Fine mapping and characterization of stripe rust resistance gene YrAYH in near-isogenic lines derived from a cross involving wheat landrace Anyuehong

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is a devastating disease in wheat worldwide.Discovering and characterizing new resistance genes/QTL is crucial for wheat breeding programs.In this study,we fine-mapped and characterized a stripe rust resistance gene,YRAYH,on chromosome arm 5BL in the Chinese wheat landrace Anyuehong(AYH).Evaluations of stripe rust response to prevalent Chinese Pst races in near-isogenic lines derived from a cross of Anyuehong and Taichung 29 showed that YrAYH conferred a high level of resistance at all growth stages.Fine mapping using a large segregating population of 9748 plants,narrowed the YRAYH locus to a 3.7 Mb interval on chromosome arm 5BL that included 61 annotated genes.Transcriptome analysis of two NIL pairs identified 64 upregulated differentially expressed genes(DEGs)in the resistant NILs(NILs-R).Annotations indicated that many of these genes have roles in plant disease resistance pathways.Through a combined approach of fine-mapping and transcriptome sequencing,we identified a serine/threonine-protein kinase SRPK as a candidate gene underlying YrAYH.A unique 25 bp insertion was identified in the NILs-R compared to the NILs-S and previously published wheat genomes.An InDel marker was developed and co-segregated with YrAYH.Agronomic trait evaluation of the NILs suggested that YrAYH not only reduces the impact of stripe rust but was also associated with a gene that increases plant height and spike length.

Fine mapping and transcriptome sequencing reveal candidate genes conferring all-stage resistance to stripe rust on chromosome arm 1AL in Chinese wheat landrace AS1676

Stripe rust, caused by Puccinia striiformis f. sp. tritici(Pst), threatens wheat production worldwide, and resistant varieties tend to become susceptible after a period of cultivation owing to the variation of pathogen races. In this study, a new resistance gene against Pst race CYR34 was identified and predicted using the descendants of a cross between AS1676, a highly resistant Chinese landrace, and Avocet S, a susceptible cultivar. From a heterozygous plant from a F7recombinant inbred line(RIL) population lacking the Yr18 gene, a near-isogenic line(NIL) population was developed to map the resistance gene. An allstage resistance gene, YrAS1676, was identified on chromosome arm 1AL via bulked-segregant exomecapture sequencing. By analyzing a large NIL population consisting of 6537 plants, the gene was further mapped to the marker interval between KA1A_485.36 and KA1A_490.13, spanning 485.36–490.13 Mb on1AL. A total of 66 annotated genes have been reported in this region. To characterize and predict the candidate gene(s), an RNA-seq was performed using NIL-R and NIL-S seedlings 3 days after CYR34 inoculation. Compared to NIL-S plants, NIL-R plants showed stronger immune reaction and higher expression levels of genes encoding pathogenesis-associated proteins. These differences may help to explain why NIL-R plants were more resistant to Pst race CYR34 than NIL-S plants. By combining fine-mapping and transcriptome sequencing, a calcium-dependent protein kinase gene was finally predicted as the potential candidate gene of YrAS1676. This gene contained a single-nucleotide polymorphism. The candidate gene was more highly expressed in NIL-R than in NIL-S plants. In field experiments with Pst challenge,the YrAS1676 genotype showed mitigation of disease damage and yield loss without adverse effects on tested agronomic traits. These results suggest that YrAS1676 has potential use in wheat stripe rust resistance breeding.

QTL mapping for grain number per spikelet in wheat using a high-density genetic map

Grain number per spikelet (GNS) is a key determinant of grain yield in wheat.A recombinant inbred line population comprising 300 lines was developed from a cross between a high GNS variety H461 and Chinese Spring from which the reference genome assembly of bread wheat was obtained.Both parents and the recombinant inbred lines were genotyped using the wheat 55K single nucleotide polymorphism(SNP) array.A high-density genetic map containing 21,197 SNPs was obtained.These markers covered each of the 21 chromosomes with a total linkage distance of 3792.71 c M.Locations of these markers in this linkage map were highly consistent with their physical locations in the genome assembly of Chinese Spring.The two parents and the whole RIL population were assessed for GNS in two consecutive years at two different locations.Based on multi-environment phenotype data and best liner unbiased prediction values,three quantitative trait loci (QTL) for GNS were identified.One of them located on chromosomes 2B and the other two on 2D.Phenotypic variation explained by these loci varied from 3.07%to26.57%.One of these QTL,QGns.sicau-2D-2,was identified in each of all trials conducted.Based on the best linear unbiased prediction values,this locus explained 19.59%–26.57%of phenotypic variation.A KASP(Kompetitive Allele-Specific PCR) marker closely linked with this locus was generated and used to validate the effects of this locus in three different genetic backgrounds.The identified QTL and the KASP marker developed for it will be highly valuable in fine-mapping the locus and in exploiting it for markerassisted selection in wheat breeding programs.

Production of aneuhaploid and euhaploid sporocytes by meiotic restitution in fertile hybrids between durum wheat Langdon chromosome substitution lines and Aegilops tauschii

Fertile F1 hybrids were obtained between durum wheat （Triticum durum Desf.） Langdon （LDN） and its 10 disomic substitution （LDN DS） lines with Aegilops tauschii accession AS60 without embryo rescue. Selfed seedset rates for hybrids of LDN with AS60 were 36.87% and 49.45% in 2005 and 2006, respectively. Similar or higher selfed seedset rates were observed in the hybrids of 1D （1A）, 1D （1B）, 3D （3A）, 4D （4B）, 7D （7A）, and 2D （2B） with AS60, while lower in hybrids of 3D （3B） ＋ 3BL, 5D （5A） ＋ 5AL, 5D （5B） ＋ 5B and 6D （6B） ＋ 6BS with AS60 compared with the hybrids of LDN with AS60. Observation of male gametogenesis showed that meiotic restitution, both first-division restitution （FDR） and single-division meiosis （SDM） resulted in the formation of functional unreduced gametes, which in turn produced seeds. Both euhaploid and aneuhaploid gametes were produced in F1 hybrids. This suggested a strategy to simultaneously transfer and locate major genes from the ancestral species T. turgidum or Ae. tauschii. Moreover, there was no significant difference in the aneuhaploid rates between the F1 hybrids of LDN and LDN DS lines with AS60, suggesting that meiotic pairing between the two D chromosomes in the hybrids of LDN DS lines with AS60 did not promote the formation of aneuhaploid gametes.

Wheat breeding in the hometown of Chinese Spring

The common wheat landrace Chinese Spring(CS) was made famous by the work of Ernie Sears, a great cytogenetist, who developed a number of CS-based aneuploid series that were used to identify individual wheat chromosomes. Based on this, a standard karyotype and nomenclature system was developed for wheat chromosomes that allowed wheat researchers to analyze and manipulate the wheat genome with unprecedented precision and efficiency. Nevertheless, not much is known about the utilization of CS at its hometown, Chengdu in Sichuan province, during early wheat breeding activity. In this review, we follow the speculation that CS is a selection from the Cheng-du-guang-tou(CDGT) landrace. We provide a description of how CDGT became a founder landrace for wheat breeding activities in early times. We show that CDGT-derived varieties were reinforced genetically by crosses to six more exotic parents. These varieties remained the major elite cultivar for several decades. Later, synthetic hexaploid wheats were introduced into the breeding program, firstly using those from CIMMYT and later using materials produced with local tetraploid wheat and goat grass. Finally, we discuss the strategies and future directions to improve wheat yield and resistance through an expanded genetic basis,especially by recapturing lost genetic variations from landraces and related wild species, a process that may set an example for wheat breeders in China and elsewhere.

Identification of loci and candidate genes controlling kernel weight in barley based on a population for which whole genome assemblies are available for both parents

Kernel weight(KW), together with kernel number per unit area, determines yield of cereal crops. Here,two barley recombinant inbred lines(RILs) populations with a shared parent were used to identify loci controlling KW. One is Baudin/AWCS276(BA) for which a linkage map was available. Several largeeffect QTL controlling KW were detected in this population. Another is Morex/AWCS276(MA). A linkage map with 5273 makers formed 1454 clusters, was constructed by the genotyping by sequence(GBS) data of 201 RILs from this population. A single marker was selected to represent each of the clusters and the linkage map constructed with these markers covers a total length of 1022.4 c M with an average interval of approximately 0.7 cM between loci. Three of the large-effect loci controlling KW(located on 2 HL, 6 HL,and 7 HL, respectively) identified from the BA population were also detected in the MA population under different environments. The locus on 6 HL was detected in each of the experiments conducted for both populations thus was selected for developing near isogenic lines(NILs). Apart from KW, the two isolines for each pair of the putative NILs obtained showed no significant difference for any of the morphological characteristics assessed. The average difference in KW between the isolines for all the NILs obtained was about 15% based on assessments under both glasshouse and field conditions. Taken advantage that high quality genome assemblies for both Morex and AWCS276 are available, we identified candidate genes underlying two of the three loci based on an orthologous analysis. The NILs developed and the candidate genes identified in this study should facilitate the cloning and functional analysis of genes regulating KW in barley.

A major vernalization-independent QTL for tiller angle on chromosome arm 2BL in bread wheat

Tiller angle(TA)strongly influences plant architecture and grain yield in cereals.However,the genetic basis of TA in wheat is largely unknown.We identified three TA-related quantitative trait loci(QTL).One of them was QTa.sau-2 B-769,a major QTL localized on chromosome arm 2 BL.QTa.sau-2 B-769 was detected in seven environments,explaining 18.1%–51.1%of phenotypic variance.We developed a linked Kompetitive Allele-Specific Polymerase chain reaction(KASP)marker,KASP-AX-108792274,to further validate this locus in three additional populations in multiple environments.QTa.sau-2 B-769 increased TA by up to 24.9%in these populations.There were significant and positive correlations between TA and flag leaf angle(FLANG).However,TA was not correlated with plant height or anthesis date,suggesting that expression of QTa.sau-2 B-769 is independent of vernalization.Traes CS2 B01 G583800,a gene known to be involved in leaf angle regulation,was identified as the most likely candidate gene for QTa.sau-2 B-769.These results enrich our understanding of the mechanisms regulating wheat TA at maturity and may support precise mapping and cloning of gene(s)underlying QTa.sau-2 B-769.

Mapping a stable adult-plant stripe rust resistance QTL on chromosome 6AL in Chinese wheat landrace Yibinzhuermai

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most important diseases threatening the yield and stability of wheat production in China and many other countries.Identification and utilization of new genes for durable stripe rust resistance are important for ongoing control of this disease.The objectives of this study were to identify quantitative trait loci(QTL)associated with adult-plant stripe rust resistance in the Chinese wheat landrace Yibinzhuermai(YBZR)and to provide wheat breeders with new sources of potentially durable resistance.A total of 117 recombinant inbred lines(RILs)(F5:8)derived from a cross between YBZR and highly susceptible cultivar Taichung 29(TC29)were assessed for stripe rust severity in field experiments at Wenjiang in 2016 and 2017 and Chongzhou in 2016,2017,2018,and 2019 in Sichuan following inoculation with a mixture of current Pst races.The RILs were genotyped using the Wheat55K single nucleotide polymorphism(SNP)array.Three QTL were identified on chromosome arms 6AL,5BL and 7DS.QYr.YBZR-6AL and QYr.YBZR-7DS conferred major effects in all field environments,explaining 10.6%to 14.7%and 11.5%to 21.2%of phenotypic variation,respectively.The QTL on 5BL and 7DS likely correspond to previously known QTL,whereas QYr.YBZR-6AL is probably novel.Haplotype analysis revealed that the resistance allele at QYr.YBZR-6AL was present in 2.8%of 324 Chinese wheat landraces.SNP markers closely linked with QYr.YBZR-6AL were converted to kompetitive allele-specific PCR markers and validated in the RIL population and a subset of 92 wheat cultivars.QYr.YBZR-6AL and its markers should be useful in breeding programs to improve the level and durability of stripe rust resistance.

Isolation and characterization of an isoamylase gene from rye

Genomic DNA and cDNA sequences of an isoamylase gene were isolated and characterized from the rye genome. The full-lengths of the rye isoamylase gene are 7351 bp for genomic DNA and 2364 bp for cDNA. There are 18 exons and 17 introns in the genomic sequence, which shares a similar organization with homologous genes from Aegilops tauschii, maize, rice and Arabidopsis. Exon regions of rye and other plant isoamylase genes are more conserved than the introns. High sequence similarity(>95%) was observed in mature proteins of isoamylase genes originating from rye, Ae. tauschii, wheat and barley. The transcript profile revealed that rye isoamylase is mainly expressed in the seed endosperm with a maximum level at the middle developmental stage(15 DPA). A phylogenetic tree based on the deduced aa sequences of mature proteins from rye and other plant isoamylases indicated that rye isoamylase is more closely related to Ae. tauschii wDBE1 and wheat iso1. This is the first report on identification and characterization of the isoamylase gene from rye, making it possible to explore the roles of this enzyme for amylopectin development in rye and triticale.

渗入杂交与小麦杂种优势

杂种优势利用是实现小麦增产稳产的重要途径.渗入杂交为小麦杂种优势利用提供了发展动力.小麦具有大量可渗入利用的近缘属种,同时基因组结构有利于外源基因渗入.目前常用的基因渗入有两种方式.一是借助染色体工程,通过不同基因组之间的部分同源染色体重组,将单个外源染色体区段以易位系为载体的方式渗入.易位系在引入外源染色体区段的同时,丢失了对应的小麦染色体区段,因此在常规育种中应用受限.但当其应用于杂交小麦,既保留了外源区段的优良基因,也不存在小麦基因缺失问题.二是通过同源染色体遗传重组,将不同染色体区段上的基因同时渗入,可实现小麦基因组遗传结构的重塑.小麦基因组缓冲性强,单个基因拷贝变化常常仅有较小的遗传效应,但多个基因的同时导入可能产生明显效果.因此,从基因组的角度丰富小麦遗传变异对杂种优势利用尤为重要.小麦3个亚基因组的遗传多样性不对称,其中D基因组的遗传多样性远低于A和B.因此,将未参与小麦进化的节节麦亚种的遗传多样性导入小麦,尽快丰富小麦D基因组的遗传多样性,是当前同源渗入的优先任务.

Synthetic hexaploid wheat and its utilization for wheat genetic improvement in China

Synthetic hexaploid wheat （Triticum turgidum x Aegilops tauschii） was created to explore for novel genes from T. turgidum and Ae. tauschii that can be used for common wheat improvement. In the present paper, research advances on the utilization of synthetic hexaploid wheat for wheat genetic improvement in China are reviewed. Over 200 synthetic hexaploid wheat （SHW） accessions from the International Maize and Wheat Improvement Centre （CIMMYT） were introduced into China since 1995. Four cultivars derived from these, Chuanmai 38, Chuanmai 42, Chuanmai 43 and Chuanmai 47, have been released in China. Of these, Chuanmai 42, with large kernels and resistance to stripe rust, had the highest average yield （〉 6 t/ha） among all cultivars over two years in Sichuan provincial yield trials, outyielding the commercial check cultivar Chuanmai 107 by 22,7%. Meanwhile, by either artificial chromosome doubling via colchicine treatment or spontaneous chromosome doubling via a union of unreduced gametes （2n） from T. turgidum-Ae, tauschii hybrids, new SHW lines were produced in China. Mitotic-like meiosis might be the cytological mechanism of spontaneous chromosome doubling. SHW lines with genes for spontaneous chromosome doubling may be useful for producing new SHW-alien amphidiploids and double haploid in wheat genetic improvement.

Synthesizing double haploid hexaploid wheat populations based on a spontaneous alloploidization process

Doubled haploid （DH） populations are useful to scientists and breeders in both crop improvement and basic research. Current methods of producing DHs usually need in vitro culture for extracting haploids and chemical treatment for chromosome doubling. This report describes a simple method for synthesizing DHs （SynDH） especially for allopolyploid species by utilizing meiotic restitution genes. The method involves three steps： hybridization to induce recombination, interspecific hybridization to extract haploids, and spontaneous chromosome doubling by selfing the interspecific Fis. DHs produced in this way contain recombinant chromosomes in the genome（s） of interest in a homogeneous background. No special equipment or treatments are involved in the DH production and it can be easily applied in any breeding and/or genetic program. Triticum turgidum L. and Aegilops tauschii Coss, the two ancestral species of common wheat （Triticurn aestivum L.） and molecular markers were used to demonstrate the SynDH method.