Molecular Marker Assisted Identification And Analysis Of Anther-culture Derived,Photoperiod-and Thermo-sensitive Male Sterile Doudoubled Haploid Lines In Rice

Rice is one of the most important crops in the world,and application of hybrid rice has made a tremendous contribution to the increase of rice productivity.Being a self-pollination crop,the success of hybrid rice is benefited from the utilization of male sterility,including cytoplasmic male sterility(CMS)and environment-conditioned genic male sterility(EGMS).Among them,the discovery of a spontaneous photoperiod-sensitive genic male sterility(PGMS)rice mutant,Nongken 58S,has established a new paradigm in breeding and production of two-line hybrid rice.Since then,Chinese scientists have been developed more than one hundred photoperiod-and thermo-sensitive genic male sterile lines and about 1/3 area of hybrid rice production has been occupied by the two line hybrid system,thereby greatly advanced the development of hybrid rice.In this study,Firstly we established a genotyping system for PGMS and TGMS gene by integrating a rapid DNA extraction protocol into CADMA-HRM(Competitive Amplification of Differentially Melting Amplicons-High Resolution Melting)analysis.And then we use this system to genotyping a total of 7200 regenerated rice plants that were obtained by using the anther culture technique.DH1 lines were further genotyped and 20 DH lines,each carrying either the PGMS gene,TGMS gene,or both genes were selected and further studied for their fertility conversion characteristics under field conditions.The main results are summarized as follows:1.Establishment of a fast genotyping system for PGMS and TGMS gene.A safe,fast and high throughput system was developed by integrating a rapid DNA extraction protocol with CADMA-HRM analysis.By using this system,a total of 981 anther-culture derived plants carrying both the PGMS and TGMS genes,and 743 lines with the PGMS gene and 2871 plants with the TGMS gene were identified from a total of about 7200 plants regenerated from anther culture.Finally,560 DH lines,carrying PGMS gene,TGMS gene,or both P/TGMS genes with good agronomic performance were selected.2.Preliminary observation of fertility conversion characteristics of DH lines carrying different PGMS genes.A total of 60 DH lines,twenty each carrying the TGMS gene,the PGMS gene,or both,were examined for their pollen fertility from field grown plants,the main findings are:(1)DH lines carrying the PGMS gene can be divided into two categories,those whose fertility are affected by day length,and those showing a low degree of sterility in the long day condition,which might be regulated by other genes;(2)DH lines carrying the TGMS gene showed sterility at high temperature,however there were some differences in the fertility of these DH lines in relation to the temperature;(3)DH lines with P/TGMS genes were affected by both day length and temperature at the same time,but there were also some variations among these DH lines.