Studies On Germplasm Resources Of Walnuts

Walnut(Juglans regia L) is a species belonging to the genus Juglan, and it is considered to have originated in China. This walnut is one of the oldest cultivated crops and a traditional edible tree crop in the world, with high fat, protein, unsaturated fatty acids, carbohydrate, P, K, Ca, Mg, Zn, Mn, Cu, Fe, B and vitamin B, C, E. It is a worldwile traditional nut. Therefore, it is vital to investigate and evaluate the genetic diversity of walnut germplasm resources for further studying its origin, distribution and evolution, for collecting the germplasm resources, for working out conservation measures for the genetic diversity, rationally exploiting walnut germplasm, and for guiding parental selection and germplasm improvement in walnut breeding program. In this study, germplasm resources of walnut were systematically evaluated at morphological, fatty acid compositons, amino acid contents and molecular levels. The studied materials were mainly collected from western regions of China, especially Sichuan province. The main results are as following:1.The morphological diversity of 160 walnut germplasm resources selected from 27 populations of Sichuan were investigated based on 17 morphological characters. The results showed that the morphological diversity was high among walnut germplasm. Nut weight varied from5.82 to 21.04g, kernel weight from 3.01 to 9.55g, kernel ratio from 0.36 to 0.67, shell thickness from 0.08 to 0.22 cm, nut width from 2.42 to 5.17 cm, nut length from 2.05 to 4.19 cm, nut thickness 2.58 to 4.08 cm, nut diameter from 2.45 to 4.17 cm, kernel fullness from 1.83 to 5.10g/cm. The nut shape of walnut among 160 walnut germplasm resources was mainly round and ovate, shell texture was mainly smooth and very smooth, Nut diaphragm was leather, Shell crest was degradation and underdevelopment, Shell suture line was low and flat, Kernel colour was light amber, Kernel sapor was fragrant and sweet, Ease of removal of kernel halves was easy. Nested variance analysis showed that there were significant variation among/within populations(P＜0.01), which inferred that diferentiation was observed among and within populations. The variation within populations was much greater than that among populations. The coefficient of variation(CV) was 7.89% to17.12%, and phenotypic differentiation coeficient(Vst) ranged from22.37% to 30.109%, the mean value of 27 populations was 23.611%. The variation within population was the main part of the phenotypic variation, variance component of within population variance was 60.0%. According to phenotypic variation within populations, the 27 populations were divided into four groups, which the morphological diversity of groupⅠand groupⅡwas much greater than groupⅢand groupⅣ.2.The fat content in 100g walnut kernel varied from 52.5 to 73.8g on a dry weight basis, Saturated fatty acid from3.76 to 7.65g, Monounsaturated fatty acid form 7.04 to 41.81g, Polyunsaturated fatty acid from 21.61 to 52.88g, Unsaturated fatty acid from 48.03 to 67.98g, Palmitic (16:0) from 2.55 to 5.82g, Palmitoleic(16:1△9c) from 0 to 0.33g, Stearic(18:0) from 0.83 to 2.74g, Oleic(18:1△9c) from 6.99 to 41.51g, linoleic (18:2△9c, 12c) from 18.51 to 47.87g, linolenic(18:3△9c, 12c, 15c) from 1.92 to 8.61g, Arachidic(20:0) from 0 to 0.33g, arachidonic(20:4△5c, 8c, 11c, 14c) from 0 to 0.28g. Nested variance analysis showed that there were significant variation among populations (P＜0.01). The fat and fatty acid compositons diversity of P13, P15, P22 and P24 were lower, the mean value of coefficient of variation(CV) was 12.95%, the diversity of P1, P2, P9 and P16 were higher, mean value of CV was 26.225%. The variation within populations was much greater than that among populations. The coefficient of variation (CV) was from 10.90% to 31.93%, and phenotypic differentiation coeficient(Vst) ranged from 18.10% to 28.60%, the mean value of 27 populations was 21.642%. The results also showed that the correlation between the fat, fatty acid content and Average Annual Relative Humidity, Annual Sunshine time, Annual Precipitation, Average temperature in July was significant at the 0.01 level respectively, and The frost-free period was significant at the 0.05 level, which indicated that they were the main factor for fat and fatty acid content of walnut.3.The total amino acids content in walnut kernel varied from 10.07 to 29.94g on a dry weight basis, Leucine form 0.74 to 1.63g, Isoleucine from 0.39 to 0.84g, Lysine from 0.31 to 0.67g, methionine from 0.15 to 0.53g, Phenylalanine from 0.46 to 0.98g, threonine from 0.35～0.79g, valine from 0.48～1.01g, histidine from 0.23～0.52g, cysteine from 0.15～0.33g, tyrosine from 0.32 to 0.71g, aspartic acid from 0.99 to 2.26g, Arginine from 1.52 to 3.81g, Glutamic acid from 2.09 to 4.76g, serine from 0.55 to 1.20g, glycine from 0.56 to 1.05g, Alanine from 0.43 to 0.95g, Proline from 0.35 to 0.73g. Nested variance analysis showed that there were significant variation among populations (P＜0.01). The amino acids diversity of P3, P12, P15, P19, P24, P27 were lower, the mean value of coefficient of variation(CV) was 6.44%, the diversity of P2, P4, P9, P10, P17, P20 were higher, mean value of CV was 14.22%. The variation within populations was much greater than that among populations. The coefficient of variation (CV) was 4.47% to 16.89%, and phenotypic differentiation coefficient (Vst) ranged from 2.94% to 45.05%, the mean value of 27 populations was 15.79%. The results also showed that the correlation between the amino acids content and Average Annual Relative Humidity, Average temperature in July, Annual Precipitation was significant at the 0.01 level respectively, and Accumulative temperature over 10℃degree was significant at the 0.05 level, which indicated that they were the main factor for the amino acids content of walnut.4. A total of 244 bands including 146 polymorphic bands was obtained. Percentage of polymorphic bands (P) was 59.84%. As for J sigillata population, the percentage of polymorphic bands (P) was 52.05%, the effective number of alleles per locus (Ae) was 1.3399, and the Nei's gene diversity index (H) was 0.1961, Shannon's information index (I) was 0.2898. While for J regia populations at species level, the estimates were P=55.33%, Ae=1.3229, H=0.1908, and I=0.2863, respectively. Although these genetic parameters showed that genetic diversity of J sigillata was slightly higher than the other species, genetic diversity level was equivalent in general. Shannon information index, Nei's genetic diversity coefficient and analysis of molecular variance showed that 85.64%, 87.4%, 88.93% genetic diversity or differentiation distributed within populations at species level, respectively. Most of variation (80.65%) consistently originated from interior of groups. Population of J sigillata possessed the greatest amount of unique bands, accounting for 4.5% of the total amplified bands, which indicated genetic variation between two species. Although there are much morphological differentiation and biological variance between the two species, the genetic differentiation coefficient (G_ST=0.0935) detected by AFLP is very low. J regia showed high genetic affinity to J sigillata. Nei's Genetic distances between each population varied from 0.0382 to 0.0692 and genetic similarities within each population ranged from 0.9332 to 0.9625, which indicated there were high similarities among populations. The results of UPGMA analysis revealed that three J regia populations were clustered firstly and genetic distance between Daduhe valley population and southern Ganzhi district population was the closest.To sum up, the results in morphological, fatty acids composition, amino acids and molecular analysis indicate that walnut genetic diversity did exist. In the meantime, this study revealed that molecular marker technology, such as AFLP could test higher level of polymorphism, and be effective tools to construct genetic maps, to identify germplasm resources and to construct DNA fingerprinting for protecting new varieties of walnnut. Based on the results from the present study and in combination with results from other researchers, breeding strategies for walnut were put forward in order that the information on genetic diversity of walnut germplasm could be fully utilited, and walnut germplasm resources could be rationally exploited for creating new excellent germplasms and improvement of walnut varieties.