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Molecular Mapping of CMS-D8 Restoration and Gene Expression Specific to D8 Restoration

Jinfa Zhang, James McD. Stewart and Rickie B. Turley


 
ABSTRACT

CMS-D8 was developed by transferring the D8 cytoplasm from the American diploid wild species, Gossypium trilobum (D8 genome) into cultivated tetraploid cottons (G. hirsutum and G. barbadense). Male fertility can be recovered by two independent dominant restorer genes; Rf1 from the D2 restorer and Rf2 from the D8 restorer. Identification of molecular markers closely linked to the restorer genes could facilitate the development of hybrid cotton.

Bulked segregant analysis was used to tag RAPD markers that were linked to the restorer genes. Three test crosses were used for mapping Rf2 and one for Rf1. Two RAPD markers, UBC 1113000 and UBC 188500, were found to be associated with Rf2 in coupling phase. UBC 188500 was closely linked to Rf2 with an average genetic distance of 2.9 cM, although the distance varied from 0 to 5.1%, depending on the segregating population. A survey for UBC 188500 in several Gossypium species and cotton cultivars revealed that this RAPD marker was absent in normal cotton cultivars. However, it was present in the D8 restorer line of G. hirsutum, and in G. raimondii (D5), G. trilobum (D8) and several wild species of Australia. This indicates that Rf2 and the DNA corresponding to the RAPD marker were both introgressed into the tetraploid cotton nuclear background from the D8 genome. A RAPD marker, UBC 169Rf, was found to cosegregate with Rf1. The two RAPD markers, UBC 188500 and UBC 169Rf, were population specific. Since Rf1 and Rf2 were tightly linked (in repulsion) with a genetic distance of 1 cM, a linkage group comprising Rf1, Rf2 and three RAPD markers was established. Construction of a high-resolution linkage map containing Rf1 and Rf2 is underway.

Differential display of mRNA was employed to compare transcript patterns between 8518, a normal upland cotton, and isogenic line 8518R that had D8 cytoplasm and the D8 restorer gene, Rf2. 8518R with D8 cytoplasm was developed by repeatedly backcrossing the original D8 restorer, D8R, with 8518 as the recurrent parent. For comparison, two G. barbadense lines, 57-4 and Pima S-1, and D8R were included in the study. Anthers from one day preanthesis buds of the five genotypes were harvested and stored in liquid nitrogen prior to RNA extraction. Differential display of cDNA transcripts was performed using 12 anchor primers (9 dT plus 3'MN) and six decamer primers. Bands differential displayed following electrophoresis were excised from the gels, reamplified, cloned and sequenced. Thus far, 26 DNA sequences have been analyzed and compared with sequence databases using the BLAST program. Ten sequenced cDNA fragments were present only in the two restorer lines, 8518R and D8R, and absent in the three normal cottons, 8518, 57-4 and Pima S-1, while 8 sequenced fragments were present only in the three normal cottons. Four fragments were absent in D8R but present in the other four lines and 2 fragments were present only in D8R. One fragment was present and another absent only in 8518. Seventeen cDNA fragments were highly homologous to known sequences in GenBank, for example, cysteine proteinase, phosphoribosylanthranilate transferase, pectinesterase, starch synthase, nuclear transport factor, 4-coumarate-CoA ligase, aminopeptidase, ankyrin-like protein, eIF-4, and calnexin. The functions of these genes as they relate to male fertility restoration in cms-D8 system require further investigation.



Reprinted from Proceedings of the 1999 Beltwide Cotton Conferences pg. 448
©National Cotton Council, Memphis TN

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Document last modified Monday, Jun 21 1999