Construction Of A Maize Genetic Map And Maize Transformation Via Pollen-Tube Pathway

Maize (Zea may L.) is an economically important crop in the world. Maize is also the best-studied and most tractable genetic system among the cereals, making it the premier model system for studying this important group of crops. Maize yield in China is next below that in the United States, which plays an important role in national economy. So both basic research and genetic improvement of maize have great theoretic and practical significance.For any crop plants, a genetic map serves as the foundation of numerous studies. Up to now, high-resolution maize genetic maps have been generated using many groups of molecular markers. However, the maize genome variations may trouble the work of gene mapping or cloning with special maize lines, if the basic genetic map was not constructed with the same parental lines. So it is necessary to construct genetic map with different maize lines, which is the fundamental step in maize genomics research. In this study, we constructed a molecular linkage map for maize with the F₂ population of Ye478×90110. Inbred line Ye478, with the characteristics of high combining ability, high yield and lodging-resistance, accounted much for maize production in China. However, this line is susceptible to maize virus disease. Inbred line 90110, with the characteristics of high combining ability, high yield and high resistance to MRDV (Maize Rough Dwarf Virus), is a member of P group, which is a new maize group and an important supplement to the old maize heterosis groups in China.The polymorphisms between parental lines (Ye478 and 90110) were detected with 317 markers covering almost whole maize genome. Of these, 215 (67.8%) showed polymorphisms between the two parents, including 59 RFLP markers (68.6% of total detected RFLP markers) and 156 SSR markers (67.5% of total detected SSR markers). RFLP analysis presented 145 combinations (33.7% of total detected combinations) of "probe/enzyme" showing polymorphisms between parents with five restriction enzymes. According to band patterns, 149 optimal markers were chosen to test 150 random individuals of a Ye478×90110 F₂ population. Through Chi-square test, 13 markers weredetected segregation distortion among five different chromosomes. We used MAPMAKERP/EXP3.0 to construct the maize linkage map. The map consisted of 134 molecular markers and spanned maize genome about 2201.1cM with the average distance of 16.4cM between markers, which formed the frame molecular linkage map of Ye478><90110. On this map, locations of most markers were identical with that on the IBM (MaizeGDB) map. Relative to IBM map, the linkage map located several markers on different positions, inclulding: umcl841 (bin7.03, IBM) was located on chromosome 2; bnlg381 (bin2.04, IBM) was located on chromosome 5; umc36 (bin2.09, IBM) was located on bin2.07; umclO4 (bin5.08, IBM) was located on bin5.03⁵.04; umc76b (bin9.03, IBM) and umcl733 (bin9.08, IBM) were located on bin9.05 9.07 respectively.The genetic diversity of parental lines was analyzed among different chromosomes and special chromosome regions. The genetic similarity (GS) of parents was 0.682, which indicated the close hereditary relation between parental lines, even if they belong to different maize groups. Furthermore, the GS of chromosomes varied from 0.564 to 0.801, and that of special chromosome regions varied from 0.145 to 0.920. Among them, the chromosome regions with higher GS include binl.01 (0.920), bin3.05 (0.916) and bin8.06 (0.894), and that with lower GS include bin9.07 (0.145), bin3.04 (0.189) and binl.06 (0.372).Maize rough dwarf disease (MRDD) is severe in recent years. Inbred line Ye478 is highly susceptible to MRDV. However, inbred line 90110 is highly resistant to MRDV. Here we not only constructed the linkage map, but also built a segregated population over generations from the cross of Ye478x90110. The population could serve as the plant materials for studying the resistance to MRDV.The segregated population was generated from 60 original F2 plants by successive self-pollination up to F6 generation, with 3⁴ plants self-pollinated in each F3 or later-generation line. During 1999-2003, the segregated population was evaluated the resistance to MRDV by natural infection. In 1999 and 2000, the resistance of parental lines, Fi, F2 and F3 populations were evaluated. The symptom or'Ye478 showed severe with the disease index of 61.1 (1999) and 71.1 (2000), whereas the 90110 line and Fi displayed completely resistant to MRDV. The mean resistances of F2 (0.5, 1999; 0.57,2000) and F3 population (0.65, 2000) were between that of parents and biased to the resistant-parent Within F2 and F3 populations, the resistant plants were significantly more than the susceptible ones, indicating major genes may control the resistance to MRDV.The genetics of resistance was analyzed with the parental lines, Fi, F2 and F3 populations. Through the single-generation and multiple-generation scale tests, the resistance was confirmed fitting for the additive-dominant genetic model. The genetic parameters, nu [d] and [h], were estimated as 0.9236, -1.1001 and -0.9794 by weighted least square method respectively. So both additive and dominant effects were detected remarkable, and the additive factor was predominant. Moreover, the broad heritability were estimated with F2 (86%, 1999; 85%, 2000) and F3 (83.3%, 2000) populations respectively.For the disease pressure reduced from 1999 to 2003, we scored the resistance scales of higher-generation lines with the relative index of disease. Along with successive self-pollination, the mean resistance tended to reduce with the populations. In F5 or F6 generation, we found the lines showing completely resistant or susceptible to MRDV, and that segregating for MRDV-resistance following Mendelian fashion. These lines could be used to map the resistant genes to MRDV.The two principal maize transformation methods are particle bombardment and Agrobacteriurn-mediated transformation. The pollen-tube pathway is a transformation method developed by Chinese scholar, which has been successfully used in many crops. In this study, we introduced the als gene, a herbicide-resistant gene from Arabidopsis thaliana, into maize inbred line Qi319 by pollen-tube pathway, and analyzed the inheritance of transgene up to T3 generation. Moreover, the characteristics of als inheritance were compared over generations among the transgenic maize plants that generated by pollen-tube pathway, particle bombardment and Agrobacterium-mediated transformation respectively.The maize transformation via pollen-tube pathway was performed by method A (applying plasmid DNA solution on the severed styles after self-pollination) and method B (applying plasmid DNA solution on the severed just prior to self-pollination). A total of 44 ears were treated, and 28 of them set seeds. 1200 treated seeds were sown in flowerpots for screening the TO plants. Through herbicide screening, 6.3% of the TOplants were herbicide resistant. By PCR and southern blot analysis, 17 TO plants were confirmed transgenic, in which 16 were obtained following transformation method B. The B-3 procedure, that is, applying 200ul plasmid DNA solution (120u.g/ml) on the severed styles prior to self-pollination, obtained the maximal positive rate (6.9%). The inheritance of als was analyzed up to T3 generation. 12 out of 16 Tl lines included the positive plants. But none of them segregated the als gene according to expected Mendelian ratio of 3:1. Out of 65 T2 lines, 47 showed the plants including als gene. Through Chi-square test, only line QP8-3 segregated the als gene according to expected Mendelian ratio. In T3 generation, 88 out of 115 lines presented the als gene in a portion of plants. Through Chi-square test, lines QP4-3-1, QP4-3-3 and QP4-4-1 segregated the als gene following the 3:1 Mendelian ratio. All plants of seven lines from QP8-3 were detected with als gene, so line QP8-3 was confirmed as a stably inherited line of transgene. Moreover, the loss of als gene was found in higher rate with pollen-tube pathway method.In this study, we compared the inheritance of als gene transformed by pollen-tube pathway, particle bombardment and Agrobacterium-mediated transformation respectively. As a result, the als gene transformed by pollen-tube pathway was not transmitted according to Mendelian fashion in most cases, especially in early generations. Otherwise, the als gene delivered by particle bombardment and Agrobacterium-mediated transformation was inherited generally according to Mendelian fashion. Moreover, the als gene was introduced into maize genome with single or lower copies (general 1-2) by Agrobacterium-mediated transformation, but that introduced by particle bombardment tended to be more copies, resulting in distortion segregation of transgene sometimes. Therefore, the Agrobacterium-mediated transformation has advantages over pollen-tube pathway and particle bombardment method in maize transformation.