FISH Localization Of Several Duplicated Loci Between Chromosomes A_h03/D_h02 And A_h03/D_h03 In Gossypium Hirsutum

Cotton is a model plant for studies on cytogenetics, genomics and evolution. Chromosome 3 is homoeologous to chromosome 14 and 17 in allotetraploid cotton, respectively. Here for Gossypium hirsutum, we name chromosome 3 as Ah03, chromosome 14 as Dh02 and chromosome 17 as Dh03. In this study, several duplicated markers between homoeologous chromosomes in allotetraploid cotton were used to screen Pima90-53 BAC library, and their chromosomal physical localizations were identified by BAC-FISH technology. The cytogenetic maps of corresponding chromosomes were constructed and they were integrated with genetic maps and Gossypium raimondii(D5) assembly map. The main results were as following:1. In this study, several BACs screened from duplicated SSR markers between Ah03 and Dh02 and Ah03 and Dh03 in G. hirsutum were used for FISH mapping. Cytogenetic maps of Dh02 and Dh03 were constructed in G. hirsutum, which included seven and one duplicated marker-derived BAC clones, respectively. The genetic map and cytogenetic map of Dh02 were integrated and the analysis revealed that localization orders of the FISH-mapped loci along the middle of the genetic map showed generally similar except the two loci(SHIN659 and BNL226). The integration of cytogenetic map and genetic map of Dh03 revealed that the physical and genetic localization of FISH-mapped loci NAU3016 showed considerable variation between the two maps.2. The FISH-mapped BACs were sent to biotechnology company for BAC-end sequencing. Using BLASTN algorithms, the sequences were compared successively to the draft map of G. raimondii(D5) assembly(Paterson et al., 2012). BACs FISH-mapped on Dh02 were all aligned to pseudo molecule D505 in G. raimondii assembly and BAC 407N13 FISH-mapped on Dh03 was aligned to D503 with E-value eaqual or nearly to 0, respectively. Physical distances between forward and reverse sequences hits of the five BACs, 57H14, 388G13, 348F12, 24F23 and 270G20 in G. raimondii genome assembly were all ~100Kb apart, which were close to the insert size of the Pima 90-53 BAC library, indicating that the lengths of these BACs were relatively conserved duing evolution. The physical distributions of BACs on the G. raimondii assembly map and cytogenetic map of Dh02 in G. hirsutum were compared, and the linear orders and positions of these BACs were congruent between two maps, which revealed the colinearity between the D subgenome and D genome in Gossypium.3. Of the seven BAC clones FISH-mapped on Dh02, the BAC clone 270G20 was homoeologous BAC because it generated FISH signals on both Ah03 and Dh02, and the signal on Ah03 was brighter than that on Dh02 in G. hirsutum. More interestingly the clone 270G20 was found also generated a pair of signals on G. arboretum and G. herbaceum while no signals on G. raimondii, and so we suggest a chromosomal duplication in the homoeologous chromosomes evolution after polyploidization.4. A positive BAC clone 350B21 was got by screening the Pima 90-53 BAC library using SSR marker CIR093 of tetraploid cotton genetic map. It was hybridized to mitotic chromosomes of five tetraploid cottons, two A-genome diploid cotton and two D-genome diploid cotton using FISH. The clone 350B21 generated signals on the terminal positions of chromosomes of A genome and A sub-genome in these cotton species while no signal on the chromosomes of D genome and D sub-genome. Herein, using the clone 350B21 as a FISH marker may be an effective tool to distinguish the whole chromosomes between A genome(sub-genome) and D genome(sub-genome).