Creation Of Sexual Hybrid Populations And Construction Of Molecular Genetic Linkage Framework Map In Citrus

Citrus is a very important fruit for agricultural economy in the world. Citrusbreeding and construction of linkage map has been hindered by several factors includingpolyembryony, female/male abortion and high hetetozygosis. In an effort to resolveabove difficults, at first, 14 different cross combinations of citrus were carried out toobtain the hybrid plants via embro rescue and molecular marker verification; Then, thelinkage framework map of citrus was constructed with molecular markers by the geneticanalysis of F₁ population from the red tangerine (C. reticulata Blanco)×trifoliate orange(Poncirus trifoliata (L.) Raf) cross and embryogenesis capacity of trifoliate orange (T)hybrid calli from embryo rescue was tested. The main results of this research are asfollows:1,A total of 1,087 fruits and 4,846 seeds were obtained from 14 different crosscombinations which was conducted in 2003 and 2004, with HB pummelo (C. grandis (L.)Osbeck 'Hirado Buntan'), 'Bendizao'tangerine (C. reticulata 'Huanong'), 'Guoqing'Satsuma (C. unshiu Marc.), Grapefruit (C. paradisi Macq.) and Red tangerine (C.reticulata Blanco) as female, and trifoliate orange, 'Nova' tangelo, 'Page' tangelo,'Fairchild' tangelo and 'Murcott' tangor as pollen parents. A total of 2,029 progenies wasobtained from those crosses via embryo culture, 1,187 seedlings successfully survivedafter transplantation. Four hundred and twenty three ( 262 for HB×T, 49 for S×T, 111for R×T and 1 for B×T) out of 990 individuals exhibiting the trifoliate phenotype oftrifoliate orange were considered to be hybrids by morphology identification and SSRverification; Six hundred and fourteen of 756 plantlets derived from other crosses wereverified to be hybrids by SSR analysis. Of them, 244 for HB×'Nova' tangelo, 179 forHB×'Page' tangelo, 172 for HB×'Fairchild' tangelo, 1 for 'Bendizao'tangerine×'Nova'tangelo, 7 for 'Bendizao'tangerine×'Page' tangelo, 3 for 'Bendizao'tangerine×'Murcott'tangor and 8 for satsuma mandarin×'Nova' tangelo. The hybrids provided the materialsfor citrus variety selection, rootstock breeding and construction of linkage map.2,Fouty five out of 84 genomic SSR primers, 30 out of 68 EST-SSR primers, 15 outof 45 RAPD primers and the Lcyb-CAPs marker exhibited the polymorphisms andsegregations among red tangerine, trifoliate orange and ten hybrids. Those polymorphic primers were screened out for amplification in the F₁ population (107 individuals) of redtangerine×trifoliate orange, and 114 segregated loci (53 SSR loci, 30 EST-SSR loci, 30RAPD loci and 1 Lcyb-CAPs loci) were obtained. Of which, 20.2% (23 loci) showed thedeviation of different Mendelian segregation type, and other loci (91 loci) segregated in1:1 or 1:1:1:1 or 1:2:1 or 3:1 ratio. The percentages of skewed loci for genomic SSR,EST-SSR and RAPD loci were 28.3%, 6.7% and 20% respectively.3,The consensus framework map of red tangerine×trifoliate orange was producedby linkage analysis using Joinmap3.0 software, 36 (15 SSR, 19 EST-SSR, 1 RAPD and 1Lcyb-CAPs) out of 91 examined loci were assigned to 9 linkage groups, with a totallength of 329 cM, and the loci linkage to the Lcyb gene were found. There was no severeeffects on the position of previous loci after adding 7 new loci(including 5 slightlyskewed loci) into the map, and the map length achieved 417 cM, which was suggestedthat the little distorted loci also could be use for map construction. Eighty one loci for redtangerine, 57 loci for trifoliate orange and 114 loci for red tangerine×trifoliate orangewere analyzed respectively, to construct the linkage maps. Eleven linkage groups with 49markers in red tangerine, 8 with 20 markers in trifoliate orange, and 14 with 56 markersin the cross pollinator (CP) consensus of both, were constructed. About 477 cM of redtangerine genome, 202 cM of trifoliate orange genome were covered. By comparison ofshared markers, a high level of colinearity existed among different linkage groups forthose three maps. This map was mainly composed of codominant markers; It will beessential for citrus comparison map, mapping further saturation and resistant genelocation.4,Immature embryos of 80, 85, 90 days after pollination (DAP) from the Satsumamandarin (S)×trifoliate orange (T) cross, 80 and 85 DAP from the Red tangerine (R)×trifoliate orange (T) cross, were cultured on MG1.0 medium consisting of MT basalmedium supplemented with 1.0 mgl^-1 GA₃ and 4% sucrose. The results showed that 80DAP was the optimal time for embryo rescue of the tested crosses. Among the eighttested media, MT medium supplemented with 0.5 mgl^-1 GA₃ was the best one forsatsuma mandarin, and MT plus 1.0 mgl^-1 GA₃ for red tangerine. By testing theembryogenesis capacity of hybrid calli derived from embryo rescue, it was observed thatno embryoids appeared on the tested seven media for hybrid calli of red tangerine×trifoliate orange, however, the capacity of embryogenesis still be possessed by hybridcalli of satsuma mandarin×trifoliate orange on M2 (MT+5% lactose), M6 (MT+500 mgl^-1 ME+5% sucrose) and M7 (MT+500 mgl^-1 ME+3.0 mgl^-1BA+5% sucrose)midia.