Mapping And Molecular Mechanism Analysis Of The Male Sterility Gene In Cotton Pgms Line ‘CCRI9106’

The use of photosensitive genetic male sterility（PGMS）to achieve heterosis can greatly improve the yield and quality of crops,and mapping male sterile genes and exploring their molecular mechanisms provide a theoretical basis for achieving this goal.Hybrid breakdown（HB）is a common type of reproductive barrier and has been observed in both plants and animals.In general,HB causes weakness and/or sterility in F₂ or later progenies.As one of the most important commercial crops in the world,cotton（Gossypium spp.）usually suffers from HB during inter-subspecific hybridization.However,the related genes and the underlying genetic mechanisms of HB in cotton remain unknown.In this study,the photosensitive genetic male sterile（PGMS）line,CCRI9106,was discovered to be a hybrid progeny of G.hirsutum and G.barbadense,and probably a product of HB.Then,we focused on mapping of the candidate genes and analyzed the potential mechanism underlying male sterility.In addition,Gh GPAT12/25 were studied for their functions on cotton anther development.The main results are as follows:1.Compared with WT,CCRI9106 was accompanied by shorter filaments and shriveled anthers,with the anthers did not dehisce,and lacked mature pollen grains at the late development stages of anther when stained with 1%I₂-KI solution.Two pairs of F₂hybridized combination were constructed using TM-1 and Hai 7124 as male parents to reveal the genetic basis of the male sterile phenotype.The results suggested that the male sterile phenotype of CCRI9106 was a recessive trait controlled by a single nuclear gene in each population.2.Based on a whole-genome sequencing-based bulked segregant analysis（BSA）and map-based cloning with the F₂ population of CCRI9106×TM-1,the male sterility gene was initially mapped on chromosome D12.Based on the data of recombination,we narrowed the locus to a 2.1Mb interval in-between QZD84 and QZD38.Among the 30 genes in the candidate region,24 of them exhibited variance site（s）in the coding DNA sequence.The re-sequencing results implied that the D12 chromosome of CCRI9106 may carry a large segment（from c.7 to 41 Mb）originating from the genetic background of G.barbadense.Based on the CCRI9106×G.barbadense F₂ populations,the male sterility was narrowed down to a c.15.9Mb region between A12＿1156 and A12＿1305 on the chromosome A12,which containing 138 genes according to the Hai 7124 reference genome.Therefore,the male sterility of CCRI9106 was controlled by two pairs of genes located on A12 and D12chromosomes.Therefore,CCRI9106 was predicted to be a hybrid progeny of G.hirsutum and G.barbadense,and its male sterility might be caused by the HB between these two subspecies.3.Paralogous gene pairs from A12/D12 chromosome were considered to be the most likely candidate genes due to the allotetraploid characteristics of G.hirsutum and G.barbadense.Based on their expression pattern and variant information,GH＿D12G1039and GB＿A12G1076 were considered to be the pivotal genes controlling male fertility in G.hirsutum and G.barbadense.These genes in Gossypium were named as Gossypium FLA19s（Go FLA19s）because of their similarity to At FLA19 which encodes a fasciclin-like arabinogalactan family protein in Arabidopsis.Based on the sequence alignment of Go FLA19s in Gossypium,specific variations occurring in fasciclin-like domain or/and AGP domain were predicted to cause the non-functionalization of Gb FLA19-D and Gh FLA19-A.CRISPR/Cas9-mediated knockout assay confirmed the effects of Gh FLA19s on male sterility.Variation analysis of a wide range of cotton accessions from various Gossypium indicated that the loss-of-functional Go FLA19s may occur after the formation of allotetraploid cottons.4.Gh FLA19-D showed persistent high expression levels in all the stages up to the mature stage,however,Gh FLA19-A was expressed at a low level in microsporocyte stage,and then dropped to an undetectable level in the subsequent stages.Further,RNA in situ hybridization demonstrated that Gh FLA19s was predominantly expressed in the tapetum and microspores during the meiosis stage,callose during the TTP stage and intine during binucleate stage.By preforming RNA-seq and q RT-PCR analysis,Go FLA19s were predicted affect the expression of genes required for tapetal development,pollen exine formation and pollen maturation.