| Adzuki bean [Vigna angularis (Willd). Ohwi & Ohashi] is a cultivated species belonging to the genus Vigna, which is considered to have originated in China. This bean is one of the oldest cultivated crops and a traditional edible legume crop in China and in East Asia, with high seed protein but low fat contents. It is used for Chinese traditional' medicine. Therefore, it is vital to investigate and evaluate the genetic diversity of adzuki bean germplasm resources for further exploring its origin, distribution and evolution, for collecting the germplasm resources, for working out conservation measures for the genetic diversity, rationally exploiting adzuki bean germplasm, and for guiding parental selection and germplasm improvement in adzuki bean breeding program. In this study, germplasm resources of adzuki bean were systematically evaluated at morphological, cytological and molecular levels. The studied materials were mainly collected from western regions, especially the southwest regions, of China. The main results are as following:1. The morphological diversity of 106 adzuki bean germplasm resources were investigated based on 14 morphological characters. The results showed that the morphological diversity was high among the cultivated adzuki bean germplasm. The average diversity index was 1.553 , and average Euclidean genetic distance was 1.369 ranging from 0.175 to 2.945. Based on the morphological data, 106 materials were clustered into 5 groups. The first group had longer growth period, semi- twining habit, longer plant height; long and thin pod, most pods/plant and seeds/pod; smallest 100-seed weight etc. The second group had the longest growth period, but short and thin pod, least pod/plant and smallest grain yield per plant etc. The third group had shorter growth period, lowest plant height, but long and wide pod, more pods/plant, more seeds/pod, most 100-seed weight, and upright growth habit etc. The fourth group had longer growth period, but longer plant height too, more pods/plant and 100-seed weight, most grain yield/plant, and semi-twining growth habit etc. The main characters of the fifth group were between the third and the fourth group. Based on the morphological characters, principal components were analyzed, and the results showed that the first and second principal components represented 47.77% and 19.74% of morphological diversity respectively. These results suggested that the analysis of principal components could be used for evaluating adzuki bean germplasm resources and selecting parent materials for breeding program.2. The karyotypes of the 12 adzuki bean germplasm and G-banding karyotypes of 3adzuki bean germplasm were analyzed by the advanced ASG G-banding method. The morphologic and constructional differences of chromosomes were observed, there were 6 karyotypes among 12 materials, and their formula are 2n=22=20m+2sm; 2n=22=18m+4sm; 2n=22=16m+6sm; 2n=22=16m+6sm(2Sat); 2n=22=14m+8sm and 2n=22=14m+8sm(2Sat), which karyotype asymmetry belong to 1A and 2A types. Multiple G-bands were able to be observed in each metaphase chromosome, which number, position of distribution, staining degree of G-bands between homologous chromosomes were basically identical; but non-homologous chromosomes were distinct in G-band patterns. G-band patterns of different adzuki bean germplasm were polymorphic, which reflected the difference of chromosome construction among materials, also revealed the polymorphisms of chromosome construction in adzuki bean germplasm resources. These results suggested that the advanced ASG G-banding technique could be an effective method for evaluating on the cytogenetic polymorphisms of adzuki bean germplasm.3. The genetic diversity of 92 adzuki bean germplasm were tested by RAPD markers. A total of 109 RAPD bands were amplified with 10 arbitrary primers, of which 101 bands (92.7%) were polymorphic. A mean of 10.1 polymorphic bands was detected for each RAPD primer ranging from 5 to 15. The total heterozygosity (Ht) of RAPD loci in the adzuki bean resources was 0.675. The genetic distance (GD) coefficients ranged from 0.073 to 0.803 with average 0.402, based on Jaccard's genetic similarity {GS). Clustering analysis was made with unweighted pair group method for arithmetic averages (UPGMA) using the symmetric matrix of similarity coefficients. The 92 materials were grouped into eleven groups. The dendrogram revealed that the cluster pattern did not show correlation with the growth characteristics and the locations of the cultivated adzuki bean.4. The genetic diversity of 94 adzuki bean germplasm were investigated by AFLP markers. A total of 855 fragments were amplified with 10 AFLP primer combinations, of which 315 bands (36.9%) were polymorphic. A mean of 31.5 polymorphic bands was detected for each AFLP primer combination ranging from 14 to 50. The total heterozygosity (Ht) of AFLP loci in the adzuki bean resources was 0.480. The genetic distance (GD) coefficients ranged from 0.003 to 0.252 with average 0.067. based on Jaccard's genetic similarity (GS). Clustering analysis was made with unweighted pair group method for arithmetic averages (UPGMA) using the symmetric matrix of similarity coefficients. The 94 materials were grouped into four groups. The dendrogram revealed that the cluster pattern showed correlation with the growth characteristics and the locations of the cultivated adzuki bean.5. Ninty-three adzuki bean germplasm, including 8 released cultivars and 85 land varieties, were analysed with random amplified microsatellite polymorphism (RAMP)markers. PCR was performed with 5' anchored primers complementary to microsatellites in combination with random primers. The results were the following: A total of 308 RAMP bands were obtained with 42 informative primer combinations, of which 297 bands (96.43%) were polymorphic. A mean of 7.1 polymorphic bands was detected for each RAMP primer combination ranging from 3 to 17. The total heterozygosity (Ht) of RAMP loci in the adzuki bean resources was 0.693. Nei and Li's genetic similarity (GS) coefficients ranged from 0.506 to 0.934. The mean genetic distance based on the GS values among the materials was 0.281 ranging from 0.066 to 0.494. It is revealed that there was sufficient genetic diversity in the studied materials but the genetic base of these materials was narrow. Clustering analysis was made with unweighted pair group method for arithmetic averages (UPGMA) using the symmetric matrix of similarity coefficients. The 93 materials were grouped into five groups. The second group included three subgroups. The dendrogram revealed that the cluster pattern showed obvious correlation with the growth characteristics and the locations of the cultivated adzuki bean.To sum up, results above in morphological, cytological and molecular analyses indicated that plentiful genetic diversity did existed, but the genetic relationship was near and the genetic base was narrow among the cultivated adzuki bean germplasm resources from different regions. The results showed that heterogeneous germplasms should be introduced into the cultivated adzuki bean to broaden the genetic base. In the meantime, this study revealed that molecular marker technology, such as RAPD, AFLP and RAMP 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 adzuki bean.Based on the results from the present study and in combination with results from other researchers, breeding strategies for adzuki bean were put forward in order that the information on genetic diversity of adzuki bean germplasm could be fully used, and adzuki bean germplasm resources could be rationally exploited for creating new excellent germplasms and improvement of adzuki bean varieties.
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