Development And Application Of Oligonucleotide Probes For Maize And Related Species

Maize（Zea mays L.,2n=2x=20）is the largest crop in the world,which can be used for food,forage and industrial raw materials and has important economic value.The high yield and quality modern maize cultivars are developed from its ancestor teosinte through long term of artificial domestication and genetic improvement.Modern cultivars play key roles in maize production,but being used a limited number of inbred lines for producing hybrid cultivars,maize genetic diversity becomes lower and thus affects further improving of yield and durable resistances.Development of new germplasm via chromosome engineering is an important approach to broaden its genetic diversity.With the rapid development of molecular biology and genomics,the origin and evolution of maize have been clarified at molecular level,but at chromosome level,such studies are still very few,mainly because of high levels of heterochromatin content and difficulty in chromosome identifying.Development of oligonucleotide fluorescence in situ hybridization（oligo-FISH）provides a simple and efficient tool for corresponding maize polymorphic chromosomes.In this study,two single-copy oligonucleotide libraries and three tandem repeat oligonucleotide probe sets have been developed and used for both identifying and corresponding all chromosomes of maize and its relative species teosinte and coix through sequential FISH,the consensus reference karyotypes of maize and teosinte were developed and used for identifying chromosomal variations of maize landraces and hybrids cultivars.The main results are as follows:Using the latest genome sequence of B73（v4）,two oligonucleotide probes Gypsy-1and Gypsy-2 were developed based on its consensus Gypsy sequences.The two probes produced clear signals in the centromeric and Knob regions after FISH.Similarly,two other probes Opje-1 and Huck-1 were developed based on the consensus sequences of Opje and Huck family,respectively,where Opje-1 produced dispersed signals across all chromosomes except centromeric regions.Combing with FAM-labeled oligonucleotides of Grass-5S-1 and Gypsy-2,and TAMRA-labeled（ACT）₁₀and MR68-3,a new oligonucleotide probe multiplex#2（Maize ONPM#2）was developed,which can distinguish all chromosomes of B73,Mo17,and B73/Mo17 F₁after a single round of FISH,which makes maize chromosome identification simple and efficient.Based on Chorus software,two oligonucleotide probe libraries（Library 1&2）were designed which covered 12 chromosome segments of seven chromosomes except 1,2 and 6according to the consensus 45 nt single-copy sequences of B73 and teosinte.The two libraries labeled with Digoxignein-11-d UTP and Biotin-1-d UTP,combined with FAM-labeled Grass-5S and TARMA-Grass-45S,can identify and correspond all chromosomes of maize and teosinte.The average mapping rate of Library 1&2 probe sequences in Mo17,W22 and CML247 was 85.31%.Library 1&2 combined with Maize ONPM#2 sequential FISH can accurately identify all chromosomes of B73,Mo17,W22,SWL01,SWL12,Shenkenuo 1 and teosinte.Consensus karyotypes within maize species and between maize and teosinte were developed,where an obvious inversion in chromosome 4 of teosinte was clarified.This result solved the problem of polymorphic chromosome correspondence and provided a powerful tool for maize.In order to reveal chromosome heterozygosity and heterogeneity and corresponding polymorphic chromosomes of maize landraces and cultivars,Library 1&2 combined with Maize ONPM#2 sequential FISH were used for analyzing two landrace accessions Huangjinxiaoyumi and Silunuo.Obvious chromosome polymorphisms,heterozygosity and heterogeneity were identified in the two landraces.Among them,26 polymorphic types were found in 28 plants of Huangjinxiaoyumi,its heterozygous rate and heterogeneity rate of chromosome 8 were both the highest,which were 53.6%and 32.1%,respectively.For Silunuo,17 polymorphic types were found.Chromosome 4 has the most heterozygous types,with a heterozygous rate of 64.3%.To understand chromosome variations of maize hybrid cultivars in Henan Province,11 commercial cultivars were analyzed and consensus karyotypes were developed.Among them,35 polymorphic types were found,all chromosomes involved heterozygosity and the mean heterozygosity rate was 62.7%,whether chromosome heterozygosity have positive effects on maize heterosis needs further studies.Through genome resequencing,genome sequences of coix and mexican teosinte were obtained and 7 types of coix-and 2 types of teosinte-specific repeats were identified by TAREAN.10 oligonucleotide probes were developed.A set of oligonucleotide probes was developed for identifying all chromosomes of coix and a high-resolution karyotype has been established.In addition,combination of Gypsy-2,（ACT）₁₀,Grass-45S and Grass-5S also established a karyotype of teosinte.In Maize ONPM#2,only Grass-5S,MR68-3 and Gypsy-2 produced signals of in coix,while the coix probe set had no signal in maize;However,Maize ONPM#2 produced rich signals in teosinte,and vice versa.This result revealed that maize is genetically much closer to teosinte than coix.