milling production line is determined by

bead milling method of cell disruption - industry news - news - shanghai ele mechanical and electrical equipment co.,ltd

bead milling method of cell disruption - industry news - news - shanghai ele mechanical and electrical equipment co.,ltd

There are many methods of cell disruption among which the mechanical methods of bead mill and high pressure homogenizer are used not only in laboratory but also in industry. Other methods are still in the laboratory, and industrial applications are still being explored.

The Bead Mill is a common equipment for breaking microbial cells. Generally, there are two types of vertical and horizontal. However, the horizontal type is superior to the vertical type in terms of efficiency and productivity. The grinding medium is loaded into the grinding chamber, and the motor drives the rotor to rotate the microorganism at a high speed. The cell suspension and the small grinding ball generate shearing force to break the cells. In the process of crushing cells by sanding, the influencing factors are as follows:

1)Tip speed:the rotor speed increases, the shear force increases, the cell breakage increases, but the high energy consumption, high heat generation and wear of the grinding ball and product inactivation due to shear force, therefore for a given Under the throughput and release requirements for the protein, there is a point of optimum efficiency at the station. In actual production, the tip speed of the rotor is controlled between 5 and 15 m/s.

2)Cell concentration: During the cell disruption process, the amount of heat generated increases with increasing concentration, increasing the cost of cooling, so the optimal cell concentration is determined experimentally. When ELEs bead mill EDW-15 was used to grind and break yeast or bacteria, the cell concentration was controlled at about 40%.

The smaller the grinding media, the faster the cell breaks. However, the grinding ball is too small to float and it is difficult to stay in the grinding chamber. Therefore, in the experimental scale sander, the ball diameter is preferably 0.2 mm. In industrial scale operation, the ball diameter is >0.4 mm, and different cells should be Choose a different ball diameter. The loading amount of the grinding medium also affects the crushing effect, and is generally controlled between 80% and 85%, and varies with the size of the ball.

4)TemperatureThe operating temperature control has little effect on the broken material in the range of 5~40. However, heat accumulation occurs during the grinding process. In order to control the temperature inside the grinding chamber, a cooling jacket is designed outside the grinding chamber, and the temperature of the chamber is adjusted by cooling water, and the ELE EDW-15 is also designed at the front end of the discharge. Cool the cavity for better cooling.

leading supplier of milling & grinding equipment | skiold

leading supplier of milling & grinding equipment | skiold

Milling equipment from SKIOLD ensures optimized nutritional feed, higher efficiency and reduced operational costs. Improve animal health and increase feed conversion rate.The feed structure should be adapted to the different animal groups, and a wide range of different milling and grinding machines are available for the production of animal feed and fine-ground feed for poultry, livestock or aquaculture. The choice of milling & grinding equipment is determined by the type of animal feed that is to be produced: For pigs and poultry, hammer mills and disc mills are commonly used for feed production. For cattle and other ruminants, the natural choice is a crusher or a SKIOLD Disc mill for feed production. Regarding the question, whether you should choose a SKIOLD Disc mill, Crusher or a Hammermill for your milling & grinding plant, first and foremost depends or your need. We are always ready to advise you which option would be the best for your feed production in your specific situation. Need to know more? We are ready to advise you on how to find the most optimal milling or grinding equipment for your animal feed production plant Contact SKIOLD for further information

Milling equipment from SKIOLD ensures optimized nutritional feed, higher efficiency and reduced operational costs. Improve animal health and increase feed conversion rate.The feed structure should be adapted to the different animal groups, and a wide range of different milling and grinding machines are available for the production of animal feed and fine-ground feed for poultry, livestock or aquaculture.

Regarding the question, whether you should choose a SKIOLD Disc mill, Crusher or a Hammermill for your milling & grinding plant, first and foremost depends or your need. We are always ready to advise you which option would be the best for your feed production in your specific situation.

The SKIOLD Disc mills are based on proven and award-winning technology and thousands of disc mills have been put into operation worldwide.Every year the Disc mills from SKIOLD produces more than 20 million tonnes of feed.

SKIOLD Disc mills are suitable for grinding of many different types of raw materials, including pellets with a diameter of up to 12 mm. One of the advantages of the SKIOLD Disc mill is that its possible to vary the grinding degree during operation, and thereby optimize the feed structure for different animal groups. Other advantages are its very low noise level in operation, an exceptionally long durability of wearing parts, and high capacity with low power consumption. With its compact construction, the SKIOLD Disc mill can easily be incorporated into new as well as existing plants.

DM is the type designationfor SKIOLD's range of hammer mills. The DM mills are made in verysolid and simple construction and have a transportation capacity of up to 80 meters. The DM3 is also available in a Prof-Line version, with longer durability for wearing parts. All wearing parts are easy to replace by original spare parts from SKIOLD. In addition to the traditional hammer mills, our range includes a gravity mill, DM6, and a WM version for grinding of moist maize (CCM).

With SKIOLD's complete range of crushers, there is a crusher for any herd size - from a few horses to many hundreds of dairy cattle. The crusher program includes two standard models, each available in different versions: Drive on two or three rollers, different motor sizes, different capacities, different outlet heights.

The simple andsolid construction of the SKIOLD crushers ensures a high capacity/kW and optimal security of operation. They are easy to operate and to adjust and are efficient for crushing of all common raw materials.Two models are available:

A magnet is usually placed in connection with the raw material intake to the feed mill, or before the grinding unit, to remove metallic impurities etc. before further processing. All the magnets are permanent. Two types of magnets are available:

the control of machining errors of tool path for unequal diameter flank milling of globoidal cam | springerlink

the control of machining errors of tool path for unequal diameter flank milling of globoidal cam | springerlink

The profile error is inevitable in the machining process of globoid cam. In order to tackle the problem of normal vector anisotropy in unequal diameter machining, the offset point optimization algorithm is served to optimize the tool position according to the error transfer relationship of equidistant surfaces and the characteristics of unequal diameter tool path generation. In this paper, a bidirectional search algorithm is devised to minimize the deviation between unequal diameter machining tool path surface and fitting ruled surface. The optimal numerical solution with high accuracy is obtained by genetic algorithm. And then the end points of the tool axis are regarded as the initial data. Inverse interpolation of the NURBS (Non-Uniform Rational B-Spline) interpolation is utilized to reconstruct the ruled surface of the flank milling tool path. The simulation results verify the effectiveness of the proposed approach. Compared with the existing methods, the presented one reduces the machining error greatly.

Zhou Y, Chen ZC, Yang X (2015) An accurate, efficient envelope approach to modeling the geometric deviation of the machined surface for a specific five-axis CNC machine tool. Int J Mach Tools Manuf 95:6777

This research was financially supported by the National Nature Science Foundation (Project No. 51775172) and The Key Scientific and Technological Project of Luoyang City, Henan Province (No. 1801006A).

Hu Dongfang contributed to the research concept. Chu Zhengkai made important contributions to the analysis and preparation of the manuscript, conducted data analysis, and wrote the manuscript. Wu Panlong had a constructive discussion and helped with the analysis.

Hu, D., Chu, Z. & Wu, P. The control of machining errors of tool path for unequal diameter flank milling of globoidal cam. Int J Adv Manuf Technol 113, 29993009 (2021).

bartlett milling investing $28m for expansion, new jobs in johnston county | wral techwire

bartlett milling investing $28m for expansion, new jobs in johnston county | wral techwire

The company, which is headquartered in Kansas City and operates a facility in Statesville that manufactures animal feed and distribution centers in Goldsboro and Shelby, plans to hire at least five new employees. It will pay salaries above the average wage in Johnston County, as well as provide benefits, according to a statement issued by Johnston County Economic Development.

The company produces commercial patent flours for bakeries, restaurants, and institutional food service operations like schools and hospitals Its current Wilson Mills facility was acquired from Midstate Mills, Inc., in 2007. The company expansion will result in opening a new production line at the facility.

We are excited about expanding our operations to better serve our customers in North and South Carolina, said Bob Knief, president of Bartlett Milling, in a statement. This new production line will primarily process soft wheat, creating increased opportunities for area farmers to market their soft wheat crop.

The expansion is expected to create a one-time economic boost of $21.5 million for the county, according to an analysis conducted by Dr. Michael Walden, WRAL TechWire contributor and theWilliam Neal Reynolds Distinguished Professor Emeritus at North Carolina State University.

The company is a part of a larger entity, Savage Enterprises, LLC, which operates in 33 states and three other countries, in a variety of industries that include energy, logistics management, agriculture, transportation, and environmental cleanup services.

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types of milling operation as precision

types of milling operation as precision

Milling operations are broadly classified as peripheral milling and face milling: Peripheral Milling. Generally, peripheral, or plain, milling is accomplished with the workpiece surface mounted to the milling machine table and the milling cutter mounted on a standard milling machine arbor. The arbor is well supported in a horizontal plane between the milling machine spindle and one or more arbor supports. For plain milling, the workpiece is generally clamped directly to the table or supported in a vise. The milling machine table should be checked for alignment before starting to make a cut. If the workpiece surface that is to be milled is at an angle to the base plane of the piece, the workpiece should be mounted in a universal vise or an adjustable angle plate. The holding device should be adjusted so that the workpiece surface is parallel to the table of the milling machine. For plain milling operations, a plain milling cutter should be used. Deeper cuts may generally be taken when narrow cutters are used than with wide cutters. The choice of milling cutters should be based on the size of the workpiece. If a wide area is to be milled, fewer traverses will be required using a wide cut. The milling cutter is positioned on the arbor with sleeves so that it is as close as possible to the milling machine spindle, while maintaining sufficient clearance between the vise and the milling machine column. This practice reduces torque in the arbor and permits more rigid support for the cutter.

If large quantities of metal are to be removed, a coarse tooth cutter should be used for roughing and a finer tooth cutter should be used for finishing. A relatively slow cutting speed and a fast table feed should be used for roughing, and a relatively fast cutting speed, and a slow table feed used for finishing. The surface should be checked for accuracy after each completed cut.

Also called slot milling, the width of the cutter is less than the width of the workpiece. It is used to make the slot in the workpiece. Thin slots can be made by using very thin milling cutters. The workpiece can be cut into two pieces by making a very thin slot throughout the depth of the workpiece. Cutting the workpiece this way is called saw milling.

When two or more parallel vertical surfaces are machined at a single cut, the operation is called straddle milling. Straddle milling is accomplished by mounting two side milling cutters on the same arbor, set apart so that they straddle the workpiece. The diagram below illustrates a typical example of straddle milling. In this case a spline is being cut, but the same operation may be applied when cutting squares or hexagons on the end of a cylindrical workpiece. The workpiece is usually mounted between centers in the indexing fixture, or mounted vertically in a swivel vise. The two side milling cutters are separated by spacers, washers, and shims so that the distance between the cutting teeth of the cutters is exactly equal to the width of the workpiece area required. When cutting a square by this method, two opposite sides of the square are cut, then the spindle of the indexing fixture or the swivel vise is rotated 90 and the other two sides of the workpiece are straddle milled.

The term applied to an operation in which two or more milling cutters are used together on one arbor when cutting horizontal surfaces. The usual method is to mount two or more milling cutters of different diameters, shapes and/ or widths on an arbor as shown in the following diagram. The possible cutter combinations are unlimited and are determined in each case by the nature of the job.

The process of machining special contours composed of curves and straight lines, or entirely of curves, at a single cut. This is done with formed milling cutters, shaped to the contour to be cut, or with a fly cutter ground for the job. The more common form milling operations involve milling half-round recesses and beads and quarter-round radii on the workpieces. This operation is accomplished by using convex, concave, and corner rounding milling cutters ground to the desired circle diameter. Other jobs for formed milling cutters include milling intricate patterns on workpieces and milling several complex surfaces in a single cut, such as produced by gang milling.

Also called conventional milling; in this case movement of cutter teeth is opposite to the direction of feed motion. Down Milling. Also called climb milling; in this case direction of cutter motion is the same so that of direction of feed motion.

Face milling cutters, end milling cutters, and side milling cutters are used for face milling operations. The size and nature of the workpiece determines the type and size of the cutter required. In face milling, the teeth on the periphery of the cutter does most of the cutting. However, when the cutter is properly ground, the face teeth remove a small quantity of stock which is left as a result of the springing of the workpiece or cutter, thereby producing a finer finish. It is important in face milling to have the cutter securely mounted and to see that all end play or sloppiness in the machine spindle is eliminated.

When face milling, the workpiece may be clamped to the table or angle plate, or supported in a vise, fixture, or jig. Large surfaces are generally face milled on a vertical milling machine with the workpiece clamped directly to the milling machine table to simplify handling and clamping operations. The following diagram illustrates face milling performed with a swivel cutter head milling machine with its spindle in a vertical position. The workpiece is supported parallel to the table in a swivel vise. Angular surfaces can also be face milled on a swivel cutter head milling machine. In this case, the workpiece is mounted to the table and the cutter head is swiveled to bring the end milling cutter perpendicular to the surface to be produced. During face milling operations, the workpiece should be fed against the milling cutter so that the pressure of the cut is downward, thereby holding the work against the table. Whenever possible, the edge of the workpiece should be in line with the center of the cutter. This position of the workpiece, in relation to the cutter, will help eliminate slippage.

When setting the depth of the cut, the workpiece should be brought up to just touch the revolving cutter. After a cut has been made from this setting, a measurement of the workpiece is taken. The graduated dial on the traverse feed is then locked and used as a guide in determining the depth of the cut. When starting the cut, the workpiece should be moved so that the cutter is nearly in contact with its edge, after which the automatic feed may be engaged. When a cut is started by hand, care must be taken to avoid pushing the corner of the workpiece between the teeth of the cutter too quickly, as this may result in cutter tooth breakage. In order to prevent time wasting during the operation, the feed trips should be adjusted to stop table travel just as the cutter clears the workpiece.

End milling primarily differs from other milling processes due to the type of tooling that is used for abrading a given material. Unlike cutters and drill bits, end mills have cutting teeth on the sides and end of the mill. Additionally, the milling applications for the end mill are unique. End mills are typically used in applications requiring profile milling, tracer milling, shape milling, face milling, and plunging. This operation is used for non-conventional non-conventional or unique applications,

This is a selective portion milling on the flat surface of the workpiece used to make shallow packets. Surface Contouring. In this operation, a ball nose cutter if feedback and forth across the workpiece along a curvilinear path at short intervals. This creates the required contours on the surface of the workpiece. This operation is used to make contours of molds and dies and this time the operation is called die sinking.

fasciclin-like arabinogalactan protein gene expression is associated with yield of flour in the milling of wheat | scientific reports

fasciclin-like arabinogalactan protein gene expression is associated with yield of flour in the milling of wheat | scientific reports

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A large portion of the global wheat crop is milled to produce flour for use in the production of foods such as bread. Pressure to increase food supplies sustainably can be address directly by reducing post-harvest losses during processes such as flour milling. The recovery of flour in the milling of wheat is genetically determined but difficult to assess in wheat breeding due to the requirement for a large sample. Here we report the discovery that human selection for altered expression of putative cell adhesion proteins is associated with wheats that give high yields of flour on milling. Genes encoding fasciclin-like arabinogalactan proteins are expressed at low levels in high milling wheat genotypes at mid grain development. Thirty worldwide wheat genotypes were grouped into good and poor millers based flour yield obtained from laboratory scale milling of mature seeds. Differentially expressed genes were identified by comparing transcript profiles at 14 and 30 days post anthesis obtained from RNA-seq data of all the genotypes. Direct selection for genotypes with appropriate expression of these genes will greatly accelerate wheat breeding and ensure high recoveries of flour from wheat by resulting in grains that break up more easily on milling.

Wheat is the leading crop in the temperate world1. It provides 20% of the total calories and proteins consumed worldwide2. Wheat is ground to flour by the process of dry milling to make various food products such as bread which are suitable for human consumption. From a milling perspective wheat grain has three main components; endosperm, bran and germ, each with different mechanical properties3. In milling, the endosperm of the grain is scraped from the bran by passing the grain between metal rollers and sieving out the flour derived from the small particles of endosperm released. Clean separation of endosperm from bran and germ is desired in this process4. The efficiency of the process as measured by the yield of flour is the subject of selection by wheat breeders developing new wheat varieties for these products. The recovery of flour is largely determined by genotype and milling process with recoveries usually in the range 70 to 80%5. However, the maximum theoretical yield expected is around 85% which is the percentage of endosperm in the wheat grain4. In wheat breeding, small scale laboratory test mills are widely used for the selection of wheat genotypes that deliver a higher flour yield. However, this process is difficult because the test has poor repeatability and still requires more than 1kg of grain. These quantities of grain are not available from single plants to allow early generation selection for this key trait.

Flour yield is a complex trait controlled by interaction between genotype, environment and processing5,6,7,8. Three main factors which influence milling performance of the wheat are grain hardness9, endosperm to bran ratio and ease of separation of endosperm from bran3. Soft wheats usually produce ~2% less flour than the hard wheats due to differences in mechanical properties of the grains4. Several QTLs associated with milling yield have been reported, on chromosome 6A10, 3B11, 1DL, 2AS, 2BS, 2DS, 3DL, 4AC, 4DS, 5AL, 6AC, 6DS12, 1B, 2A, 2B, 3B, 6A, 7A, 7B13. Co-relation between starch granule size distribution (SGSD) and milling yield has been demonstrated by Edwards et al.14 in hard wheats. It was suggested that an even size distribution (increased volume of B- and C- type small granules and reduced % volume of A-type large granules) of starch granules is genetically controlled and associated with higher flour yield. Edwards et al.9 showed that 68% of the variation in hard wheats (Pina-D1a/Pinb-D1b) could be explained by combined effect of grain hardness and SGSD. Endosperm strength and stiffness measured by SKCS has also been shown to correlate with milling yield15. Much of this may explain environmental rather than genetic variation in milling performance. Despite of all this analysis the genetic and environmental components of variation and their molecular basis remain unclear.

Genetics controls a large part of the variation in flour yield. For example, Laidig et al.16 (2017) reported that genotype was a large contributor to variation in yield of wheat in Germany (19832014). Knowledge of the molecular basis17 of the intense human selection for high flour yield in breeding modern wheat genotypes would greatly accelerate the breeding of wheat combining high productivity and the grain quality required by consumers. This capability will be of great value in rapidly adapting wheat to changing and variable climates. The main objective of this study was to identify candidate genes which may control flour yield of wheat. RNA Seq of 30 diverse worldwide wheat genotypes was conducted at 14 and 30 days post anthesis (DPA) and gene expression was analysed relative to flour yield obtained on laboratory milling of the mature grain.

Milling yield of the thirty wheat genotypes measured as % of total flour yield is shown in Table1. Among the thirty genotypes, 17 were identified as high milling wheat genotypes (HM-group) (>/=77%) and 13 were identified as poor milling wheat genotypes (PM-group) (<77%). The milling yield of genotype NW51A was the lowest (71.4%) while that of Ellison was the highest (79.7%), respectively (Table1). The milling yield of Gregory and Bobwhite could not be determined due to lack of grain samples from these trails. However, these genotypes are commercially known as high milling wheats and thus were placed into the HM-group.

Among the thirty genotypes, 14 genotypes had Pina-D1a/Pinb-D1a alleles of puroindoline genes within which 7 genotypes showed soft grain texture (SKCS HI<50) and 7 showed hard grain texture (SKCS HI>50), 8 genotypes were hard with Pina mutation (Pina-D1b/Pinb-D1a), and 7 were hard wheats with Pinb mutation (Pina-D1a/Pinb-D1b) (Table1)18. Genotypes in HM-group and PM-group were mixture of soft and hard wheats containing different set of Pin alleles. All the hard wheats with Pinb mutation were good millers, except Giza 139.

RNA-Seq analysis of genotypes corresponding to the PM-group and HM-group, followed by application of the Baggerleys test for identification of significant differences led to the identification of twelve genes at 14 DPA and eight genes at 30 DPA that were statistically differentially expressed at a false discovery rate (FDR) p-value of <0.01 (Tables2 and 3). Sorting the gene list based on proportion difference led to the identification of both up- and down- regulated genes. At 14 DPA, highly significantly differentially expressed transcripts GH726097 and TC379245 showed high homology with a fasciclin-like arabinogalactan-8 (FLA-8). GH726097 and TC379245 were expressed at much lower levels in wheat genotypes with high flour yield; 28.2 and 6.1 fold, respectively (Fig.1). These two transcripts were also down regulated at 30 DPA in the HM-group. A third transcript (TAGI; TC429180) with homology with FLA-8 was expressed at higher levels in high milling wheats at 30 DPA. This transcript was 4.5 fold up-regulated in the HM-group. The top two up-regulated gene indices at 14 DPA, TC400069 and TC414662, at a fold change of 20.78 and 10.70 were annotated as Trypsin alpha amylase inhibitor and as wheat Serpin 3 gene, respectively. The top two gene indices at 30 DPA, TC373342 and CJ624986, at a fold change of 9.3 and 45.3 respectively, were annotated as uncharacterized protein from Triticum urartu and as 40s ribosomal protein from Aegilops tauschii.

Fasciclin like arabinogalactan-8 (TAGI; GH726097 and TC379245) expression is down-regulated in developing wheat grains at 14 days post anthesis of wheat genotypes giving a high flour yield. Expression of GH726097 and TC3792456 is measured in reads per kilo base per million mapped reads by analysing Illumina sequencing data on CLC genomic workbench. On the X-axis genotypes are arranged in order of increasing flour extraction rate scores shown within brackets. NA, not analysed; PM, poor milling wheat genotypes giving poor flour yield; HM, high milling wheat genotypes giving high flour yield; Pin a, puroindoline-a gene allele; Pin b, puroindoline-b gene allele.

The expression of GH726097 and TC379245 was significantly up-regulated in all of the genotypes of the PM-group but significantly down-regulated in all genotypes from HM-group except for the genotype Jing Hong No.1. Jing Hong No.1 showed very high up-regulation of -amylase genes (TAGI; CA713114 and CA717472) compared with all other good millers. CA713114 and CA717472 were 707 and 80 fold up-regulated in Jing Hong No.1.

The alignment of three differentially expressed putative FLA genes GH726097 (536bp), TC379245 (1267bp) and TC429180 (1252bp) is shown in (Fig.2). When BLASTn was performed on these three genes using Ensemble with IWGSC survey sequences, two transcripts Traes_2BS_D44E4B43A (cDNA; 1026bp) and Traes_4BS_03EB5D160 (594bp) showed the highest similarity. Traes_4BS_03EB5D160 sequence is exactly identical to Traes_2BS_D44E4B43A from base 433 to base 1026. The first of these is located on chromosome 4BS and the later on 2BS. A single FAS domain unique to FLA genes was identified in both these sequences. Limited annotation is available for these genes to date ( NCBI BLASTn identified DQ872381 as a highly similar gene to GH726097, TC379245 and TC429180. DQ872381 has been annotated as a fasciclin-like arabinogalactan-8 19.

Nucleotide sequence alignments of three Fasciclin like arabinogalactan gene sequences corresponding to tentative sequences from the Triticum Aestivum Gene Indices (TAGI), Expression of transcripts corresponding to TC379245 and GH726097 is significantly-down regulated at 14DPA, while that of TC429180 is significantly up-regulate at 30 DPA, in high milling wheat genotypes. Red boxes indicate nucleotides encoding for amino acids comprising the AGP Glyco-modules comprising of Alanine and Proline. Black boxes indicate nucleotides encoding for amino acids comprising the FAS domain comprising of H1, [YF]H and H2 conserved regions. Blue Boxes indicate start and stop codons. Global alignment was carried out using Clone Manager (Sci Ed, USA).

Nucleotide and the polypeptide sequence alignment performed on Clone Manager (Sci Ed, USA) showed that TC379245 shares 99% sequence identity with Traes_2BS_D44E4B43A and DQ872381 in the coding region (Figs3 and 4). In TC379245 conserved regions were identified in the FAS domain (Fig.2), H1:Leu-Thr-Val-Phe-Cys-Pro-Glu-Asp-Lys-Val, [YF]:Val-Leu-Leu-Tyr-His-Gly-Ala-Ala-Val-Cys and H2: Val-Tyr-Val-Ile-Asp-Val-Ile-Ile-Pro. Two notable conflicts were observed in this alignment. First, nucleotide substitution of C (cytosine) in the cDNA of TC379245 at position 486 (425 in CDS) was observed to cause a frame shift mutation. However, none of the reads mapped to TC379245 for any of the wheat genotypes showed that substitution. Therefore, the nucleotide C at the position 486 was removed from the TC379245 sequence. Second, a deletion of a nucleotide codon was observed in Traes_2BS_D44E4B43A at position between 91^^^92 (21^^^22 in CDS) (corresponding position in TC379245 CDS; 22, 23, 24). This missing codon codes for Leucine at the 7th position in the polypeptide chain of DQ872381 and Traes_2BS_D44E4B43A. All the nucleotide conflicts and the corresponding changes in the amino acid polypeptide are shown in Table4.

Nucleotide sequence alignments of three Fasciclin like arabinogalactan gene sequences TC379245, DQ872381 and, Traes_2BS_D44E4B43A. For TC379245 (Triticum Aestivum Gene Indices), DQ872381 was the top hit on NCBI BLAST and Traes_2BS_D44E4B43A on Ensembl BLAST. Red boxes indicate nucleotides encoding for amino acids comprising the AGP GLyco-modules comprising of Alanine and Proline. Black boxes indicate nucleotides encoding for amino acids comprising the FAS domain comprising of H1, [YF]H and H2 domain. Blue Boxes indicate start and stop codons. Multiway sequence alignment was carried out using Clone Manager (Sci Ed, USA).

Amino acid sequence alignments of three Fasciclin like arabinogalactan polypeptide sequences TC379245, DQ872381 and, Traes_2BS_D44E4B43A. For TC379245 (Triticum Aestivum Gene Indices), DQ872381 was the top hit on NCBI BLAST and Traes_2BS_D44E4B43A on Ensembl BLAST. Red boxes indicate the AGP GLyco-modules comprising of Alanine and Proline. Black boxes indicate amino acids comprising the FAS domain comprising of H1, [YF]H and H2 conserved regions. Red dots indicate conflict positions. Multiway sequence alignment was carried out using Clone Manager (Sci Ed, USA). Note TC379245 ORF was corrected (C at position 425bp removed) before translating the nucleotide sequence.

The TC429180 nucleotide sequence showed 87% identity with TC379245. Deletion of a 78bp sequence was observed in TC429180, from position 461 to 541, corresponding to TC379245. TC429180 RNA-seq mapping files from all the wheat genotypes were manually checked to determine coverage at the conflict region. In none of the files were reads observed to span the region of conflict. In none of the hits obtained for TC429180 on NCBI and Ensemble BLAST was the 78bp deletion observed. Therefore, true identity of this deletion was not confirmed. A FAS domain was identified in the nucleotide sequence of TC429180 (Fig.2). The translated amino acid sequence shows 99% sequence similarity to the TC379245 polypeptide before the start of deletion site but the later part of the sequence shows no similarity due to the apparent frameshift in the TC429180 polypeptide. However, as the 78bp deletion couldnt be confirmed the true sequence of the TC429180 polypeptide couldnt be identified.

GH726097 showed 90% sequence similarity to the TC379245 in the aligned region (Fig.2). The GH726097 sequence is less than half the length of TC379245 and no open reading frame was identified within the sequence and thus it is likely to be an incomplete transcript sequence. No FAS domain was identified in this nucleotide sequence. Reads mapped to these two transcripts were sequence specific/unique which provides the evidence for them to be two different genes.

Analysis of published wheat transcriptome data was used to examine the tissue specificity of expression of these genes. In the grain, both transcripts have been reported in pericarp (inner and outer) but not in endosperm tissue (Fig.5, transcriptome data from20).

Tissue-specific expression (RPKM) of FLA transcripts in developing wheat seeds. The mean of three biological replicates20 is presented for different tissues of the developing wheat grain at 12 days-post-anthesis in the cultivar, Holdfast. ESP: Endosperm; IP: inner pericarp; OP: outer pericarp; TC378245 and GH726097: transcript IDs correspond to FLA genes from the TAGI database Raw reads obtained from data reported by Pearce et al. (2015)20 ( were processed by RNA-Seq analysis to deduce FLA expression, using CLCbio Workbench (Qiagen Bioinformatics, Denmark).

Variation in the gene pool provides the resource for breeding improved genotypes17. The relationship between gene expression and flour yield in milling was analysed for 30 diverse wheat genotypes. Gene expression varies throughout seed development21 with genes expressed at different stages contributing to the final composition and properties of the grain. Much of the variation in hardness of the wheat genotypes was explained by differences in Pin genes18 and these loci have been associated with flour yield variation in some germplasm8 but hardness did not explain most of the variation in milling performance (Fig.1).

Down regulation of expression of transcripts (TAGI; TC379245 and GH726097) encoding cell adhesion proteins, FLA-8, was found at the critical mid grain development stage (14 DPA) and also at a later stage, close to maturity (30 DPA) in genotypes that gave high flour yield (Table2). Reduced expression of these genes may be associated with a reduction in the structural strength of the grain. These changes are likely to result in a grain that breaks up more easily in the mill and yields greater quantities of flour.

The arabinogalactan proteins (AGPs) are highly glycosylated proteins that are rich in hydroxyproline and found in plant cell wall and plasma membrane22. AGPs that contain putative cell adhesion domain known as fasciclin domain are known as fasciclin like arabinogalactan proteins. FLA has been implicated in cell adhesion and may link the cell membrane and cell wall. AGPs carry glycosylphosphotidylinositol (GPI) anchor at the C-terminus which signals the peptide to the plasma membrane. However, wheat FLA-8 has a shorter GPI-anchor which raises a question about its binding to plasma membrane19. But it might have a role in cell to cell and cell to extracellular environment interaction as suggested by Faik et al.19. Modification of a FLA in poplar23 has been shown to reduce the mechanical strength of plant tissues24,25. Human selection for higher flour yield has apparently resulted in the selection of wheat genotypes with greatly reduced levels of expression of this gene. Nanospherical arabinoxylan proteins have been recently identified as the adhesive component secreted by the climbing plant, English ivy26.

A third transcript (TAGI; TC429180) with homology with FLA-8 was expressed at higher levels in high milling wheats at 30 DPA. The tissue specificity of expression is not known in this system but varies significantly in other plants25. Most gene expression at 30 DPA may be in the embryo as the endosperm is terminally differentiated by this stage.

Recently Wilkinson et al.27 (2017) reported that the product of the granule softness protein gene was an AGP. This would complicate any attempts to relate FLA levels to milling yield. Methods for distinguishing the FLAs from other AGPs would be required. Hard wheats are generally considered to be better milling wheats14 and a reduced expression of AGP encoded by the Gsp-1 gene at the hardness locus might contribute to improved flour yield. However this study showed that flour yield was not associated with the pin genotype at the hardness locus (Fig.1).

FLA-8 related genes were found to be located on chromosome 2BS and 4BS in the Chinese Spring wheat genome assembly with limited annotation to date ( A major QTL for flour yield has been reported from chromosome 2B7,12. The QTL on chromosome 2BS had the highest overall LOD score (13.1) in analysis of the progeny of a Sunco X Tasman cross, explaining 14.431.3% of the variation in flour yield at different sites7. Sunco was shown to have low expression of the FLA gene in the current study and was the source of the high flour yield in the Sunco X Tasman cross7. A QTL on chromosome 4B was the second most significant flour yield QTL in this cross. However in another cross, Katepwa X CD87, the 4B QTL was the most important and explained 13.623.8% of variation in flour yield. The coincidence of the structural genes encoding FLA and the chromosomal locations of the major know QTLs for milling yield in these crosses on chromosomes 4B and 2A provides strong confirmation of the significance of these associations.

The strength of the endosperm may be influenced by properties of the protein bodies and starch granules28 that fill the endosperm cells and account for most of the grain content. The expression of storage protein genes was significantly altered in wheat with high flour yield. Several genes may contribute to changes in protein synthesis in the better milling wheat genotypes. The expression of 40S ribosomal protein was significantly higher in the better milling wheat genotypes at both 14 and 30 DPA (Tables2 and 3). This protein may regulate protein synthesis and down regulation of this gene has been reported in a late ripening citrus genotype29. Selection for protein composition that better suits end uses such as bread making may have been more intense in the development of genotypes also selected for milling quality.

Earlier studies have suggested that starch granule size distribution may be associated with flour yield differences9,14. This suggests that modification of starch may contribute to flour yield. Jing Hoang No.1 had a very high level of expression of -amylase and was the only genotype giving high flour extraction without significantly reduced expression of the FLA-8 genes. High flour yield in wheat may be the result of unintended human selection for different types of genetic variation in different genotypes. New deliberate combinations of these individual genes may have application in development of wheat genotypes with very high flour yields. Other wheat is processed by directly grinding the grain. This type of flour is used to make products such as chapatti. Wheat genotypes in regions where these products predominate may not have been subjected to selection for flour yield in roller milling.

The tissue specificity30 of the genes identified in this study may be important as changes in the expression of many of these genes may have been achieved by human selection for flour yield without perturbing critical gene expression in other parts of the plant. These genes are specifically expressed in the pericarp where they may influence the adherence of the pericarp to the Aleurone/starchy endosperm. In hexaploid wheat modification of sub-genome specific expression (in the A, B or D sub-genomes) may allow tissue specific alteration of function. Recent analysis of expression of defence genes and response to pathogen infection has been shown to result in highly sub-genome specific expression31.

Analysis of the wheat grain transcriptome has identified a gene controlling bread quality32 and new pathways for carbon assimilation33. Although the gene controlling bread quality has at least five copies in the wheat genome with high sequence homology, the cause of the differential expression of this gene was identified to be the promoter region of a specific allele of this gene. A simple PCR-test was develop which allowed the identification of wheat genotypes for the presence or absence of the specific allele of gene controlling bread quality32 which has been used by CIMMYT34. A similar strategy can be used to identify the presence or absence of the specific FLA allele which corresponds to high FLA-8 expression and thereby select for wheat genotypes for good milling.

Selection for these genes and the flour yield genes identified in the present study provides a new set of tools for wheat breeders that should reduce the constraints of selection for wheat quality35 on the rate of genetic gain in wheat breeding and assist the selection of new genotypes to support the adaptation of wheat production to climate change36,37.

Wheat grains of a worldwide set of wheat genotypes were sourced from Australian Winter Cereal Collection (AWCC), Tamworth, NSW, Australia; now known as Australian Grains Genebank (AGG), Horsham, Victoria, Australia ( Plants of all thirty wheat genotypes were grown under field conditions at Narrabri (NSW, Australia) and harvested at maturity. Another trial was grown at Biloela (Qld, Australia). The conditions at this site were harsher resulting in some varieties not performing well. Milling of these samples gave flour yield data that showed a highly significant (P=0.01) correlation with that from the Narrabri site but the data was not included in the association analysis because it was considered a far less reliable measure of the flour milling qualities of these genotypes when grown in unfavourable environments.

Milling yield of harvested grain, identified by as % of total flour yield, was determined using a Buhler mill as explained by Edwards et al.9. Wheat genotypes were grouped as poor milling (PM) wheats and high milling (HM) wheats if their milling yield was equal to/above or below 77%, respectively.

Grain hardness for all the genotypes was measured using Single Kernel Characterization System (SKCS) as described by Nirmal et al.18. Puroindoline alleles in all the genotypes were also characterized as described in Nirmal et al.18.

Total RNA was isolated from the whole caryopsis at 14 days post anthesis (DPA) and at 30 dpa38. cDNA was prepared and used to produce indexed Illumina NGS libraries which were then multiplexed to allow the sequencing of eight indexed libraries in one lane on a GA IIx Illumina sequencing platform to obtain 100bp paired-end reads. RNA sequencing of the thirty wheat genotypes generated a total of ~2.5million to ~7.2million reads varying with the genotype.

All NGS data was analysed using CLC Genomic Workbench Version 9.0 (CLC Bio, Qiagen). All the reads were trimmed for quality to exclude calls with Fred quality scores less than 20. Following trimming all sequence data was subjected to quality analysis. After quality check reads were subjected to RNA-seq analysis. Triticum aestivum gene index (TAGI) database ( which contains 221,925 tentative consensus sequences obtained from EST, cDNA and mRNA libraries was used as a reference database for RNA-seq analysis. Read alignment parameters length fraction and similarity fraction were set to 0.8 and 0.9, respectively. Paired reads were counted as two. Gene expression was normalised by calculating reads per kilo base per million mapped reads (RPKM).

Tissue specificity of expression of FLA genes in wheat grain was investigated in transcriptome data available from published data20 for outer pericarp, inner pericarp, and endosperm tissues at 12 days-post-anthesis in developing wheat grains (cv. Holdfast) by RNA-Seq analyses using the TAGI reference in CLC genomics workbench. The data included three biological replicates and since there is was no significant difference between the replicates the mean values are shown in Fig.5.

RNA-seq data of the PM-group and the HM-group was subjected to proportion-based Baggerleys test39 within CLC workbench to identify differentially expressed genes (DEGs) in the HM-group when compared to the PM-group. Differentially expressed genes with FDR (false discovery rate) p-value of <0.01 were considered as statistically significant candidate genes associated. A further sorting of the DEGs was undertaken based on Baggerleys weighted proportion difference, to identify those genes with consistent expression difference in genotypes within the PM and HM wheat genotypes.

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Nirmal, R.C., Furtado, A., Rangan, P. et al. Fasciclin-like arabinogalactan protein gene expression is associated with yield of flour in the milling of wheat. Sci Rep 7, 12539 (2017).

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