Creation Of Excellent Introgression Lines From The Hybrids Of Maize And Zea Mays Ssp. Mexicana And Cytological Analysis For These Lines

Maize is an important food and feed crop in the world today. The "domestication bottleneck" effect and the subsequent artificial selection for improvement reduce the genetic diversity on maize relative to the wild progenitors. Moreover, contemporary breeders prefer current, elite inbreds with known combining ability as sources for inbred development and for the improvement of hybrid performance, making maize genetic base narrower than before. China is not the original area of maize domestication and short of germplasm for maize breeding. No great progress in product for single cross hybrids has been made in China in recent years. It is unanimous that there is no breakthrough for maize improvement without germplasm enrichment and creation, so exploring and utilizing the wild relatives plays a key role in this strategy.Zea mays ssp. mexicana, an annual teosinte, is a wild relative of maize. It has some valuable traits, such as strong growth vigor, high protein content in the kernel and notable immune ability or resistance to multiple fungal diseases, making it beneficial to maize quality and resistance improvement. Ye515, an elite inbred line widely used in China, has good agricultural traits such as high combining ability and large kernel rows, however, it also has some disadvantages, such as imperfect kernel quality, high infection of maize stalk rot, maize rough dwarf disease and brown spot. In present study, F₁ plants were produced by hybridization with maize elite inbred Ye515 as female parent and Z. mays ssp. mexicana as pollen parent. Then, the filial populations of BC₁F₃ and BC₂F₂ (population I and III) were obtained under conventional breeding scheme. Meanwhile, another two corresponding populations (population II and IV) were constructed via conventional methods combining with tissue culture. Immature embryos from selfed and backcrossed BC₁ plants were inoculated on modified MS medium to induce embryogenic calli. After 8-9 months of culture, plantlets were regenerated from embryogenic calli. The regenerated plants were self-pollinated to develop BC₁F₃ and BC₂F₂ generations. After successive self-pollination for several generations, the uniform progeny lines resembled cultivated maize in plant type, kernel yield and other traits, of which some presented excellent agronomic traits.Morphological and cytological analysis of different populationsPhenotypic characters of thirty randomly selected ear-lines from each population were evaluated. The agronomic traits were obviously different among lines. Some teosnite specific characters were still manifested in some lines of each population, such as ratooning or ramifying ability, leaf-like bracts and lanceolate leaf. Population II possessed the highest frequency of 63.3% for the lines with wild parental specific characters. Among these four populations, the infection frequency of stalk rot and rough dwarf diseases was lower than 10%, indicating economically important genes from wild relative might have introgressed into them. Five lines were randomly selected from each population for karyotype analysis. The chromosome number was consistent with the euploid number of 20 and no numerical chromosome changes were observed. Constitutive heterochromatin by C-banding mainly presented in terminal or subterminal regions of the chromosome arms. The C-banding polymorphisms were a result mainly of the presence/absence of particular bands. Variation was also observed for band size and staining intensity. Ten pairs of chromosomes in examined lines were apportioned to either metacentric or submetacentric chromosomes (m or sm), according to arm ratios. The variation of arm ratios and kayotypic formula were obvious within and between populations.Ye515 (18m+2sm) possessed metacentric chromosomes (m) except for chromosome 6 being a sm. In line I-12 (population I) (10 m +10 sm), chromosome 4, 7, 8, 9 exhibited obviously increased arm ratios and changed into sm chromosomes from m chromosomes. Karyotypic formulae for line II-96 (population II), III-77(population III), IV-18 (population IV) were 2n=12m+8sm, 2n=14m+6sm, 2n=10m+10sm respectively. For arm ratios, 26% (Population I) to 38% (population II) of chromosomes from the population differed significantly from that of Ye515. GISH analysis was conducted using Z. mays ssp. mexicana genomic DNA labeled by digoxigenin as the probe and Ye515 genomic DNA as a blocker. Obvious yellow-green alien chromosome segments, rather than entire chromosome (s) or chromosome arm (s) were observed in the metaphase cells, and some lines didn't show any hybridization signals. So the karyotypic variation for these lines was not the result of introgressed entire alien chromosomes. The average number of chromosomes with hybridization signal was 3.3 in the cell from population I (progeny of BC1F3). For population II, derived from tissue culture scheme, alien chromosome segments were present in all the lines analyzed, with averaged 6.5 chromosomes per line carrying hybridization signals. The average number of chromosomes introgressed alien chromatin of population III, IV was 2.5 and 5.9 per line, respectively. The introgression frequencies among the 10 chromosomes were different. In the four populations, the most signals were detected on chromosome 5 (above 50%), followed by chromosome 6. Hybridization signals were mainly distributed in the terminal or subterminal regions of the chromosomes.Tissue culture facilitated the introgression of genetic material from Z. mays ssp. mexicana into cultivated maizeThe frequencies of lines with teosinte specific traits in population II and IV that derived from tissue culture were 63.3% and 36.7%, respectively, vs. 40.0% and 20.0% in the corresponding conventional population. The mean number of chromosomes per line carrying hybridization signals for population II and IV was twice as much as that of the corresponding traditional population I and III, suggesting that tissue culture promoted the introgression of alien chromatin.The signal sizes were notably different among populations. Three signal sizes (S, M, L) were classed according to the ratio of hybridization signal area to that of the chromosome, which it resided on. The ratio for S was less than 20%, M from 20 to 30% and L larger than 30%. The frequency was 54.5% for M type, followed by 27.3% for L type, and 18.2% for S type in population I vs. 33.3%, 24.3% and 42.4% , respectively, for population II. In the population III, M was the major introgression type (57.2%), while S was the least (9.5%). Of the signals from population IV, 45.5% were L; 34.1% were M and 20.4% were S type. From these results we concluded that the population II, the self-pollinated progeny of BC₁F₃ via tissue culture scheme, was the best population in the purpose of getting small-segment introgressants by wild hybridization and the linkage drag effect of alien chromosome segment would be much reduced. Population IV, also derived from tissue culture, showed rather high introgression frequency, but the ratio of introgressants with small alien segments was not ideal.Creation of excellent maize inbred lines using alien introgression from Zea mays ssp. mexicanaThe advanced introgression lines selected were crossed to testers Ye478, Ye502, P138 and Huangzao4, respectively; all of the testers were elite inbred lines in China. Total of 152 testcross hybrids were evaluated in 2004 at Jinan and Weifang, respectively, with three commercial hybrids Yedan12 (Ye478×Ye515), Nongda108 and Zheng958 as controls for agricultural traits and yield test. 83 (54.6%) hybrids (Weifang) had greater testcross grain yield than Yedan12. 26 (17.1%) and 15 (9.9%) hybrids yielded competitive or greater grain than that of commercial hybrids Nongdal08 and Zheng958, respectively. Among them, SD00333×Huangzao 4 had higher yield than that of Nongda108 and Zheng958, respectively, with significant difference at 0.01 level. These results indicated that some excellent alleles/genes from wild parent were reserved in the introgression lines.A few of good introgression lines with high yield and good agriculture traits were crossed to elite inbred lines B-9 and Chang 7-2 for performance test. Three hybrids with higher yield than control hybrids Denghai9 and Zheng958 showed promising prospect for future application.The value of Zea mays ssp. mexicana for maize quality improvement The endosperm protein and seed protein content of Zea mays ssp. mexicana were 22.51% and 21.47%, respectively, higher than that of Ye515. There were great differences in total protein content in different introgression lines. The protein content of some inbred lines was significantly higher than that of Ye515, but lower than that of Zea mays ssp. mexicana. These results suggested that some genes involved in high protein content from Zea mays ssp. mexicana might have introgressed in these lines.Endosperm, which occupies more than 80% of the whole seeds, is the main deposition location of many reserves. The protein quality and quantity of endosperm is the main factor of grain quality. The endosperm proteins were extracted from Ye515 and Zea mays ssp. mexicana by TCA/ acetone and Tris buffer methods, respectively, and were analyzed by two-dimensional gel electrophoresis. Protein expression maps of them were analyzed using PDQuest software (version 7.2.0; Bio-Rad). 253 and 215 protein spots were detected from Zea mays ssp.mexicana and Ye515, respectively, using TCA/ acetone extraction method. There were 58 spots changed significantly (P < 0.01), which included 28 up-regulation spots (8.7%), and 11 specific spots (4.35%) to Zea mays ssp. mexicana compared to that of Ye515. While 255 and 231 protein spots were detected using Tris buffer extraction method in Zea mays ssp. mexicana and Ye515, respectively. There were 48 significantly different protein spots between them, including 17 (6.67%) up-regulation spots and 13 (5.10%) specific spots to Zea mays ssp. mexicana. These results indicated that some genes, which influenced grain protein content and quality, might have lost or been repressed during the course of maize domestication. So it is significant to enrich maize germplasm through re-introducing these genes into maize genome by crossing with wild progenitor.The present work explored effective pathway and methods for transferring useful genes from Zea mays ssp. mexicana to cultivated maize for the creation of excellent materials for maize breeding. The results confirmed the important value of Zea mays ssp. mexicana for maize improvement and obtained some excellent introgression lines. In the present study, the introgression frequency and the size of alien chromomatin were determined in different derivative lines and populations, which offered vital information for getting alien introgression lines with high efficiency and affirmed that tissue culture could increase introgression frequency. Two-dimensional gel electrophoresis results showed that Zea mays ssp. mexicana is an important germplasm for the improvement of maize grain protein. Some introgression lines produced by this work were ideal materials for identifying and mapping useful genes from Zea mays ssp. mexicana.