes a sample tray platform804adapted to securely retain a plurality of sample trays14in fixed positions and orientations. Each sample tray14includes a plurality of sample wells22, each of which are adapted for receiving a collected aqueous sample. The sample tray platform804is mounted to an X-Y stage808. The X-Y stage808is a two-dimensional translation mechanism, including a first translating track812and a second translating track816. The X-Y stage808additionally includes a first linear actuator818operable to bidirectionally move a first carriage (not shown) along the length of the first translating track812. The X-Y stage808further includes a second linear actuator820operable to bidirectionally move a second carriage (not shown) along the length of the second translating track816. The second translating track816is mounted to the first carriage and the sample tray platform804is mounted to the second carriage.

The first and second linear actuators818and820are controlled by the system controller to precisely move the sample tray platform804in two dimensions. More particularly, the first and second actuators818and820move the sample tray platform804within an X-Y coordinate system to precisely position any selected well22of any selected sample tray14at a target location beneath the CTP device608holding the collection tube624containing the collected aqueous sample. The target location is the location in the X-Y coordinate system that is directly below the collection tube tip672when the collection tube624is in the load and deposit position above the sample tray platform804. Thus, once the CTP device608is positioned above the sample tray platform804and the respective collection tube624is placed in the load and deposit position, with the tip672pointing at the target location, the system controller positions a selected well22, of a selected sample tray14at the target location. The aqueous sample is then deposited into the selected well22by providing positive pressure to the proximal end628of tube mount616.

As the sample trays14are placed on the sample tray platform804, a tray identification number, e.g., a bar code, for each sample tray14and the location of each sample tray14on the platform804is recorded. Additionally, as each aqueous solution is deposited in a well22, an X-Y location of the well, i.e., the target location, on the sample tray platform804can be recorded. The recorded tray and well positions on the sample tray platform804can then be compared to the X-Y locations of each deposited aqueous sample, to identify the specific aqueous sample in each well22of each sample tray14.

Once each aqueous sample is deposited into a selected well22, the system controller advances the rotating platform604to position a subsequent CTP device608, holding a collection tube624containing a subsequent aqueous sample, above the sample deposit subsystem800. Additionally, the CTP device608holding the used, empty collection tube624is advanced to a collection tube discard station850(shown inFIG.1) where the used collection tube624can be removed or ejected from the respective tube mount616and discarded. Referring briefly toFIG.1, in various embodiments, the collection tube discard station850includes a collection tube removal device854mounted to a linear actuator858operable to extend and retract an automated gripper862. When a CTP device608holding a used collection tube624is positioned adjacent the collection tube removal device854, the system controller commands the linear actuator858to extend and gripper862to grasp the used collection tube624. The system controller then commands the linear actuator858to retract, thereby removing the used collection tube624from the respective tube mount616. The gripper862can then be commanded to release the used collection tube624allowing it to fall into a discard container (not shown).

Referring now toFIG.14, in various embodiments, after a seed has had a sample extracted at the sampling station500, the system controller may advance the turntable308to position the respective seed holder304adjacent a seed treatment station900. The seed treatment station900includes a treatment dispenser904mounted to system support structure above the perimeter area of the turntable308. The treatment dispenser904includes an applicator908configured to apply a seed treatment such as a sealant to the exposed portion of the respective seed, i.e., the area of the seed crown where the seed coat has been removed and the sample extracted. The seed treatment can be any substance designed to enhance one or more properties of the seed or to protect the seed from bacteria or other harmful elements that could damage the seed and destroy the germination viability of the seed. For example, in various embodiments, the seed treatment is a sealant comprising a fungicide and/or polymer delivered to the seed by the treatment dispenser904via the applicator908. The applicator908can be any device suitable to apply the desired seed treatment to the seeds, for example, a brush, needle or nozzle. In various embodiments, the applicator908comprises a spray nozzle and the treatment dispenser904includes a fluid port912coupled to a metering valve916. In such embodiments, the treatment dispenser904is connected to liquid seed treatment supply source (not shown) via the fluid port912. Accordingly, when a seed holder304is positioned at the seed treatment station900, beneath the treatment dispenser904, the system controller commands the treatment dispenser904spray a metered amount of seed treatment on the respective seed.

Referring now toFIGS.15and16, after sampling and the optional seed treatment, the system controller advances the turntable308until the respective seed holder304is positioned adjacent a second clamp head spreader1004of the seed deposit subsystem1000. The clamp head spreader1004is mounted to system support structure and includes a pair of fork tangs1008coupled to a fork base1012. The clamp head spreader1004is substantially identical in form and function as the clamp head spreader340described above with reference toFIG.5. Accordingly, upon activation of the clamp head spreader1004, the fork base1012is extended toward the seed holder304such that the tangs1008are inserted into the fork passageways336. As the tangs1008slide into the respective fork passageways336, the clamp heads312of the respective seed holder304are retracted, as similarly described above. As the clamp heads312retract, the respective seed is allowed to fall through the coaxially aligned holes in the bottom of the seed holder seed channel318and the turntable308into a funnel1016of a seed conveyor1020.

The seed conveyor1020comprises a first tube section1024coupled at a first end to the funnel1016and to an inlet of a first venturi device1028at a second end. A second tube section1032is connected at a first end to an outlet of the first venturi device1028and at a second end to an inlet of a second venturi device1036. An outlet of the second venturi device1036is connected to seed dispenser1040that is mounted to system support structure above a seed tray platform1044. The first venturi device1028is operable to induce an air flow in the first and second tube sections1024and1032toward the seed dispenser1040. At the same time, the second venturi device1036is operable to induce an air flow toward the funnel1016. Thus, the air flow induced by the first venturi device1028will draw the seed into the first funnel1016and first tube section1020. Additionally, as the seed enters the first tube section1024it is propelled toward the seed dispenser1040by the air flow provided by the first venturi device1028. Subsequently, as the seed nears the seed dispenser1040, the seed is slowed down by the air flow provided by the second venturi device1036so that the seed is gently dispensed from the seed dispenser1040, into a seed tray18without damaging the seed. In various embodiments, the air flow provided by the second venturi1036actually stops the movement of the seed, allowing the seed to drop under gravity into a seed tray18. Various position sensors (not shown) can be provided on the first and second tube sections1024and1032to detect the presence of the seed, and provide input to the system controller to control operation of the seed conveyor1020.

Referring particularly toFIG.16, the seed deposit subsystem1000additionally includes a seed tray platform1044adapted to securely retain a plurality of seed trays18in fixed positions and orientations. Each seed tray18includes a plurality of seed wells26, each of which are adapted for receiving a seed dispensed from the seed dispenser1040. The seed dispenser1040is mounted to system support structure above the seed tray platform1044such that seeds can be dispensed from the seed dispenser1040into selected seed wells26of selected seed trays18.

The seed tray platform1044is mounted to an X-Y stage1048. The X-Y stage1048is a two-dimensional translation mechanism, including a first translating track1052and a second translating track1056. The X-Y stage1048additionally includes a first linear actuator1060operable to bidirectionally move a first carriage (not shown) along the length of the first translating track1052. The X-Y stage1048further includes a second linear actuator1064operable to bidirectionally move a second carriage (not shown) along the length of the second translating track1056. The second translating track1056is mounted to the first carriage and the seed tray platform1044is mounted to the second carriage.

The first and second linear actuators1060and1064are controlled by the system controller to precisely move the seed tray platform1044in two dimensions. More particularly, the first and second actuators1060and1064move the seed tray platform1044within an X-Y coordinate system to precisely position any selected well26of any selected seed tray18at a target location beneath the seed dispenser1040. The target location is the location in the X-Y coordinate system that is directly below a tip1068of the seed dispenser1040. Once a seed holder304is positioned above the funnel1016, the system controller positions a selected well26, of a selected seed tray at the target location. The seed in the seed holder304is released into the funnel1016and transported to seed dispenser1040, as described above, and gently deposited into the selected well.

As the seed trays18are placed on the seed tray platform1044, a tray identification number, e.g., a bar code, for each seed tray18and the location of each seed tray18on the seed tray platform1044is recorded. Additionally, as each seed is deposited in a well26, an X-Y location of the well, i.e., the target location, on the seed tray platform1044can be recorded. The recorded tray and well positions on the sample tray platform1044can then be compared to the X-Y locations of each deposited seed, to identify the specific seed in each well26of each seed tray18.

As described above, each of the seed trays18and the sample trays14include a plurality of wells26and22, respectively. In various embodiments, the number and arrangement of the wells26in the seed trays18corresponds to the number and arrangement of the wells22in the sample trays14. This facilitates a one-to-one correspondence between a seed and its extracted sample. However, in some embodiments, it may be desirable to provide multiple wells22in the sample trays14for each well26in the seed trays18, for example, where multiple tests may be run on the samples, or where different samples may be taken from the same seed (e.g. samples from different depths).

Referring now toFIG.17, in various embodiments, the seed sampler system10additionally includes a collection tube loading station1100for mounting the collection tubes624on the tube mounts616of each CTP device608. The tube loading station1100includes a hopper1104having a shaped surface and a vibrating feeder chute1108extending from an open bottom of the hopper1104. Large amounts of collection tubes624can be deposited into the hopper1104where the vibrating feeder chute1108feeds the collection tubes624into a vibrating bowl feeder1112. A gravity based feed track1116is connected to an outlet1118of the vibrating bowl feeder1112at a first end1116A. A second end of the feed track1116terminates at a collection tube ram device1120. The ram device1120extends orthogonally downward from the feed track second end1116B and includes a longitudinal lift channel1124extending along the length of the ram device1120. The ram device1120additionally includes a push mechanism (not shown) internal to the ram device1120. The push mechanism can be any mechanism operable to push a collection tube624, longitudinally positioned within the lift channel1124, out an upper end1120A of the ram device1120. For example, the push mechanism can include a linear actuator that drives a ram shaped to receive at least a portion of a collection tube624.

As the vibrating feeder bowl1112vibrates, collection tubes624migrate toward the outlet1118of the vibrating bowl feeder1112. At the outlet1118, the collection tubes624fall into the feed track first end1116A that is shaped to cause the collection tubes624fall into a tube slot (not shown) that extends the length of the feed track1116. More specifically, the collection tubes624are caused to fall tip-down into the tube slot and hang within the tube slot by a lip620A of the collection tube base620(shown inFIG.10). Gravity and vibration from the vibrating feeder bowl1112cause the collection tubes624to travel the length of the feed track1116and accumulate, single-file, at the feed track second end1116B. As the collection tubes624accumulate, single-file at the second end1116the lead collection tube624will be longitudinally oriented within the longitudinal lift channel. The ram device1120is then actuated such that the push mechanism pushes the lead collection tube624out the upper end1124A of the ram device lift channel1124.

Prior to actuating the ram device1120, the system controller will advance the rotating platform604to position a CTP device608above the second end1116B of the feed track1116. The system controller will further command the pivot bar actuator632to position the tube mount616in the load and deposit position, such that the tube mount distal end618is directly above the lift channel upper end1124A. Therefore, as the lead collection tube is pushed, or lifted, out of the lift channel upper end1124A the collection tube base620is pushed onto the tube mount distal end618. The tube mount distal end618is sized such that there will be a friction fit between the collection tube base620and the tube mount distal end618. Accordingly, the collection tube624is lifted out of the ram device1120and mounted on the respective tube mount. The next collection tube624in the feed track1116will then be positioned within the lift channel1124and a subsequent tube mount distal end618positioned to receive the collection tube624.

Referring now toFIG.18, in various embodiments, the collection tubes624can comprise commercially available pipettes, referred to herein as pipettes624′. In such embodiments, the pipettes624′ may require a portion of the tip672′ be removed to allow for proper extraction of the sample, flushing of the pipette, and depositing of the aqueous sample in the sample trays14. Therefore, in such embodiments, the seed sampler system10can include a collection tube preparation subsystem1150operable to cut off a portion of each pipette tip672′ after each pipette624′ has been mounted on a respective tube mount616. The collection tube preparation subsystem1150includes a linear actuator1154operable to extend and retract a base1158A of a cutter1158along a linear axis P. The linear actuator1154is mounted to system support structure below the rotating platform604such that when a newly mounted pipette624′, i.e., the pipette624′ has just been mounted on the respective tube mount616, is advanced to the collection tube preparation subsystem1150, the pipette tip672′ is positioned within a cutting chamber1162.

The cutting chamber1162is formed between the cutter base1158A and a cutting recess1166formed in a head1158B of the cutter1158. As illustrated inFIG.18, when the newly mounted pipette624′ is advanced from the collection tube loading station1100, the cutter base1158A is in the retracted position and the tip672′ is positioned within the cutting recess1166. Subsequently, the system controller commands the linear actuator1154to extend the cutter base1158A. The cutter1158includes a cutting instrument1170, e.g., a knife blade, fixedly coupled with, or held to, the cutter base1158A by a cutting instrument bracket1174. The cutting instrument is fixedly positioned such that when the linear actuator1154extends the cutter base1158A, the cutting instrument will sever the pipette tip672′ thereby removing a portion of the tip672′.

Referring now toFIG.19, in various embodiments, after the sampled seed has been deposited in a selected well26of a selected seed tray18, the system controller advances the turntable308and positions the now empty seed holder304at a cleaning station1200. The cleaning station1120is operable to clean and remove any residual seed sample and/or seed treatment, e.g., sealant, from the respective seed holder304after the sampled seed has been conveyed to a seed tray18and before a new seed is oriented and placed in the seed holder304. The cleaning station comprises a roller brush1204and a vacuum1208. The vacuum1208is connected to a vacuum source (not shown) to provide a vacuum at vacuum nozzle1212positioned in close proximity to the seed holder seed channel318when the respective seed holder304is advanced to the cleaning station1200. The provided vacuum will remove any residual sample material and/or seed treatment that may have collected on the seed holder304. Additionally, the roller brush1204is driven, e.g., electrically or pneumatically, to rotate on or with a roller shaft1216. Simultaneous with providing the vacuum at the vacuum nozzle1212, the system controller rotates the roller brush1204to remove any residual sample material and/or seed treatment that may have collected on the seed holder304.

Applications

The present disclosure provides methods for analyzing seeds having a desired trait, marker or genotype. In one aspect of the disclosure, the analytical methods allow individual seeds to be analyzed that are present in a batch or a bulk population of seeds such that the chemical and/or genetic characteristics of the individual seeds can be determined.

Samples prepared by the present disclosure can be used for determining a wide variety of physical, morphological, chemical and/or genetic traits. Generally, such traits are determined by screening the samples for one or more chemical or genetic characteristics indicative of the traits. Non-limiting examples of chemical characteristics include proteins, oils, starches, fatty acids, and metabolites. Accordingly, non-limiting examples of chemical traits include protein content, starch content, oil content, determination of fatty acid profiles, determination of metabolite profiles, etc. Genetic characteristics may include, for example, genetic markers, alleles of genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, quantative trait loci (QTL), 5′UTR, 3′UTR, satellite markers, transgenes, mRNA, ds mRNA, transcriptional profiles and methylation patterns.

In some embodiments, the methods and devices of the present disclosure can be used in a breeding program to select plants or seeds having a desired trait or marker genotype. The methods of the present disclosure can be used in combination with any breeding methodology and can be used to select a single generation or to select multiple generations. The choice of breeding method depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F1hybrid cultivar, pureline cultivar, etc.). Selected, non-limiting approaches for breeding the plants of the present disclosure are set forth below. It is further understood that any commercial and non-commercial cultivars can be utilized in a breeding program. Factors such as, for example, emergence vigor, vegetative vigor, stress tolerance, disease resistance, branching, flowering, seed set, seed size, seed density, standability, and threshability etc., will generally dictate the choice.

In various embodiments, the methods of the present disclosure are used to determine the genetic characteristics of seeds in a marker-assisted breeding program. Such methods allow for improved marker-assisted breeding programs wherein nondestructive direct seed sampling can be conducted while maintaining the identity of individuals from the seed sampler to the field. As a result, the marker-assisted breeding program results in a “high-throughput” platform wherein a population of seeds having a desired trait, marker or genotype can be more effectively bulked in a shorter period of time, with less field and labor resources required. Such advantages will be more fully described below.

In other embodiments, the present disclosure provides a method for analyzing individual seeds within a population of seeds having genetic differences. The method comprises removing a sample comprising cells with DNA from seeds in the population without affecting the germination viability of the seeds; screening the DNA extracted from the sample for the presence or absence of at least one genetic marker; selecting seeds from the population based upon the results of the DNA screening; and cultivating plants from the selected seed.

As described above, the sampling systems and methods of this disclosure protect germination viability of the seeds so as to be non-destructive. Germination viability means that a predominant number of sampled seeds (i.e., greater than 50% of all sampled seeds) remain viable after sampling. In some particular embodiments, at least about 75% of sampled seeds, and in some embodiments at least about 85% of sampled seeds remain viable. It should be noted that lower rates of germination viability may be tolerable under certain circumstances or for certain applications, for example, as genotyping costs decrease with time because a greater number of seeds could be sampled for the same genotype cost.

In yet other embodiments, germination viability is maintained for at least about six months after sampling to ensure that the sampled seed will be viable until it reaches the field for planting. In some particular embodiments, the methods of the present disclosure further comprise treating the sampled seeds to maintain germination viability. Such treatment may generally include any means known in the art for protecting a seed from environmental conditions while in storage or transport. For example, in some embodiments, the sampled seeds may be treated with a polymer and/or a fungicide to protect the sampled seed while in storage or in transport to the field before planting.

In various embodiments, the samples of the present disclosure are used in a high-throughput, non-destructive method for analyzing individual seeds in a population of seeds. The method comprises removing a sample from the seed while preserving the germination viability of the seed; and screening the sample for the presence or absence of one or more characteristics indicative of a genetic or chemical trait. The method may further comprise selecting seeds from the population based on the results of the screening; and cultivating plants from the selected seed.

DNA may be extracted from the sample using any DNA extraction methods known to those of skill in the art which will provide sufficient DNA yield, DNA quality, and PCR response. A non-limiting example of suitable DNA-extraction methods is SDS-based extraction with centrifugation. In addition, the extracted DNA may be amplified after extraction using any amplification method known to those skilled in the art. For example, one suitable amplification method is the GenomiPhi® DNA amplification prep from Amersham Biosciences.

The extracted DNA is screened for the presence or absence of a suitable genetic marker. A wide variety of genetic markers are available and known to those of skill in the art. The DNA screening for the presence or absence of the genetic marker can be used for the selection of seeds in a breeding population. The screening may be used to select for QTL, alleles, or genomic regions (haplotypes). The alleles, QTL, or haplotypes to be selected for can be identified using newer techniques of molecular biology with modifications of classical breeding strategies.

In other various embodiments, the seed is selected based on the presence or absence of a genetic marker that is genetically linked with a QTL. Examples of QTLs which are often of interest include but are not limited to yield, lodging resistance, height, maturity, disease resistance, pest resistance, resistance to nutrient deficiency, grain composition, herbicide tolerance, fatty acid content, protein or carbohydrate metabolism, increased oil content, increased nutritional content, stress tolerance, organoleptic properties, morphological characteristics, other agronomic traits, traits for industrial uses, traits for improved consumer appeal, and a combination of traits as a multiple trait index. Alternatively, the seed can be selected based on the presence or absence of a marker that is genetically linked with a haplotype associated with a QTL. Examples of such QTL may again include, without limitation, yield, lodging resistance, height, maturity, disease resistance, pest resistance, resistance to nutrient deficiency, grain composition, herbicide tolerance, fatty acid content, protein or carbohydrate metabolism, increased oil content, increased nutritional content, stress tolerance, organoleptic properties, morphological characteristics, other agronomic traits, traits for industrial uses, traits for improved consumer appeal, and a combination of traits as a multiple trait index.

Selection of a breeding population could be initiated as early as the F2breeding level, if homozygous inbred parents are used in the initial breeding cross. An F1generation could also be sampled and advanced if one or more of the parents of the cross are heterozygous for the alleles or markers of interest. The breeder may screen an F2population to retrieve the marker genotype of every individual in the population. Initial population sizes, limited only by the number of available seeds for screening, can be adjusted to meet the desired probability of successfully identifying the desired number of individuals. See Sedcole, J. R. “Number of plants necessary to recover a trait.”Crop Sci.17:667-68 (1977). Accordingly, the probability of finding the desired genotype, the initial population size, and the targeted resulting population size can be modified for various breeding methodologies and inbreeding level of the sampled population.

The selected seeds may be bulked or kept separate depending on the breeding methodology and target. For example, when a breeder is screening an F2population for disease resistance, all individuals with the desired genotype may be bulked and planted in the breeding nursery. Conversely, if multiple QTL with varying effects for a trait such as grain yield are being selected from a given population, the breeder may keep individual identity preserved, going to the field to differentiate individuals with various combinations of the target QTL.

Several methods of preserving single seed identity can be used while transferring seed from the chipping lab to the field. Methods include, but are not limited to, transferring selected individuals to seed tape, a cassette tray, or indexing tray, transplanting with peat pots, and hand-planting from individual seed packets. Multiple cycles of selection can be utilized depending on breeding targets and genetic complexity.

The screening methods of the disclosure may further be used in a breeding program for introgressing a trait into a plant. Such methods comprise removing a sample comprising cells with DNA from seeds in a population, screening the DNA extracted from each seed for the presence or absence of at least one genetic marker, selecting seeds from the population based upon the results of the DNA screening; cultivating a fertile plant from the seed; and utilizing the fertile plant as either a female parent or male parent in a cross with another plant.

Examples of genetic screening to select seeds for trait integration include, without limitation, identification of high recurrent parent allele frequencies, tracking of transgenes of interest or screening for the absence of unwanted transgenes, selection of hybrid testing seed, and zygosity testing.

The identification of high recurrent pair allele frequencies via the screening methods of the present disclosure again allows for a reduced number of rows per population and an increased number of populations, or inbred lines, to be planted in a given field unit. Thus, the screening methods of the present disclosure may also effectively reduce the resources required to complete the conversion of inbred lines.

The methods of the present disclosure further provide quality assurance (QA) and quality control by assuring that regulated or unwanted transgenes are identified and discarded prior to planting.

The methods of the present disclosure may be further applied to identify hybrid seed for transgene testing. For example, in a conversion of an inbred line at the BCnF1stage, a breeder could effectively create a hybrid seed lot (barring gamete selection) that was 50% hemizygous for the trait of interest and 50% homozygous for the lack of the trait in order to generate hybrid seed for testing. The breeder could then screen all F1seeds produced in the test cross and identify and select those seeds that were hemizygous. Such method is advantageous in that inferences from the hybrid trials would represent commercial hybrid genetics with regard to trait zygosity.

Other applications of the screening methods of this disclosure for identifying and tracking traits of interest carry the same advantages identified above with respect to required field and labor resources. Generally, transgenic conversion programs are executed in multi-season locations which carry a much higher land and management cost structure. As such, the impact of either reducing the row needs per population or increasing the number of populations within a given field unit are significantly more dramatic on a cost basis versus temperate applications.

Still further, the screening methods of this disclosure may be used to improve the efficiency of the doubled haploid program through selection of desired genotypes at the haploid stage and identification of ploidy level to eliminate non-haploid seeds from being processed and advancing to the field. Both applications again result in the reduction of field resources per population and the capability to evaluate a larger number of populations within a given field unit.

In various embodiments, the disclosure further provides an assay for predicting embryo zygosity for a particular gene of interest (GOI). The assay predicts embryo zygosity based on the ratio of the relative copy numbers of a GOI and of an internal control (IC) gene per cell or per genome. Generally, this assay uses an IC gene that is of known zygosity, e.g., homozygous at the locus (two IC copies per diploid cell), for normalizing measurement of the GOI. The ratio of the relative copy numbers of the IC to the GOI predicts the GOI copy number in the cell. In a homozygous cell, for any given gene (or unique genetic sequence), the gene copy number is equal to the cell's ploidy level since the sequence is present at the same locus in all homologous chromosomes. When a cell is heterozygous for a particular gene, the gene copy number will be lower than the cell's ploidy level. The zygosity of a cell at any locus can thus be determined by the gene copy number in the cell.

In some particular embodiments, the disclosure provides an assay for predicting corn embryo zygosity. In corn seed, the endosperm tissue is triploid, whereas the embryo tissue is diploid. Endosperm that is homozygous for the IC will contain three IC copies. Endosperm GOI copy number can range from 0 (homozygous negative) to 3 (homozygous positive); and endosperm GOI copy number of 1 or 2 is found in seed heterozygous for the GOI (or hemizygous for the GOI if the GOI is a transgene). Endosperm copy number is reflective of the zygosity of the embryo: a homozygous (positive or negative) endosperm accompanies a homozygous embryo, heterozygous endosperm (whether a GOI copy number of 1 or 2) reflects a heterozygous (GOI copy number of 1) embryo. The endosperm GOI copy number (which can range from 0 to 3 copies) can be determined from the ratio of endosperm IC copy number to endosperm GOI copy number (which can range from 0/3 to 3/3, that is, from 0 to 1), which can then be used to predict zygosity of the embryo.

Copy numbers of the GOI or of the IC can be determined by any convenient assay technique for quantification of copy numbers, as is known in the art. Examples of suitable assays include, but are not limited to, Real Time (TaqMan®) PCR (Applied Biosystems, Foster City, CA) and Invader® (Third Wave Technologies, Madison, WI) assays. Preferably, such assays are developed in such a way that the amplification efficiency of both the IC and GOI sequences are equal or very similar. For example, in a Real Time TaqMan® PCR assay, the signal from a single-copy GOI (the source cell is determined to be heterozygous for the GOI) will be detected one amplification cycle later than the signal from a two-copy IC, because the amount of the GOI is half that of the IC. For the same heterozygous sample, an Invader® assay would measure a GOI/IC ratio of about 1:2 or 0.5. For a sample that is homozygous for both the GOI and the IC, the GOI signal would be detected at the same time as the IC signal (TaqMan®), and the Invader assay would measure a GOI/IC ratio of about 2:2 or 1.

These guidelines apply to any polyploid cell, or to haploid cells (such as pollen cells), since the copy number of the GOI or of the IC remain proportional to the genome copy number (or ploidy level) of the cell. Thus, these zygosity assays can be performed on triploid tissues such as corn endosperm.

The description herein is merely exemplary in nature and, thus, variations that do not depart from the gist of that which is described are intended to be within the scope of the teachings. Such variations are not to be regarded as a departure from the spirit and scope of the teachings.