[Development of new PCR markers specific to a Thinopyrum intermedium chromosome 2Ai-2 and cloning of the St-specific sequences].
To meet the need of selecting translocation lines, some new RAPD markers for 2Ai-2 chromosome of Th. intermedium were identified in the paper. Out of 320 RAPD primers, 2 specific primers, OPO05 and OPM04, can amplify respectively a specific band with size of about 650 bp and 1400 bp in the BYDV resistant materials containing the chromosome 2Ai-2, including Th. intermedium, wheat-Th. intermedium partial amphipoild Zhong 4 Awnless, addition lines Z1, Z2 and Z6, F1(Z2/Wan7107) and substitute line ZD28 etc, but absent in all the materials lacking the 2Ai-2 chromosome, including susceptible wheat parents and other wheat-Th. intermedium addition lines L1 and Z4. The RAPD markers specific to chromosome 2Ai-2, OPO05(650) and OPM04(1400), may be located on the St genomic region of 2Ai-2 chromosome by PCR analysis on Th. intermedium (E1E2St), Pseuderogneria strigosa (St), Th. elongatum (E), Haynaldia villosa (V), Secale cereale (R), Hordeum vulgare (H), Aegilops squrrosa (D) and Triticum aestivum (ABD) genomic DNA. The specific bands of RAPD markers OPO05(650) and OPM04(1400) were isolated and cloned. After the clones were subjected to restrict digestion analysis, PCR and Southern hybridization analysis, some clones were sequenced. Based on the sequences, 1 pair of primers SC-O5(U + L) and 1 pair of primers SC-M4(U + L) were designed, synthesized and used to amplify the materials with and without 2Ai-2 chromosome. The results showed that the SCAR markers of chromosome 2Ai-2, SC-O5 and SC-M4, were converted successfully from the RAPD markers OPO05(650) and OPM04(1400). The Th. intermedium fragments amplified by the primers of SC-O5 (U + L) and SCM4(U + L) were cloned and analyzed. The results of Southern hybridization indicated that TiSCO5, the cloned fragment of Th. intermedium amplified by primers of SC-O5(U + L) was a low-copy sequence specific to St genome, and another sequence TiSCM4, the cloned fragment of Th. intermedium amplified by primers of SC-M4(U + L) was a repeat sequence specific to St genomic. The sequences will be new probes to detect St genomic chromatin.
Zhang
Zeng-Yan
ZY
Key Lab. of Crop Genetics & Breeding, Ministry of Agriculture, Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences, Beijing 100081, China. zengyanzhang@yahoo.com
Wang
Li-Li
LL
Xin
Zhi-Yong
ZY
Lin
Zhi-Shan
ZS
D002875:Chromosomes / Q000235:genetics*; D003001:Cloning, Molecular; D017343:Genes, Plant; D016133:Polymerase Chain Reaction; D019105:Random Amplified Polymorphic DNA Technique; D014908:Triticum / Q000235:genetics*