Molecular Cytogenetic Characterization Of New Wheat-secale Africanum Germplasm With Novel Disease Resistance

Genus Secale （Secale L.,2n=2x=14, RR） belongs to Poaceae （syn. Gramineae）, inthe tribe Triticeae, and is a member of tertiary gene pool of bread wheat. It constitutes avast source of genetic diversity and is an important source of genes associated with highprotein content, high lysine, resistant to many cereal diseases, sprouting, drought, winterhardiness, and other morphological and biochemical traits that can be used in wheatbreeding program. Studies in Secale are focused on cultivated rye （S. cereale L.）, whilerelatively few studies have involved gene transfer from the wild Secale species to wheat.Wild Secale species, Secale africanum Stapf.(R^afrR^afr), is an important species in genusSecale, widely distributed in southern Africa and now verging on extinction. S.africanum showed short plant height and excellent resistance to wheat diseases, and wasthus essential to conserve the species and study its potential value for wheatimprovement. This study focused on development of a variety of Secale chromosomespecific molecular markers, identification of a series of new wheat–S. africanumintrogression lines by molecular cytogenetic analysis, investigation of the effects ofdisease resistance and agronomic performance of S. africanum chromatin in wheatbackground and comparision of the differences between cultivated rye and S. africanumchromosomes.The research results showed as follows:1. Development of new Secale chromosome specific molecular markers.（1） A totalof490pairs of primers, including138pairs of PLUG （PCR-based Landmark UniqueGene） primers and258pairs of EST-SSR （Expressed sequences tags-Simple sequencerepeats） primers,38pairs of STS （Sequence-taged site）primers, and56pairs of COS（conserved orthologous set） primers were utilized to develop Secale chromosomespecific molecular markers. Total97chromosome specific molecular markers wereassigned to Secale chromosomes1R-7R.（2） The wheat–S. africanum introgeressionlines were identified by cytological methods including Giemsa C-banding, genomic insitu hybridizaion （GISH） and fluoresence in situ hybridizaion （FISH）. Above newlydeveloped Secale specific markers could be used to track S. africanum chromatin inwheat background.（3） Using the reported Secale genome-specific and newly developed chromosome specific markers, we primarily screenned300wheat–S. africanumintrogressed progenies. It was found that chromosome1R^afrtransmitted with the highestfrequency, while6R^afrwas the second, and2R^afrwas followed.2. Characterization of a set of wheat–S. africanum1R^afrintrogression linesrevealed1R chromosome evolutionary trend.（1） Wheat–S. africanum chromosome1R^afraddition,1R^afr（1D） substitution, T1BL.1R^afrS and T1DS.1R^afrL translocation, and1R^afrL monotelocentric addition lines were indentified from a serial of wheat–S.africanum1R^afrintrogression lines by Giemsa C-banding and in situ hybridization.Disease resistance screening revealed that chromosome1R^afrS carried stripe rustresistance gene（s）.（2） Fifteen1R specific molecular markers were localized onchromosome arms of cultivated rye1R and S. africanum1R^afrby T1BL.1RS andDTA1RL and also the newly identified S. africanum1R^afrintrogression lines. Combinedthe reported fourteen1R specific molecular markers, we studied the amplificationpattern between S. cereale chromosome1R and S. africanum1R^afrderivatives.（3）Thecomparison of the cytogenetic analysis, target bands of polymorphic markers andnucleotide sequences of these unigene polymorphic markers suggested that accumulatedtrends of highly repetitive sequences and the satellite sequences, gene duplication andsequence divergence might have occurred among Secale species during its evolutionand domestication.3. Molecular cytogenetic characterization of new wheat–S. africanum chromosome2R^afrintrogression lines with novel disease resistance and agronomic performances.（1）A stable, highly fertile wheat–S. africanum2R^afr（2D） substitution line LF24was derivedfrom the F6generation of the cross between Mianyang11（MY11） and T. durum-S.africanum amphiploid （YF）, which was identified through C-banding, in situhybridization and molecular markers analysis. When inoculated with stripe rust isolatesof wheat, S. africanum and LF24, we found that the stripe rust resistance of LF24wasderived from S. africanum chromosome2R^afr.（2） Based on the screening of theoffsprings from the crosses between LF24and MY11, the T2DL.2R^afrS andT2DS.2R^afrL translocations, mono-2R^afrS and2R^afrL addition lines and other S.africanum chromosome2R^afrintrogression lines were identified by GISH and FISH.（3）Combined the C-banding, GISH, FISH patterns and molecular marker analysis of S.africanum2R^afrwith2R from different S. cereale, it was suggested that genomic variation such as telomeric heterochromatin reduction, highly repetitive sequencesreduction or variation, telomeric regions deletion or recombination occured in S.africanum2R^afrcompared with cultivated2R.（4） Disease resistance and agronomicperformance effects of wheat–S. africanum chromosome2R^afrderivatives linesindicated that chromosome2R^afrL possessed gene（s） for stripe rust resistance anddwarfing, while the2R^afrS translocation line was the most favorable for agronomicperformance when compared with the field performance of the substitution, and2R^afrLtranslocation lines.4．Molecular cytogenetic characterization of a new wheat–S. africanum6R^afr（6D）substitution line and the study of Sec2locus on6R^afr.（1） After screening the wheat–S.africanum introgression lines, we obtained a stable6R^afr（6D） substitution line HH41.The excellent stripe rust and moderate powdery mildew resistance might be originatedfrom S. africanum6R^afrchromosome.（2） GISH, FISH pattern, molecular markersrevealed the apparent polymorphism between S. africanum chromosome6R^afrandcultivated rye chromosome6R.（3） The SDS-PAGE （Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis） and75K γ-secalin gene specific marker testrevealed that Sec2locus encoding75K γ-secalin was located on6R^afr（6D） substitutionline. Molecular cloning and sequence comparison confirmed that the S. africanum6R^afrderived Sec2sequences matched well with75K γ-secalin gene sequences.（4）Phylogenetic analysis using the S. africanum Sec2sequences and other reported Secale75K γ-secalin gene sequences indicated that the S. africanum Sec2sequences clusteredinto the same group, while the cultivated rye Sec2clustered in defferent groups. Theresults suggested that there was a close relationship between S. africanum and S.sylvestre, and the S. cereale Sec2showed higher variation than those of wild Secalespecies.