Genetic Dissection Of Tiller Angle In Bread Wheat And Development Of The Molecular Markers

Tiller angle（TA）is an important component of plant architecture,influencing heavily on the photosynthetic efficiency,stress resistance and grain yield.However,few genetic studies focusing TA has been reported in wheat.Therefore,identification of loci and/or genes for TA,and development of the closely linked molecular markers or functional markers are important for TA and yield improvement.In the present study,a recombinant inbred line（RIL）population derived from a cross between Zhongmai 871（ZM871）and its sister line Zhongmai 895（ZM895）,a doubled haploid（DH）population created by cross Yangmai 16（YM16）and ZM895,and a diverse panel of 166 elite wheat varieties from the Yellow and Huai River Valleys Winter Wheat Zone of China were used to detect stable loci for TA through linkage analysis or genome-wide association studies（GWAS）.In addition,Ta TAC-D1,a member of the conserved genes regulating branch growing orientation across plant species,was cloned and a functional marker was developed based on the polymorphisms found in the sequences,and the associations between the Ta TAC-D1 alleles and the differences in tiller angle and yield-related traits were investigated using two sets of germplasm and a set of near-isogenic lines.The main results were summarized as follows:1.Two hundred and sixty-six F₆ RILs from the ZM871/ZM895 population were evaluated for TA in eight environments.Two pools involving 30 lines from each tail（high and low）of the TA distribution were genotyped by bulked segregant RNA-sequencing（BSR-Seq）and subsequently used for preliminary QTL mapping.Two quantitative trait loci（QTL）on chromosomes 1AL and 5DL were confirmed by inclusive composite interval mapping（ICIM）with the alleles from ZM895 contributing a reduced tiller angle.QTA.caas-1AL was detected in six environments,explaining 5.4～11.2%of the phenotypic variances.The major stable QTL,QTA.caas-5DL,was identified in all eight environments,accounting for 13.8～24.8%of the phenotypic variances.These previously uncharacterized QTL were further validated using BC₁F₄ populations derived from backcrosses ZM871/ZM895//ZM871（121 lines）and ZM871/ZM895//ZM895（175 lines）.2.TA of the 174 lines from the YM16/ZM895 DH population was investigated in four environments.Seven QTL on chromosomes 2B,2DS,4A,4D,6BS,7AL and 7D were identified using a high-density 660K SNP chip genetic linkage map,which could explain3.3～21.7%of the phenotypic variances.The tiller-angle-reducing alleles of QTA.caas-2B,QTA.caas-4AL,QTA.caas-4D and QTA.caas-7D were contributed by ZM895,whereas those of QTA.caas-2DS,QTA.caas-6BS and QTA.caas-7AL were from YM16.QTA.caas-2B was further validated by genotyping the natural population using KASP-2B.3.A natural population with 166 cultivars（advanced lines）were evaluated in four environments with irrigation and non-irrigation treatments and were genotyped by the high-density 90K and 660K SNP arrays.GWAS identified 18 stable marker-trait associations（MATs）on chromosomes 1B,2A（3 loci,the same below）,2B（3）,2D,4A,4B,5B（2）,5D（2）,6D,7A（2）and 7B,explaining 10.5～18.0%of the phenotypic variances.The frequency of excellent alleles ranged from 0.06 to 0.94.KASP markers for four loci of5B（2）,7A（2）and 7B were developed and validated.The QTA.caas-5DL of ZM871/ZM895,QTA.caas-2B and QTA.caas-4D of YM16/ZM895 were detected in natural population under P<0.001 level.4.Traes CS5D02G322600,located in the QTA.caas-5DL region and designated as Ta TAC-D1,is an ortholog of rice TA control gene Os TAC1.Sequence analysis of Ta TAC-D1 revealed a SNP in the third exon with‘A’and‘G’in ZM871 and ZM895,respectively,resulting in a Thr169Ala amino acid change.A KASP marker,designated as KASP-TAC-D1,was developed to discriminate the two alleles and was validated in two sets of germplasm.Over 70%of the 156 and 93 cultivars genotyped by KASP-TAC-D1 had the Ta TAC-D1-A allele and exhibited significantly larger TA than those with the Ta TAC-D1-G allele.5.Near-isogenic lines of Ta TAC-D1,NIL⁸⁷¹ and NIL⁸⁹⁵（with homozygous alleles from ZM871 and ZM895,respectively）,were constructed with a residual heterozygous line in the ZM871/ZM895 RIL population.Yield tests conducted under environments with irrigation and non-irrigation treatments showed that NIL⁸⁷¹ had a significantly（P<0.05）larger TA than NIL⁸⁹⁵ regardless of water status.NIL⁸⁹⁵ exhibited a significantly（P<0.05）higher spike number per square meter and grain yield than NIL⁸⁷¹ under irrigation environments,whereas no significant difference was observed in the non-irrigation environments.There was no significant difference in plant height,grain number per spike and thousand grain weight between NIL⁸⁷¹ and NIL⁸⁹⁵ under both treatment environments.The present study provides a more comprehensive genetic analysis of TA.Several novel and stable loci were identified to be associated with TA,the Ta TAC-D1 gene was cloned and the effects on TA and yield-related traits were investigated.Tightly linked markers for the loci mentioned above and a functional marker for Ta TAC-D1 were developed and validated.These laid the foundation for further exploring on the genetic control of TA and applying marker-assisted selection in breeding programs targeting TA improvement.