Inner Structure Analysis And Variety Identification Of Different Wx Protein-deficient Wheat Grains Based On Infrared/Raman Spectroscopy

Wheat is an important agricultural crop.Which is rich in nutrients such as starch,protein,and minerals.Starch is the main substance in wheat grains,accounting for 67.5%-75%of the dry weight of the grains.There are 3 waxy gene loci Wx-A1,Wx-B1 and Wx-D1 in hexaploid wheat,which are related to amylose synthesis.It is located on the wheat chromosome and encodes granule-bound starch synthase.Wx protein has three protein subunits,namely Wx-A1,Wx-B1 and Wx-D1.The amylose content of wheat is highly positively correlated with Wx protein.The amylose content is significantly reduced,thereby changing the wheat starch composition,flour processing performance and edible quality.Due to the expression or deletion of the Wx gene,the proportion of amylose and amylopectin in wheat grains will be different.Amylose and amylopectin have different physical and chemical properties due to the difference in molecular structure.The ratio directly affects the yield and quality of starch products,so it is of great significance to study the effect of different Wx protein deletions on the content and morphology of wheat grains.In this paper,eight kinds of Wx protein-deficient wheat were taken as the research object,using light microscope,polarizing microscope and scanning electron microscope and other microscope technologies combined with infrared microchemical imaging technology to detect different wheat samples in situ,and observe the starch and protein in different parts of the sample grain.The size,morphology,and distribution information of the microstructure are studied.The effect of different Wx protein deletions on the physicochemical structure of wheat grain components is analyzed.From the microscopic perspective,the effect of changes in the internal structure of wheat grain on the processing performance of wheat and wheat products is analyzed.Finally,the use of near infrared/Raman spectroscopy detection technology and hyperspectral imaging technology combined with chemometrics methods for the identification of eight different types of wheat,provides a new method for the identification of wheat varieties.The main conclusions of this paper are as follows:1.After wild type wheat starch granules are dyed by KI/I₂,the starch granules are all blue,and the starch granule structure inside the grain is relatively uniform.After the waxy wheat was dyed with KI/I₂,some particles had a pink outer periphery around the blue core,and the blue particles had different shades of blue.It may be that the lack of Wx protein leads to increased particle heterogeneity.The starch granules in wild-type and glutinous wheat grains can be divided into large and small starch granules,and the granules are single granules.The large granular starch is elongated oval or round on the wheat slice.Most of the small-grain starch is round or irregular and is distributed around the large-grain starch in an aggregated form.The content of small-grain starch in waxy wheat is higher than that in wild-type wheat.2.The starch granules in the grains of wild-type and waxy wheat have polarized crosses produced under a polarizing microscope,and the intersection of the polarized crosses is located at the umbilical point,indicating that these two types of wheat starch particles are spherulites.The content of waxy small starch body is higher than that of wild type,and the brightness of starch polarized cross is slightly higher than the latter.3.According to the scanning electron microscopy test chart,the central area of the starch granules is convex,the umbilical point is depressed downward into a deep pit.The particle surface has a pore structure.The small-grain starch is mostly spherical or irregular polyhedron,and the small-grain starch is aggregated and distributed around the large-grain starch.The surface of wild-type wheat starch granules is smooth and flat,and the surface of some starch granules in waxy wheat is not smooth,and obvious microporous structure can be observed.4.By comparing the infrared spectra of four wheat samples of waxy wheat,wild-type wheat,single-gene deletion wheat and double-gene deletion wheat,it was found that the extent of the three Wx gene deletions affects the amount of amylose synthesis in the grain.The analysis results of the infrared spectra of the four wheat samples after the second derivative treatment showed that the peak intensity of starch characteristic peaks was:PI9090-1<PI 675518<PI 9048<PI 675517.By comparing the infrared spectra of the three double-gene deletion wheat samples,it was found that the three Wx proteins（Wx-A1,Wx-B1,Wx-D1）deletion have an effect on the synthesis of amylose in wheat grains:Wx-B1>Wx-D1>Wx-A1.By comparing the infrared spectra of eight different Wx gene-deficient wheat samples,it was found that the characteristic peak-peak intensity of starch in single-gene-deficient wheat samples was higher than that in double-gene-deficient wheat samples.That is,the greater the degree of Wx gene deletion,the lower the amylose synthesis in wheat grains.5.Infrared spectroscopy and Raman spectroscopy detection technology was used to qualitatively analyze eight different Wx protein-deficient wheat samples to distinguish different wheat varieties.Through spectral detection technology combined with spectral pretreatment method,multivariate analysis method can effectively divide samples of different varieties,indicating that spectral detection technology is an effective sample variety identification technology.Principal component analysis method was used to process the infrared and Raman spectral data of wheat samples.The obtained first and second principal component score maps also showed that some samples had an obvious clustering trend,and the first and second The two principal component score map is more obvious than the infrared spectrum map clustering,and the separation between different samples is higher.According to the classification judgment accuracy rate,it can be seen that the classification effect of Raman spectroscopy is superior to infrared spectroscopy detection technology,which is consistent with the results obtained by principal component analysis.Spectral detection technology has the advantages of time saving,high sensitivity,non-destructive detection and other advantages to provide a fast and effective identification method for the breeding of wheat varieties.6.Obtain the spectral data matrix of wheat samples through a hyper-spectrometer and use different spectral preprocessing methods to establish a classification model.The SVM model based on the original spectral data matrix processed by SNV is used for wild-type wheat samples and three single gene deletion types.The classification results of wheat samples are the best.The classification accuracy of the four wheat samples reaches 100%,and the classification effect of the eight wheat samples is the best.The SVM model based on the original spectral data matrix processed by the first derivative is the best classification model group for wild-type wheat samples and three types of double-gene deletion wheat samples.The possible reason for the low accuracy of the single/double gene deletion type wheat and wild type wheat classification group classification models is that the samples in the sample group are all from the same place of origin,and they are produced in the same year and are bred by the same parent,which results in very small differences between the samples.For the classification analysis of wild-type wheat samples and waxy-type wheat samples,based on the original spectral data matrix processed by three different sample spectral preprocessing methods and the three classification models established by the original spectral data matrix,due to the large genotype differences between samples,As a result,the difference in material content within the grain is also large,so the correct rate of the model for the sample prediction set classification is 100%,and the samples are well classified.