High Amylose Maize Germplasm Cluster Analysis And Development Of Molecular Markers For Sbeâ…  And Sbeâ…¡b Genes

Maize (zea mays L.) is a widely planted crop in the world. As a raw material, maize amylose is on a more wide range of uses in industrial. But in the conventional maize, low amylose content and high costs of amylose extracting and separating from kernel limits amylose application. Therefore high amylose maize breeding is very important. ae is the major amylose content controling gene. Meanwhile there were modifier genes affecting amylose content. In this study, we cloned the ae gene and sbeI gene based on the published sequence in the NCBI, developed the molecular and functional markers , and applied the markers to the selection of breeding in high amylose content maize, respectively. The results are as follows:1. 65 allelic genes were amplified from 16 high amylose maize lines and 2 conventional maize with 25 pairs SSR molecular makers. The genetic similarity coefficient was 0.446⁰.923. These 16 high amylose maize lines were divided into two categories based on the UPGMA results. Among them, GMES-0067 were divided into NSS Group. GMES-0067 is a very important germplasm resource because amylose content of it is more than 70%. We studied the heterosis groups of the selected inbred lines through analyzing the genetic diversity of the high amylose maize inbred lines. The results will provide information for accelerating high amylose maize breeding and germplasm improved.2. We designed the primer according to the ae gene sequences (AF072725) that were published in NCBI and cloned the cDNA of the ae gene of GEMS-0067,LH324,BC300,I115 and Chang7-2. Through gene sequencing and comparative analysis, we found high homology between the high amylose corn and convention corn. However, the cDNA sequences of the 9^th exon were deleted completely and some base-pairs mutated in high amylose maize.3. Since all ae mutants had the 9th exon deleted,an ae-InDel primer, targeting the sequences flanking the 84-bp deletion spectific for ae was designed. Forward primer from the 8th exon and reverse primer from the 10th exon gave rise to a 750-bp fragment from all ae mutants and a 1576-bp fragment from 10 normal maize inbreds though PCR amplification. This ae-InDel marker also was co-dominant and two bands were detected in heterogous Aeae genotype.4. We also designed the primers according to the sbeI gene sequences (NM-001111900) published in NCBI and cloned the cDNA of the sbeI gene of 16 high amylose maize materials. After gene sequencing and comparative analysis, the result showed that high homology between the high amylose maize and sequences published in the database. There were 15 base-pair mutations in the sebi gene of GMES-0067, and 5 of them leaded to the amino acids mutations. While other high amylose maize materials showed no difference with the sequences published in the NCBI.5. SbeI from GEMS-0067 had 15 SNPs and all of them could be used as potential markers. The SNP that was positioned at the 1330th nucletiode on the 6th exon happened to be the enzymatic site of AluI which is very convenient to detect this SNP. SbeI-SNP amplified a PCR product of 906-bp from all 17 inbred lines used in this study. When completely digested by AluI, only the product from GEMS-0067 was not cut while the products from all other inbred lines were cut into two smaller fragments (617bp and 289bp), confirming the SbeI cDNA sequences of ae mutants. The co-dominat nature of SbeI -SNP was reflected by digesting PCR product amplified from F1 heterogous Aeae genotype, which yielded three bands.