Association Analysis Of Glutamine Synthetase Cenes, Gln1-3 And Gln1-4, With Nitrogen Use Efficiency In Maize

Maize (Zea mays L.) is one of the most important crops as food, feed and industry use. Nitrogen is the important factor in the crop production. The low nitrogen use efficiency of cereals is difficult to handle in the agricultural research around the world. Therefore, it has become of major importance to improve nitrogen use efficiency of maize. Development of high nitrogen use efficiency varieties using maize diverse germplasm is an available way to resolve the yield loss caused by nitrogen stress. Discovering those inbred lines with high nitrogen use efficiency is the basis of maize breeding. Gln1-3 and Gln1-4 are members of GS (Glutamine synthetase)gene family that encoded the isozyme expressed in stalk and ear of maize. To generate new information for maize breeding programs, association mapping is an effective way to identify functional natural allelic variations related to high nitrogen use efficiency traits. Gln1-3 and Gln1-4 were chosen as the candidate genes in this study, which were certified to responsible for the kernel number and kernel weight of maize respectively. 189 maize inbred lines used in breeding programs of China were chosen for SNP (Single nucleotide polymorphism) genotyping and nitrogen use efficiency phenotyping. The objectives of this study were to identify functional alleles and haplotypes of high nitrogen use efficiency. The major results obtained were as follows:1. A total of 189 maize inbred lines were grown in the field , to evaluate for nitrogen use efficiency, on two levels of nitrogen fertilization (low nitrogen and normal nitrogen) in Hainan province over two consecutive years (2007 and 2008). The 19 phenotypic traits including chlorophyll content in pre-tassel stage, chlorophyll content of three leaves of the ear in pollination stage, chlorophyll content of three leaves of the ear in ten days after pollination stage, percentage of plant with seed, ASI (Anthesis-silking interval), plant height, ear height, kernel weight per ear, ear length, ear width, ear row number, kernel number, bared tip, ear weight, a hundred kernel weight and grain yield of the plot were investigated in these trials. The descriptive statistics from 19 traits investigated showed that 6 traits, chlorophyll content in pre-tassel stage, chlorophyll content of the top leave of the ear in pollination stage, plant height, a hundred kernel weight, kernel number and grain yield of the plot, were identified to evaluate inbred lines for nitrogen use efficiency. Based on factor analysis, integrated NTSI (Nitrogen tolerance synthesis index) of each inbred line was calculated to classify the maize inbreds tested into 4 different Nitrogen-tolerant types: highly nitrogen tolerance, medium nitrogen tolerance, medium nitrogen susceptible and highly nitrogen susceptible. 59 maize inbred lines had the same responses for nitrogen tolerance, including 17 highly nitrogen tolerant inbreds, 19 medium tolerant, 12 medium susceptible and 11 hghly susceptible.2. 4571 bp of genomic region of Gln1-3 was sequenced using PCR combined PCR walking strategy. The 3062 bp coding region of the gene was comprised of ten exons that were separated by nine introns. At the same time, 3824 bp of genomic region of Gln1-4 was sequenced using the same method. The full length of the coding region was 2858 bp, which was comprised of ten exons separated by nine introns. Gene structure of these two genes were analysed and two conserved domains were found through blast, this is the research base for association analysis.3. Software ClustalX was used to analyse the sequence polymorphism of these two candidate genes in 180 maize inbred lines. 1) A total of 140 SNPs and 42 Indels (Insertion and deletion) were identified in Gln1-3 gene. The number of sites'frequency more than 5% were 80, including 58 SNPs and 22 Indels, of which there was one SNP variation every 39 bp and one Indel every 104 bp, respectively. 2) A total of 148 SNPs and 38 Indels were identified in Gln1-3 gene. The number of sites'frequency more than 5% were 58, including 32 SNPs and 26 Indels, of which there was one SNP variation every 71 bp and one Indel every 88 bp, respectively. Relative higher LD (Linkage disequilibrium, r2>0.5) was identified in the region of 5' UTR, ammonium ion binding domain and ATPase domain of these two genes, association analysis should be focused on these regions.4. The results of candidate gene association analysis between genotypes and phenotypes of nitrogen use efficiency were: 1) A total of 19 polymorphism sites of Gln1-3 gene were identified significantly to be associated with 5 traits (LOD>2.5), chlorophyll content of the top leaves of the ear in pollination stage, plant height, hundred kernel weight , kernel number and grain yield. Of them, 10 polymorphism sites were identified significantly to be associated with grain yield, 8 polymorphism sites were identified significantly to be associated with plant high. Nucleotide variation C/T on site 2069 was most significantly associated with grain yield with contribution of 26.8%.In total, the contribution of the 10 polymorphism sites associated with grain yield was 32.7%. Based on five sites significantly associated with grain yield, 11 haplotypes were identified among 180 maize inbred lines. Haplotype 7 contained three positive contribution sites associated significantly with grain yield was speculated to be candidate nitrogen tolerance haplotype, which was included in nitrogen tolerant inbreds, such as Zheng30, CA339, CA156, etc. 2) A total of 14 polymorphism sites of Gln1-4 gene were identified significantly to be associated with 5 traits (LOD>2.5), chlorophyll content in pre-tassel stage, chlorophyll content of the top leaves of the ear in pollination stage, plant height, kernel number and grain yield. Of them, 11 polymorphism sites were identified significantly to be associated with kernel number and chlorophyll content in pre-tassel stage. 2 SNP sites were identified significantly to be associated with plant high respectively. SNP variation T/A on site -218 was most significantly associated with kernel number with contribution of 22.1%. In total, the contribution of the 11 polymorphism sites associated with kernel number was 36.7%. Based on five sites significantly associated with kernel number and chlorophyll content in pre-tassel stage, 10 haplotypes were identified among 180 maize inbred lines. Haplotype 1 contained three positive contribution sites associated significantly with kernel number was speculated to be candidate nitrogen tolerance haplotype, which was included in nitrogen tolerant inbreds, such as Qi205, 7595-2, Ji1037, Ji495, etc. 3) A total of 33 polymorphism sites of Gln1-3 and Gln1-4 were identified significantly to be associated with related trait of nitrogen use efficiency, the total contribution of some of the polymorphism sites were more than 20%, these results will be used to develop functional molecular markers in molecular assisted breeding in the future.