Simple bgc nulled

DNA was prepared from seedling leaves using the methodology based on Fulton et al. The deletion mutant population was screened for deletions in genes orthologous to those flanking the previously-identified Aegilops QTL for B-granule content on the short arm of chromosome 4S. Selection was conducted by genetic analysis of six genes Table 1 one of which was later identified as the candidate Bgc-1 gene, amplified by marker KT Primers were designed to the six wheat genes.

KT71 primers were designed to amplify all homoeologues whilst the other primers were designed to be homoeologue-specific. All the selected genes had three homoeologues except KT71 which had four. The KT71 primers were designed to amplify fragments of the KT71 homoeologues that differed in size. Plants lacking one or more of the four genes lack the corresponding PCR product s identified by peak s on the electrophoretogram. Plants lacking A1 always also lack A2 suggesting that these two genes are encompassed by the same deletion on 4AL.

Some or all of the markers developed for all six genes shown in Table 2 were used to screen the Paragon deletion-mutant population. Deletion mutants of the A and D genomes were obtained and the genotype of the markers closely flanking cBgc-1 revealed the extent of the deletions Table 3. All the deletions discovered were extensive, encompassing not only KT71 but also many of the flanking genes.

Primers were designed to wheat genes. For 4N5. Primer sequences and PCR conditions are shown. The markers are ordered left to right in the order in which they occur on the chromosome arm telomere to centromere. Stacking was conducted by repeated rounds of crossing and selection to incorporate multiple suitable deletions into a single plant. Thus, the B-genome copy of Bgc-1 is postulated to be dysfunctional in these wheat plants. Specifically, to generate plants with deletions in the Bgc-1 regions of both the A- and D-genomes, mutant lines A were crossed pairwise to lines D, in all combinations.

The F 1 plants were grown and allowed to self-fertilize. For each F 2 family, 24 grains were sown. This double-deletion mutant plant derived from a cross between lines A1 and D4. The observed proportion of double mutants in the F 2 was 0. The F 2 plants with single homozygous deletions on either the A- or D-genomes were also under-represented in the population.

This suggests that the deletions are deleterious and that chromosomes carrying these large deletions are transmitted from one generation to the next with a lower frequency than wild-type non-deleted chromosomes.

To screen for additional double-deletion mutant plants, selected F 2 plants with homozygous single deletions on either 4AL or 4DS were allowed to self-fertilize and the F 3 seeds were each cut in half. Plants recovered from the embryo-halves of the F 3 seeds were screened for deletions using the KT71 primer pair.

Four additional double mutant plants were discovered. Only one of the additional double mutants survived and was fertile. This second double mutant plant, like the first double mutant, derived from a cross between mutant lines A1 and D4.

The first double mutant and its progeny only, were used in subsequent experiments. Thus, two double-deletion mutant lines survived. The mutant lines are therefore independent and this suggests that it is the combination of deletions of Bgc-1 regions on 4AL and 4DS rather than the combination of loci on other chromosome arms background deletions that is responsible for the lack of B-granules in the B-less mutants.

An F 2 plant which gave progeny that were all PCR-positive i. Granule analysis was conducted by microscopic examination. The starch granules from the single deletion mutants were examined microscopically and, like the normal wheat cultivar Paragon, all were found to possess A- and B-type granules see FIG. Starch from the control wild-type segregant also had both A- and B-type granules data not shown. The starch granules in the non-embryo halves of the four additional double mutant plants discovered in the F 3 screen were also found to lack B-granules.

This shows that stacking deletions of the Bgc-1 regions of both chromosomes 4AL and 4DS in the same plant prevents the formation of B-type starch granules in wheat. The progeny of the double mutant B-less plant were grown together with replicate wildtype segregant Control and Paragon plants. The height of the primary tillers of both the Control and B-less plants was statistically significantly less than that of Paragon.

However, there was no difference in tiller height between the Control and B-less plants. Grain weight, length, width and area were compared between the three genotypes. As with Paragon tillers, the Paragon grains were bigger than either the Control or B-less mutant grains.

However, the difference in grain weight between Paragon and the Control was not statistically significant. There was no difference in grain size or weight between Control and B-less mutant grains. Similarly, the starch content of both Control and B-less plants was statistically significantly less than that of Paragon but there was no statistically significant difference in starch content between the Control and B-less plants.

This analysis suggests that the background deletions in both Control and B-less plants are deleterious for plant growth. However, the lack of B-type starch granules due to deletions in the Bgc-1 region specifically, has no detectable effect on plant growth, grain size or starch content. Starch was purified from the grains of the three genotypes: Paragon, Control and B-less mutant. Several starch functional properties were examined and for the following, there were no differences between the Control and B-less starches: protein content, moisture content, the size of the A-type starch granules and most of the DSC parameters enthalpy of starch gelatinization, onset and peak temperatures.

However, some functional properties were different between the Control and B-less starches. The lack of B-type granules was obvious when starch from mature grains was examined microscopically and was confirmed by quantitative analysis of starch-granule size distribution using image analysis.

This is also the case in the B-less Aegilops examined previously using a similar method. We assume that these small granules in the B-less mutant are small A-type granules rather than true B-type granules and this assumption is supported by the fact that the average size of this category of granules is larger in the B-less starch than in the wild-type Control. Starch from B-less wheat grains has different functional properties than Control starch with B-granules.

To some extent this is predicted from published data on the properties of purified A and B-type granules from normal wheat and barley, which can vary Lindeboom et al, , Starch 56, However, the precise differences observed in our B-less mutant starch are mainly not as predicted. First, the amylose content of purified A-type granules has been found to be either greater Peng et al, , Cereal Chemistry ; Takeda et al, , Carbohydrate Polymers or the same Evers et al, , Starch ; Myllarinen et al, , Journal of the Institute of Brewing, as that of purified B-type granules.

Thus, the amylose content of the B-less wheat starch might be predicted to be higher or the same as that of the Control.

However, we observed a slightly lower amylose content. Second, the swelling power of B-less mutant starch is predicted to be lower than normal Wei, , Acta Physiologiae Plantarum ; Chiotelli et al, , Cereal Chemistry but surprisingly was observed it to be higher. These data suggests that the A-type granules in the B-less mutant wheat differ in composition from the A-type granules in normal wheat.

The gelatinization enthalpy of B-less mutant starch was found to be the same as that of Control starch. This result is predicted by the work of Eliasson et al , Physicochemical behaviour of the components of wheat flour. In: Cereals in breadmaking: a molecular colloid approach which showed that the gelatinization enthalpy of wheat starch is independent of the granule-size distribution.

However, others have found higher gelatinization enthalpies for A-type than for B-type starch granules in wheat Peng et al. The deletion mutant plants both Control and B-less mutant grew less well than Paragon. This inhibition of growth is likely to be due to deletions of genes at locations in the genome other than the Bgc-1 region background deletions. If the Bgc-1 gene was specifically manipulated, then B-granules could be eliminated without any or with far fewer side effects on plant growth.

The lack of any detectable decrease in grain weight or size suggests that yield of the B-less mutant may not be adversely affected.

This together with the novel functional properties, indicate that B-less wheat is commercially useful. For example, uniform and on average larger-than-normal starch granules may lead to improvements in the yield of purified starch and gluten. Reduced grain hardness could lead to reduced milling energy. Increased swelling power could lead to increased bread softness and prolonged shelf life reduced staling. B-granules are also predicted to be detrimental for malting and distilling suggesting that B-less wheat may be preferred for alcohol production.

Table 5 below gives the markers on chromosome 4S of Aegilops, and their corresponding genes in wheat used to identify the Flo6 gene in wheat. The mutations chosen were ones which were likely to affect the function of the FLO6 protein i.

Both of the Kronos A-genome mutants K and K have a premature stop codon in the coiled-coil domain. The supplied Kronos plants were grown and homozygous mutant plants selected. Starch from the single-mutant grains was observed microscopically. All lines showed a bimodal starch granule-size distribution. However, analysis of granule size distribution showed a small reduction in the proportion of small B granules in the two 4A nonsense mutants K and K and in one of the 4B mutants K when compared with wild-type controls FIG.

Homozygous double mutant lines and their corresponding wild-type segregant lines were selected from a cross between the A-genome mutant, K and the B-genome mutant, K Starch from double mutant grain showed a drastic reduction in small-granule content FIGS. The apparent discrepancy between the phenotypes of the Kronos double mutant lacking B-granules and Franubet barley heterogeneous granule morphology is not completely understood.

Of the two single mutant lines used to generate the Kronos double mutant line, the B-genome line, K was a missense mutant Va—IIe which might not completely lack FLO6 activity. The Franubet mutation is in the same relative position in the protein as the mutation in the K To test whether a total elimination of FLO6 activity in wheat would cause a phenotype similar to that in Franubet barley i.

A triple mutant could be selected from the progeny of this cross. This indicated that there may be a dosage effect of the Flo6 gene of B-granule content. This was done by taking grains from a plant that was heterozygous for at least one genome-copy of TaFlo6, cutting the grains in half, germinating and genotyping the embryo half-grain and extracting and phenotyping the starch from the non-embryo half-grain.

We noticed that the phenotype of some grain from the Paragon deletion mutant population was intermediate between that of the wildtype normal B-granule content and that of the Paragon AD double mutant B-granule-less.

An F 2 plant from the Paragon deletion mutant population generated by crossing the A- and D-genome single mutants was studied. This plant was homozygous mutant for the 4D-genome deletion but segregating for the 4A-genome deletion AaBBdd.

The genotype of the progeny of this plant was assessed from phenotype of their grains as follows: three A-genome wildtype plants AABBdd were identified with grains that all had normal B-granule content; three mutant plants aaBBdd were identified with grains that all lacked B-granules and three heterozygous plants AaBBdd were identified with grains that varied in B-granule content. The B-granule content of individual grains from these nine plants is shown in FIG.

We reasoned that the grains with intermediate phenotype must be heterozygous grains having the genotype AaBBdd heterozygous for the 4A-genome deletion, homozygous wildtype for the 4B-genome and homozygous mutant for the D-genome deletion. All documents referred to herein are incorporated by reference. Whilst the invention has been described herein with reference to certain specific embodiments and examples, it should be understood that the invention is not intended to be unduly limited to these specific embodiments or examples.

The position of amino acid changes are shown in bold and boxed. The wheat plant as claimed in claim 1 wherein the number, weight or volume of B-type granules is reduced relative to the unmodified form of the grain.

The wheat plant as claimed in claim 1 which is able to produce a grain which substantially lacks B-type granules. The wheat plant as claimed in claim 3 which has a non-functional or reduced activity form of the gene Flo6.

The wheat plant as claimed in claim 1 wherein the number, weight or volume of B-type granules is increased relative to the unmodified form of the grain. The plant as claimed in claim 1 which is common wheat Triticum aestivum.

The grain as claimed in claim 7 wherein the number, weight or volume of B-type granules is reduced relative to the unmodified form of the grain. The grain as claimed in claim 7 which substantially lacks B-type granules. The grain as claimed in claim 7 wherein the number, weight or volume of B-type granules is increased relative to the unmodified form of the grain.

The grain as claimed in claim 7 which is a common wheat grain. Grain harvested from a wheat plant as claimed in claim 1. Flour nulled. Downloads for 8-bit boards Firmware upgrade Starting from version 2.

Another options: you can upgrade firmware using XLoader or AvrdudeR utility. Run utility, select COM port where controller is connected, and specify hex file to upload.

Leave all other settings at default baud is , device is "SimpleBGC". If device is restarted after upload - it means all done. Run GUI and check if firmware version has changed.

You need to do the following steps: Download the special unprotected version of firmware, and upload it from the GUI. If it's failed to upload or to start - most probably you need to upgrade a bootloader. If motors were turned OFF before ACC and GYRO calibration, they keep their state after calibration finished otherwise it was problem to make fast 6-axis calibration After executing 'Motors ON' command, reset gimbal to home position More stable work with low serial speeds when airlink connection is used 3DR modems, etc.

In this case, its recommended to reduce frame rate in the Realtime Data tab to not freese the program. GUI : new setting to enable estimation of the frame angle from motor's poles in the Lock mode, like in the Follow mode before. Required Tools.

Sep 29, — 3 Janvier 0. Jan 5, — May 14, No comments Fldigi v3. November April Nov 3, — SimpleBGC bit v3. Version Windows. Step 6 after dropping phoenixdll. You will receive a pop up on screen with this message diiregisterserver in c Inventoria Stock Manager v 3. By Kimchi Look in the fridge, then keep cracking eggs until there are none left. Skip 10 full scenes when you're replaying the game, not just us.