Application Of Biotechniques And NIRS In Peanut Breeding

As a good source of edible oil and dietary vegetable protein, the cultivated peanut, Arachis hypogaea L, is a main cash crop worldwide. Remote hybridization and induced mutagenesis are useful ways to broadening its narrow gene base, providing opportunities to change the stagnant scenario in peanut breeding and achieve big breakthroughs in the development of varieties with high yields, good quality and stress resistance. Molecular marker and transgenic techniques may greatly hasten the processs of peanut improvement.Peanut interspecific hybrid derivatives and chemical mutants were characterized for main agronomic traits. The quality of the incompatible hybrids resulting from in vitro peg culture and post-pollination phytohormones treatment, and chemical mutants obtained from injection of mutagens into flower organs, was analyzed using conventional methods and NIR spectroscopy. Peanut materials with high sucrose (14.65%),>76% oleate,>30% protein or >55% oil were identified. Through field screening, bacterial resistant bold-seeded peanut germplasm accessions were obtained. Huayu 31, the first peanut variety with wild incompatible Arachis species in its ancestry, was formally released. L36, a line developed with backcrossing, produced 321.49kg kernels per 666.6m2, outyielding Fenghua No.1 by 34.97%. 08-test-A2, a mutant developed following injection of EMS into peanut flower organs, exhibited a significant yield increase over Luhua 11 and Fenghua No.1.To facilitate the utilization of wild Arachis relatives, phylogenic trees based on nuclear rDNA internal transcribed spacers (ITS) of Arachis species were constructed. The results corroborated the broad sub-classification of the Arachis genus. The bootstrap trees showed that sections Extranervosae, Heteranthae and Triseminata were most primitive, and section Arachis was most advanced, whereas the rest sections are intermediate in evolutionary terms. Since phylogenic analysis based on nuclear rDNA ITS is a widely accepted and reliable strategy, the inference drawn from the present study should be more convincing than previous reports.SSRs have proved to be the most powerful tool for variety identification in peanut of similar origin, and have much potential in genetic and breeding studies. To facilitate SSR discovery in peanut, we proposed a highly simplified SSR isolation protocol based on multiple enzyme digestion/ligation, mixed biotin-labeled probes and streptavidin coated magnetic beads hybridization capture strategy. Totally,123 primer pairs were designed.Techniques for breeding high oleate peanuts were developed. Fast DNA extraction protocols using leaflets, plumules or a slice of cotyledonary tissues were successfully used in cloning of high oleate related genes, identification of true hybrids and screening of transformants. Based on the experience in chemical mutagenesis, effects of different DNA concentrations, injection time, and target tissues on transformation were studied using plant expression vectors including FAD2B and PEP RNAi constructs. When done in late June, injection of 1200ng per ml DNA into calyx tubes a day before anthesis resulted in a high transformation percentage, where the ratio of the number of PCR positive seeds to the number of flower ranged from 11.33%-32.00%. Several randomly selected PCR positive seeds were confirmed to be true transformants by direct sequencing of the EGFP PCR products. The differences in FAD2A and FAD2B genes between high oleate and normal oleate peanut genotypes were revealed and used to identify true hybrids in normal oleate X high oleate crosses by the occurrence of overlapped peaks in trace files of real hybrids. Using peanut germplasm accessions of diverse origin, including large and small seeded genotypes with various seed coat colours, NIRS calibration models for bulk seed samples were established for prediction of oleic, linoleic and palmitic acid contents. The optimized spectrum pretreatment method was first derivative plus vector normalization. The spectrum range, rank, R2 and RMSECV for oleic acid were 8717.1-5446.3cm'1,9,89.16 and 2.62; for linoleic acid, these values were 9,666-5,785.7cm-1,9,90.85, and 2.00; for palmitic acid, they were 8,717.1-5,446.3cm-1,8,79.21 and 0.525, respectively. NIRS models for oleic acid, linoleic acid, palmitic acid and 4 bad saturated fatty acids were also established using F1:2 single seeds from normal oleate X high oleate crosses. These models have higher R2, being more robust than the above-mentioned NIRS models for bulk samples, and thus may facilitate related genetic studies and aid selection for high oleate, low bad fatty acids peanut segregants. The NIRS models for bulk seed and single seed samples were successfully used to screen for high oleate chemical mutants. Comparison of the FAD2B gene sequence from normal oleate peanut cultivar Huayu 22 and from high oleate peanut seeds showed a C to T transition in 281 position of the coding region, which caused an I94T (isoleucine to threonine) mutation in the amino acid sequences of the desaturase in high oleate mutants, which differed from prevous reports.Based on extensive quality evaluation of diverse peanut germplasm accessions, NIRS models for predicting protein and oil contents in sun-dried bulk and single peanut seed samples were also established. These models along with other NIRS models developed in the present study enable multiple internal quality traits to be selected simultaneously, rapidly and non-destructively. Pyramiding multiple quality traits in a single adapted high yielding peanut cultivar may pave the way to a breakthrough in peanut quality breeding.