关键词: 柳树, 耐盐, 连锁分析, 特定位点参数扩增测定, 单核苷酸多态性(SNP)

Abstract:

【Objective】Due to the rapid reduction of non-agriculture and non-grain land, the afforestation area in China has been drastically decreased in the past two decades and efforts were extended to the afforestation in saline-alkali wastelands in the coastal region. Thus, cultivation and identification of salt tolerance forest germplasm appear to be of particular the importance. However, the salt tolerance in plants is a complex physiological and biochemical process involving with numerous genes, proteins and other multiple coordinative mechanisms. In order to provide a certain significant reference for an early identification and variety selection of salt tolerance shrub willow, we designed this study to screen the SNP loci related with salt tolerance and to develop corresponding SNP markers. 【Method】A hybrid cross was made using salt-tolerance and salt-sensitivity parents identified in the previous study. Cuttings from 1 505 selected seedlings were used for the salt tolerance treatment, of which 50 salt-tolerance and 50 salt-sensitivity individuals were screened for the gene mixing-pool construction. Four libraries, including the parents and two gene pools, were established and sequenced using the SLAF strategy on the Illuminna HiSeq 2500 platform. After filtering and mapping, the polymorphic SLAF tags among the two parents were selected and utilized for an association analysis based on the SNP-index method. The sequences of candidate SLAF tags related tightly with salt tolerance were blasted against multiple databases in NCBI for the functional annotation. The primer pairs for the SNP loci in candidate SLAF tags were designed, and the PCR products were re-sequenced for a loci validation. 【Result】The mean sequencing depth, Q₃₀ value, and GC content of the four samples including two parents and two gene mixing-pools, were 48.57×, 94.36% and 39.39%, respectively. Through the digestion of restriction endonuclease, a total of 175 468 SLAF tags were obtained, of which 25 675 SLAF tags were polymorphic, accounting for 14.63% of the total SLAF tags. According to the genotype and sequencing information of the two parents, 1 774 SLAF tags were determined for a further association analysis. Based on the SNP-index method, 18 SLAF tags containing 25 candidate SNP loci that related closely with salt tolerance were identified. Each three SNP loci were discovered in Marker 116156 and Marker 88668 tags, and each two SNP loci were found in Marker 118929, Marker 108616 and Marker 68843 tags, respectively. Six sequences retrieved proteins with the highest sequence similarity in the Nt database, and four or three sequences were assigned with the NOG and KEGG databases, respectively. Only one sequence had a hit in Swiss-Prot database. Base substitutions among ten SNP markers were detected between salt-tolerance and salt-sensitivity individuals, which was consistent with the original sequence of SLAF-seq. 【Conclusion】 The target trait could be rapidly and effectively located and identified using the bulked segregant analysis combined with the SLAF-seq method in this study, which could provide a theoretical basis and technical support for the development of functional markers and marker-assisted selection in willow.

Key words: willow, salt tolerance, association analysis, specific-locus amplified frangment sequencing (SLAF-seq), single nucleotide polymorphism(SNP)