Sterility Characteristics And Gene Localization Of Nuclear Male Sterile Mutant GMS104 In Pepper

Pepper（Capsicum annuum L.）is an important vegetable crop in the world.As a condiment,it has been widely planted in tropical and temperate regions around the world.Chili pepper is native to Latin America and was introduced into China by sea or land from Europe.Pepper has become the largest vegetable crop in China because of its high planting efficiency and nutritional quality,as well as its unique spicy taste.The yield of pepper is related to the safety of vegetables in our country.Pepper has strong heterosis,hybrid offspring can significantly improve fruit quality,increase economic yield,and significantly improve disease resistance and storage tolerance of pepper,which brings huge economic benefits to pepper industry.Pepper cross breeding has been popularized in the 1990s,but artificial castration consumes a lot of manpower,and the seed purity can not be guaranteed.Therefore,the use of male sterile line breeding has become one of the important ways of new pepper variety breeding.Much attention has been paid to the breeding of male sterile lines,the utilization of heterosis and the application of hybrid techniques in pepper.In recent years,a series of achievements have been made on the relationship between morphological characteristics and physiological and biochemical male sterility.With the progress of molecular technology and the improvement of instrument level,molecular marker-assisted breeding has become an important direction of pepper breeding.In this study,the nuclear male sterile mutant GMS104 of pepper was used to observe the anther morphological characteristics of the genic male sterile mutant GMS104,the microspore abortion process was studied by paraffin section,the dynamic changes of soluble protein,proline and MDA content with flower bud development and the effect of SOD activity on male sterility were determined.The genetic analysis of sterile traits was carried out by constructing F₂genetic segregation population,and the candidate interval was determined by BSA analysis and PARMS molecular marker technology.By analyzing the expression of candidate genes in the candidate interval,it was found that the candidate gene Capana10g001221 showed obvious specific expression in flower tissue.The 3D protein structure of the candidate gene was predicted.The main results are as follows:1.Morphological observation and cytological study on Flower Bud of Genic male sterile Mutant GMS104 in PepperThe flower buds were divided into five stages:sporogenic cell stage,pollen mother cell stage,tetrad stage,uninucleate microspore stage and pollen formation stage.It was found that microspore abortion occurred in the late stage of tetrad,during which tapetum cells delayed disintegration,obviously later than fertile plants,and overexpanded.The abnormal development of tapetum prevents the tetrad from developing into normal mononuclear microspores.In the pollen mature stage,the anther chamber is narrow and there are no fertile mature pollen grains,the anther chamber is covered with cell decomposition debris,and there are no pollen grains after anther maturation and cracking.2.Study on physiological and biochemical dynamic changes of flower bud of genic male sterile mutant GMS104 in pepperIn the same growth period,the MDA content of male sterile material GMS104 in pepper was higher than that of fertile material ST-8.There were significant differences in pollen mother stage,tetrad stage and binucleate microspore stage.The SOD activity of GMS104 reached the lowest value in uninucleate microspore stage,while the lowest SOD activity of ST-8 occurred in binucleate microspore stage.The soluble protein content of male sterile material GMS104 decreased gradually in uninucleate microspore stage and pollen grain mature stage,which was opposite to that of fertile material ST-8.The proline content in GMS104 was lower than that in ST-8,and there was a very significant difference in proline content between ST-8 and GMS104 at pollen maturity stage.3.Location of candidate interval of nuclear male sterile mutant GMS104 in capsicum.Using wild type S8 and nuclear male sterile mutant GMS104 induced by EMS as parents,a total of 321 isolates of F₂generation were constructed.The fertility and sterility characteristics of F₂segregated population were observed,and the extreme mixed pool was selected for BSA sequencing.A significantΔSNP-index peak region was found on chromosome 10（50.06-190.17 Mb）by BSA analysis.TheΔSNP-index value of this region was above 0.5 and close to 1,and a total of 10090 candidate sites were obtained in this region.Through designing PARMS molecular markers for many times and verifying the effectiveness of typing,22 groups of effective PARMS molecular markers were obtained.Finally,the candidate interval of GMS104 genic male sterile gene is reduced to the range of 111 Mb-117.3 Mb in Chr10,and the candidate interval distance is 6 Mb.4.Analysis of candidate genes of Genic male sterile Mutant GMS104 in PepperBy analyzing the expression of 12 candidate genes in 6 Mb candidate interval（111 Mb-117.3 Mb）on Chr10 in different tissues and periods,it was found that the candidate gene.10g001221 showed obvious flower tissue-specific expression in flower tissues.The 3D protein structure prediction of the candidate gene is helpful to the identification and mining of pepper male sterile genes.