Fine Mapping Of Stem Glandless Gene Gl₁ In Cotton And Gossypol Gland Development Related Genes Screening

Cotton is not only an important fiber and oil plant in the world, it is also the second important source of vegetable protein. However, the seeds of cotton cultivars have glands which contain gossypol and its derivatives toxic to humans and monogastric animals, therefor become the biggest obstacle to the utilization of cottonseed. On the other hand, as a natural phytoalexin, the presence of high levels gossypol have a certain antagonism topathogens and pests. Therefore, identification of key genes and the molecular mechanism of the gossypol glandformation, breeding for low gossypol in cottonseed and high gossypol in plant is an important direction. T582, which carrys gl₁, showed no glands on stems, petioles, and boll shell, wherease other parts of the plant have glands. The glandless trait is controlled by a single recessive gene. In this study, gl₁ gene was fine mapped through constructing a plurality of mapping population, combined with the development of chromosome specific SSR markers, furthermore we screened some genes close related to the development of gossypol glands by transcriptomics methods, the main results are as follows:1. F2 population was constructed using TM-1 as female parent, T582 as male parent. Another three BC1 and three F2 population were constructed using three Island Cotton（H7124, Pima90,3-79） as female parents, T582 as male parent. Three BC1 population were selfed to BC1F2 population. The total number of all individules is up to 8627. Genetic analysis of gl₁ using all these populations showed that the segregation ratio of F2 population is 3:1, the segregation ratio of BC1 population is 1: 1. This result further confirmed that the glandless character was controled by a pair of single recessive genes.2. SSR markers were developed from chromosomal sequences of two diploid genome. 200,744 SSRs were found in 13 chromosomes of G. arboreum genome, 142,409 SSRs were found in 13 chromosomes of G. raimondii genome. Chromosome specific SSRs were developed using flanking sequence BLAST based on the development of the whole genome SSR. 115,108 chromosome specific SSRs were found in G. arboreum, average 8854 each chromosome. 93,780 chromosome specific SSRs were found in G. raimondii, average 7213 each chromosome.3. Two homologous chromosomes of tetraploid cotton was used for the varification of chromosome-specific character of the developed SSRs. 459 and 448 SSRs were designed in chr01（corresponding to tetraploid chr07） of G. arboreum and chr01（corresponding to tetraploid chr16） of G. raimondii. Polymorphic SSR from chr01 of G. arboreum were only linked to chr07, and polymorphic SSR from chr01 of G.raimondii were only linked to chr16, indicated that these SSRs had chromosome specific character.4. 1691 pairs of primers were synthesized using the chromosome specific SSRs on 26 chromosomes. 6 glanded and 6 glandless cotton were used for linkage analysis of gl₁ gene, and 18 SSRs were obtained to be linked with gl₁ gene. gl₁ gene was mapped to an interval between 3786 and 3864 with 5.9cM, about 3.2Mb, using one BC1 population with 113 individules. Two SSR markers（4023 and 4101） in the interval were completely linked with gl₁ gene.5. 100 pairs of SSR primers were designed in the 3.2Mb interval, gl₁ gene was mapped to an interval with 800 kb between 4023 and 4101. Then, all the 86 SSRs in the 800 kb interval were designed, gl₁ gene was mapped to 420 kb between 3-18 and 3-64. Since all SSRs had been developed, another 99 pairs of primers were designed for the amplification of random sequences, gl₁ gene was eventually mapped to an interval between 4-3 and 4-81 with 330 kb. We also tried to narrow the mapping interval by using BC1F2 population with about 4000 individuals and "TM-1 × T582" F2 population with 3427 individuals, but only confirmed the mapping interval is reliable.6. The mapping interval contains seven genes through candidate gene analysis. Expression analysis of these genes revealed that their expression showed no difference between glanded and glandless cottons. We also constructed the virusinduced gene silencing（VIGS） vectors of these genes for functional verificationof gl₁ gene.7. Transcriptome sequencing and comparison was conducted using five homozygous glanded cotton and 5 homozygous glandless cotton. These 25 repetitions of comparison were used to find genes most closely related to gland development. We found 45 differentially expressed genes and 102 differentially alternative spliced genes. These two kinds of genes were independent of each other, and no intersection was observed. Functional analysis revealed that these gene were aminly related to gossypol biosynthesis, development of gland cell and plant pest-resistant characteristics.