Three-dimensional Pooling And HvGW2Gene Screening Of The Barley (Hordeum Vulgare) BAC Library

Object：The aim of this project is to construct the high quality three-dimens io npools us ing the barle y BAC library (HV_MBb) which we have built, and to explorea high-throughput PCR screening method, to screen the positive clo ne whichconta ins the HvGW2gene from147456BACs, and to analyze the genetic diversityof HvGW2in some extreme traits materials.Methods：A high qua lity three-dimens iona l pool of BAC was designed bymathe matics, which can minimum PCR work in gene screening. Thethree-dimens iona l pools of BAC, includ ing Floor-Pools (F-pools), Row-Pools(R-pools) and Column-Pools (C-pools), were built by Q-Pix2machine. Thecand idate clo nes of mixed BAC pools were obtained by mod ified PCR us ing thespecific primers. The pos itive BAC clones contained HvGW2gene and thenegatives were analyzed. Collinear of extre me width traits a mong differentmaterials were analyzed by PCR.Results：In this paper,160mixed pools (48F-pools,64R-pools and48C-pools) were constructed. The numerations and quantities of the negativemonoc lines were ana lyzed and the rate of the ne gative was1.52%.196M02c lone,among of t hree positive BACs, conta ined the comp lete sequence of HvGW2gene.Sequence analys is of co llinear regio ns of196M02and OsGW2and TaGW2showedthat the sequence variation in the second exon related to grain width and thousandkernel weight (TKW) in barley (Hordeum vulgare).Conclus ions：A3-D mixed pool, with160mixed poo ls and a1.52%negative,could met the screen of functiona l ge ne. By the mod ified PCR, a platform, whichwere the three-dime ns iona l pooling and modified PCR method, was exploredfor study of the functional genes in barley.