Development Of Near Infrared Spectroscopy Models Predictive Of Vitamin E Content And Sequence Analysis Of γ-TMT In Peanut

Peanut is the main cash crop in China.Vitamin E has anti-oxidation properties,and may protect the organism from damage caused by free radicals.The methods for determining vitamin E content thus far have relied heavily on chromatography techniques.Near infrared spectroscopy（NIRS）has been widely used in the quality improvement in many crops including peanut.In peanut,NIRS models predictive of oleate,oil and protein with adequate accuracy have been developed;however,no such modelsfor vitamin E has been reported.Tocopherol methyltransferase（γ-TMT）is key enzyme in tocopherol biosynthesis pathway,determining the relative content ofα-tocopherol.Twoγ-TMTgenes may be identified in the cultivated peanut,a crop with two sub-genomes;but only one was reported.The objective of the present study is to develop NIRS models for vitamin E for use as selection tools in peanut high vitamin E breeding,clone theγ-TMT genes from both sub-genomes of the cultivated peanut,and analyze the relationship of its differences in coding sequence andα-tocopherol content,to pave the way to highα-tocopherol molecular breeding in peanut.Main research results are listed below:Vitamin E content in the 48 peanut seed samples was determined by reverse phase HPLC（RP-HPLC）.The results showed that,vitamin E content ranged from8.2124 to 17.066 mg/100 g with an average of 10.985,α-tocopherol content ranged from 4.121 to 7.383 mg/100 g with an average of 5.487 mg/100 g,γ-tocopherol content was 1.545 to 9.375 mg/100 g with an average of 4.87 mg/100 g,andδ-tocopherol content ranged from 0.262 to 1.2 mg/100 g,and the average value was0.627 mg/100 g.α-tocopherol was found to be negatively correlated toγ-tocopherol（%,as percentage of total）（r=-0.99,significant at 0.01 level）.Prior to RP-HPLC analysis,the spectral data of the 48 sun-dried peanut samples,bulk or single seeds,were collected.These data along with chemical values from RP-HPLC,were used to develop through cross-validation,the NIRS models predictive of total vitamin E andα-tocopherol content for bulk seed samples,and a NIRS model forα-tocopherol content in single seeds.After optimization,for total vitamin E in bulk seed samples,the optimal spectral pretreatment method wasfirst derivative plus multiplicative scattering correction（MSC）,the spectrum range was7506 to 6094.3 cm^-1（centimeter wave number）and 5454 to 4242.8 cm^-1,the dimension was 8,the model’s coefficient of determination（R²）was 88.34,andRoot Mean Square Error of Cross Validation（RMSECV）was 0.423;forα-tocopherolin bulk seed samples,the best spectral pretreatment method was first derivative plus vector normalization,the spectral range was 7506-4242.8 cm^-1,the dimension was 10,R²was 90.05,and RMSECV was 0.203;forα-tocopherol in single seed samples,the best spectral pretreatment method was first derivative plus MSC,and the spectral range,dimension,R² and RMSECV was 7506-4242.8 cm^-1,8,82.87 and 0.28,respectively.Twoγ-TMTgenes fromthe A-and B-sub-genomes of the cultivated peanut were successfully cloned,which was named as Ahγ-TMT1andAhγ-TMT2.Bioinformatics analysis indicated that thecompletecoding sequence of Ahγ-TMT1and Ahγ-TMT2 was3111bp and 3124bp,respectively,both genes containing6 exons and 5 introns,withan open reading frame（ORF）of 1059bp,encoding 352 amino acids.Phylogeneticanalysis based onγ-TMTamino acid sequences indicated a close relationship with two other legume species,soybean andLotus japonicus.Based on the vitamin E phenotyping results,representative samples were selected and genotyped forγ-TMT genes,and nucleotide and amino acid sequence differences noted.Nodifference was detected inthecoding sequence of Ahγ-TMT2among the materials studied,while nucleotide difference was noted in 3 positions of the coding sequence of Ahγ-TMT1 that caused amino acid changes.However,these changes had no relationship with vitamin andα-tocopherol,γ-tocopherol andδ-tocopherol content.Whether as in other crop plants,there is nucleotide difference in upstreamregion ofγ-TMT leading to increasedα-tocopherol still needs further investigation.