Genome-wide identification of the NLR gene family in Haynaldia villosa by SMRT-RenSeq.

BACKGROUND: Nucleotide-binding and leucine-rich repeat (NLR) genes have attracted wide attention due to their crucial role in protecting plants from pathogens. SMRT-RenSeq, combining PacBio sequencing after resistance gene enrichment sequencing (RenSeq), is a powerful method for selectively capturing and sequencing full-length NLRs. Haynaldia villosa, a wild grass species with a proven potential for wheat improvement, confers resistance to multiple diseases. So, genome-wide identification of the NLR gene family in Haynaldia villosa by SMRT-RenSeq can facilitate disease resistance genes exploration. RESULTS: In this study, SMRT-RenSeq was performed to identify the genome-wide NLR complement of H. villosa. In total, 1320 NLRs were annotated in 1169 contigs, including 772 complete NLRs. All the complete NLRs were phylogenetically analyzed and 11 main clades with special characteristics were derived. NLRs could be captured with high efficiency when aligned with cloned R genes, and cluster expansion in some specific gene loci was observed. The physical location of NLRs to individual chromosomes in H. villosa showed a perfect homoeologous relationship with group 1, 2, 3, 5 and 6 of other Triticeae species, however, NLRs physically located on 4VL were largely in silico predicted to be located on the homoeologous group 7. Fifteen types of integrated domains (IDs) were integrated in 52 NLRs, and Kelch and B3 NLR-IDs were found to have expanded in H. villosa, while DUF948, NAM-associated and PRT_C were detected as unique integrated domains implying the new emergence of NLR-IDs after H. villosa diverged from other species. CONCLUSION: SMRT-RenSeq is a powerful tool to identify NLR genes from wild species using the baits of the evolutionary related species with reference sequences. The availability of the NLRs from H. villosa provide a valuable library for R gene mining and transfer of disease resistance into wheat.

Fine mapping of powdery mildew and stripe rust resistance genes Pm5V/Yr5V transferred from Dasypyrum villosum into wheat without yield penalty.

KEY MESSAGE: The novel wheat powdery mildew and stripe rust resistance genes Pm5V/Yr5V are introgressed from Dasypyrum villosum and fine mapped to a narrowed region in 5VS, and their effects on yield-related traits were characterized. The powdery mildew and stripe rust seriously threaten wheat production worldwide. Dasypyrum villosum (2n = 2x = 14, VV), a relative of wheat, is a valuable resource of resistance genes for wheat improvement. Here, we describe a platform for rapid introgression of the resistance genes from D. villosum into the wheat D genome. A complete set of new wheat-D. villosum V (D) disomic substitution lines and 11 D/V Robertsonian translocation lines are developed and characterized by molecular cytogenetic method. A new T5DL·5V#5S line NAU1908 shows resistance to both powdery mildew and stripe rust, and the resistances associated with 5VS are confirmed to be conferred by seedling resistance gene Pm5V and adult-plant resistance gene Yr5V, respectively. We flow-sort chromosome arm 5VS and sequence it using the Illumina NovaSeq 6000 system that allows us to generate 5VS-specific markers for genetic mapping of Pm5V/Yr5V. Fine mapping shows that Pm5V and Yr5V are closely linked and the location is narrowed to an approximately 0.9 Mb region referencing the sequence of Chinese Spring 5DS. In this region, a NLR gene in scaffold 24,874 of 5VS orthologous to TraesCS5D02G044300 is the most likely candidate gene for Pm5V. Soft- and hard-grained T5DL·5V#5S introgressions confer resistance to both powdery mildew and stripe rust in diverse wheat genetic backgrounds without yield penalty. Meanwhile, significant decrease in plant height and increase in yield were observed in NIL-5DL·5V#5S compared with that in NIL-5DL·5DS. These results indicate that Pm5V/Yr5V lines might have the potential value to facilitate wheat breeding for disease resistance.

Long-range assembly of sequences helps to unravel the genome structure and small variation of the wheat-Haynaldia villosa translocated chromosome 6VS.6AL.

Genomics studies in wild species of wheat have been limited due to the lack of references; however, new technologies and bioinformatics tools have much potential to promote genomic research. The wheat-Haynaldia villosa translocation line T6VS·6AL has been widely used as a backbone parent of wheat breeding in China. Therefore, revealing the genome structure of translocation chromosome 6VS·6AL will clarify how this chromosome formed and will help to determine how it affects agronomic traits. In this study, chromosome flow sorting, NGS sequencing and Chicago long-range linkage assembly were innovatively used to produce the assembled sequences of 6VS·6AL, and gene prediction and genome structure characterization at the molecular level were effectively performed. The analysis discovered that the short arm of 6VS·6AL was actually composed of a large distal segment of 6VS, a small proximal segment of 6AS and the centromere of 6A, while the collinear region in 6VS corresponding to 230-260 Mb of 6AS-Ta was deleted when the recombination between 6VS and 6AS occurred. In addition to the molecular mechanism of the increased grain weight and enhanced spike length produced by the translocation chromosome, it may be correlated with missing GW2-V and an evolved NRT-V cluster. Moreover, a fine physical bin map of 6VS was constructed by the high-throughput developed 6VS-specific InDel markers and a series of newly identified small fragment translocation lines involving 6VS. This study will provide essential information for mining of new alien genes carried by the 6VS·6AL translocation chromosome.

Predominant wheat-alien chromosome translocations in newly developed wheat of China.

Founder wheat lines have played key role in Chinese wheat improvement. Wheat-Dasypyrum villosum translocation T6VS·6AL has been widely used in wheat breeding in recent years due to its high level of powdery mildew resistance and other beneficial genes. Reference oligo-nucleotide multiplex probe (ONMP)-FISH karyotypes of six T6VS·6AL donor lines were developed and used for characterizing 32 derivative cultivars and lines. T6VS·6AL was present in 27 cultivar/lines with 20 from southern China. Next, ONMP-FISH was used to study chromosome constitution of randomly collected wheat cultivars and advanced breeding lines from southern and northern regions of China: 123 lines from the regional test plots of southern China and 110 from northern China. In southern China, T6VS·6AL (35.8%) was the most predominant variation, while T1RS·1BL (27.3%) was the most predominant in northern China. The pericentric inversion perInv 6B derived from its founder wheat Funo and Abbondaza was the second most predominant chromosome variant in both regions. Other chromosome variants were present in very low frequencies. Additionally, 167 polymorphic chromosome types were identified. Based on these variations, 271 cultivars and lines were clustered into three groups, including southern, northern, and mixed groups that contained wheat from both regions. Different dominant chromosome variations were seen, indicating chromosome differentiation in the three groups of wheat. The clearly identified wheat lines with T6VS·6AL in different backgrounds and oligonucleotide probe set will facilitate their utilization in wheat breeding and in identifying other beneficial traits that may be linked to this translocation. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01206-3.

Research progress of plant cytogenetics in Jiangsu province.

Cytogenetics was established based on the "Chromosome theory of inheritance", proposed by Boveri and Sutton and evidenced by Morgan's lab in early stage of the 20 th centrary. With rapid development of related research areas, especially molecular genetics, cytogenetics developed from traditional into a new era, molecular cytogenetics in late 1960s. Featured by an established technique named DNA in situ hybridization (ISH), molecular cytogenetics has been applied in various research areas. ISH provids vivid and straightforward figures showing the virtual presence of DNA, RNA or proteins. In combination with genomics and cell biology tools, ISH and derived techniques have been widely used in studies of the origin, evolution, domestication of human, animal and plant, as well as wide hybridization and chromosome engineering. The physical location and order of DNA sequences revealed by ISH enables the detection of chromosomal re-arrangments among related species and gaps of assembled genome sequences. In addition, ISH using RNA or protein probes can reveal the location and quantification of transcripted RNA or translated protein. Since the 1970s, scientists from universities or institutes belonging to the Jiangsu Society of Genetics have initiated cytogenetics researches using various plant species. In recent years, research platforms for molecular cytogenetics have also been well established in Nanjing Agricultural University, Yangzhou University, Nanjing Forestry University, Jiangsu Xuhuai Academy of Agricultural Sciences, and Jiangsu Normal University. The application of molecular cytogenetics in plant evolution, wide hybridization, chromosome engineering, chromosome biology, genomics has been successful. Significant progresses have been achieved, both in basic and applied researches. In this paper, we will review main research progresses of plant cytogenetics in Jiangsu province, and discuss the potential development of this research area.

Dissection and cytological mapping of chromosome arm 4VS by the development of wheat-Haynaldia villosa structural aberration library.

KEY MESSAGE: A cytological map of Haynaldia villosa chromosome arm 4VS was constructed to facilitate the identification and utilization of beneficial genes on 4VS. Induction of wheat-alien chromosomal structure aberrations not only provides new germplasm for wheat improvement, but also allows assignment of favorable genes to define physical regions. Especially, the translocation or introgression lines carrying alien chromosomal fragments with different sizes are useful for breeding and alien gene mapping. Chromosome arm 4VS of Haynaldia villosa (L.) Schur (syn. Dasypyrum villosum (L.) P. Candargy) confers resistances to eyespot and wheat yellow mosaic virus (WYMV). In this research, we used both irradiation and the pairing homoeologous gene (Ph) mutant to induce chromosomal aberrations or translocations. By using the two approaches, a structural aberration library of chromosome arm 4VS was constructed. In this library, there are 57 homozygous structural aberrations, in which, 39 were induced by the Triticum aestivum cv. Chinese Spring (CS) ph1b mutant (CS ph1b) and 18 were induced by irradiation. The aberrations included four types, i.e., terminal translocation, interstitial translocation, deletion and complex structural aberration. The 4VS cytological map was constructed by amplification in the developed homozygous aberrations using 199 4VS-specific markers, which could be allocated into 39 bins on 4VS. These bins were further assigned to their corresponding physical regions of chromosome arm 4DS based on BLASTn search of the marker sequences against the reference sequence of Aegilops tauschii Cosson. The developed genetic stocks and cytological map provide genetic stocks for wheat breeding as well as alien gene tagging.

Structural chromosome rearrangements and polymorphisms identified in Chinese wheat cultivars by high-resolution multiplex oligonucleotide FISH.

KEY MESSAGE: High-resolution multiplex oligonucleotide FISH revealed the frequent occurrence of structural chromosomal rearrangements and polymorphisms in widely grown wheat cultivars and their founders. Over 2000 wheat cultivars including 19 founders were released and grown in China from 1949 to 2000. To understand the impact of breeding selection on chromosome structural variations, high-resolution karyotypes of Chinese Spring (CS) and 373 Chinese cultivars were developed and compared by FISH (fluorescence in situ hybridization) using an oligonucleotide multiplex probe based on repeat sequences. Among them, 148 (39.7%) accessions carried 14 structural rearrangements including three single translocations (designated as T), eight reciprocal translocations (RT), one pericentric inversion (perInv), and two combined variations having both the deletion and single translocations. Five rearrangements were traced to eight founders, including perInv 6B detected in 57 cultivars originating from Funo, Abbondanza, and Fan 6, T 1RS∙1BL in 47 cultivars derived from the Lovrin series, RT 4AS∙4AL-1DS/1DL∙1DS-4AL in 31 varieties from Mazhamai and Bima 4, RT 1RS∙7DL/7DS∙1BL in three cultivars was from Aimengniu, and RT 5BS∙5BL-5DL/5DS∙5DL-5BL was only detected in Youzimai. In addition to structural rearrangements, 167 polymorphic chromosome blocks (defined as unique signal patterns of oligonucleotide repeat probes distributed within chromosomes) were identified, and 59 were present in one or more founders. Some specific types were present at high frequencies indicating selective blocks in Chinese wheat varieties. All cultivars and CS were clustered into four groups and 15 subgroups at chromosome level. Common block patterns occurred in the same subgroup. Origin, geographic distribution, probable adaptation to specific environments, and potential use of these chromosomal rearrangements and blocks are discussed.

Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next-generation sequencing.

BACKGROUND: Haynaldia villosa (L.) Schur (syn. Dasypyrum villosum L. Candargy, 2n = 14, genome VV) is the tertiary gene pool of wheat, and thus a potential resource of genes for wheat improvement. Among other, wheat yellow mosaic (WYM) resistance gene Wss1 and a take-all resistance gene were identified on the short arm of chromosome 4 V (4VS) of H. villosa. We had obtained introgressions on 4VS chromosome arm, with the objective of utilizing the target genes. However, monitoring these introgressions has been a daunting task because of inadequate knowledge as to H.villosa genome, as reflected by the lack of specific markers. RESULTS: This study aims to develop 4VS-specific markers by combination of chromosome sorting and next-generation sequencing. The short arm of chromosome 4VS of H.villosa was flow-sorted using a FACSVantage SE flow cytometer and sorter, and then sequenced by Illumina sequencing. The sequence of H. villosa 4VS was assembled by the software Hecate, and then was compared with the sequence assemblies of wheat chromosome arms 4AL, 4BS and 4DS and Ae. tauschii 4DS, with the objectives of identifying exon-exon junctions and localizing introns on chromosome 4VS of H. villosa. The intron length polymorphisms suitable for designing H. villosa primers were evaluated with criteria. Consequently, we designed a total of 359 intron targeting (IT) markers, among which 232 (64.62%) markers were specific for tracing the 4VS chromatin in the wheat background. CONCLUSION: The combination of chromosome sorting and next-generation sequencing to develop specific IT markers for 4VS of H. villosa has high success rate and specificity, thus being applicable for the development of chromosome-specific markers for alien chromatin in wheat breeding.

Pm55, a developmental-stage and tissue-specific powdery mildew resistance gene introgressed from Dasypyrum villosum into common wheat.

KEY MESSAGE: Powdery mildew resistance gene Pm55 was physically mapped to chromosome arm 5VS FL 0.60-0.80 of Dasypyrum villosum . Pm55 is present in T5VS·5AL and T5VS·5DL translocations, which should be valuable resources for wheat improvement. Powdery mildew caused by Blumeria graminis f. sp. tritici is a major wheat disease worldwide. Exploiting novel genes effective against powdery mildew from wild relatives of wheat is a promising strategy for controlling this disease. To identify novel resistance genes for powdery mildew from Dasypyrum villosum, a wild wheat relative, we evaluated a set of Chinese Spring-D. villosum disomic addition and whole-arm translocation lines for reactions to powdery mildew. Based on the evaluation data, we concluded that the D. villosum chromosome 5V controls post-seedling resistance to powdery mildew. Subsequently, three introgression lines were developed and confirmed by molecular and cytogenetic analysis following ionizing radiation of the pollen of a Chinese Spring-D. villosum 5V disomic addition line. A homozygous T5VS·5AL translocation line (NAU421) with good plant vigor and full fertility was further characterized using sequential genomic in situ hybridization, C-banding, and EST-STS marker analysis. A dominant gene permanently named Pm55 was located in chromosome bin 5VS 0.60-0.80 based on the responses to powdery mildew of all wheat-D. villosum 5V introgression lines evaluated at both seeding and adult stages. This study demonstrated that Pm55 conferred growth-stage and tissue-specific dependent resistance; therefore, it provides a novel resistance type for powdery mildew. The T5VS·5AL translocation line with additional softness loci Dina/Dinb of D. villosum provides a possibility of extending the range of grain textures to a super-soft category. Accordingly, this stock is a new source of resistance to powdery mildew and may be useful in both resistance mechanism studies and soft wheat improvement.

Characterization of a Triticum aestivum-Dasypyrum villosum T2VS·2DL translocation line expressing a longer spike and more kernels traits.

KEY MESSAGE: By using 2V-specific EST-PCR markers and sequential GISH/FISH analysis, we identified four homozygous CS-2V translocation lines, including a novel compensating T2VS·2DL translocation line NAU422. This translocation line has longer spikes and produces more grains per spike than its recurrent parent CS and three other translocation lines, which could be a valuable resource in wheat yield improvement. Dasypyrum villosum (2n = 14, VV), the wild relative of wheat, possesses novel and superior alleles at many important loci and should be utilized to improve the genetic diversity of cultivated wheat and may be very helpful for the improvement of wheat yield. In this study, four homozygous Chinese Spring (CS)-D. villosum translocation lines containing different fragments of chromosome 2V were characterized from a pool, including 76 translocations that occur in chromosomes 1 V through 7 V of D. villosum by both molecular markers and cytogenetic analysis. A rough physical map of 2V was developed which included nine markers in three segments of the short arm and ten markers in the long arm. The photoperiod response gene of D. villosum (Ppd-V1) was physically mapped to the FL 0.33-0.53 region of 2VS, while the gene controlling bristles on the glume ridges (Bgr-V1) was mapped to 2VS FL 0.00-0.33. A novel compensating Triticum aestivum-D. villosum Robertsonian translocation line T2VS·2DL (NAU422) with good plant vigor and full fertility was further characterized by sequential genomic in situ hybridization and fluorescent in situ hybridization and the use of molecular markers. Compared to its recurrent parent CS and three other translocation lines, the T2VS·2DL translocation line has longer spikes, more spikelets and more grains per spike in two season years, which suggested that the alien segment may carry yield-related genes of D. villosum. The developed T2VS·2DL translocation line with its morphological and co-dominant molecular markers could be utilized as a novel germplasm for high-yield wheat breeding.

Chromosome engineering, mapping, and transferring of resistance to Fusarium head blight disease from Elymus tsukushiensis into wheat.

KEY MESSAGE: This manuscript describes the transfer and molecular cytogenetic characterization of a novel source of Fusarium head blight resistance in wheat. Fusarium head blight (FHB) caused by the fungus Fusarium graminearum Schwabe [telomorph = Gibberella zeae (Schwein. Fr.) Petch] is an important disease of bread wheat, Triticum aestivum L. (2n = 6x = 42, AABBDD) worldwide. Wheat has limited resistance to FHB controlled by many loci and new sources of resistance are urgently needed. The perennial grass Elymus tsukushiensis thrives in the warm and humid regions of China and Japan and is immune to FHB. Here, we report the transfer and mapping of a major gene Fhb6 from E. tsukushiensis to wheat. Fhb6 was mapped to the subterminal region in the short arm of chromosome 1E(ts)#1S of E. tsukushiensis. Chromosome engineering was used to replace corresponding homoeologous region of chromosome 1AS of wheat with the Fhb6 associated chromatin derived from 1E(ts)#1S of E. tsukushiensis. Fhb6 appears to be new locus for wheat as previous studies have not detected any FHB resistance QTL in this chromosome region. Plant progenies homozygous for Fhb6 had a disease severity rating of 7 % compared to 35 % for the null progenies. Fhb6 has been tagged with molecular markers for marker-assisted breeding and pyramiding of resistance loci for effective control of FHB.

Serine/threonine kinase gene Stpk-V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat.

Powdery mildew resistance gene Pm21, located on the chromosome 6V short arm of Haynaldia villosa and transferred to wheat as a 6VS·6AL translocation (T6VS·6AL), confers durable and broad-spectrum resistance to wheat powdery mildew. Pm21 has become a key gene resource for powdery mildew resistance breeding all over the world. In China, 12 wheat varieties containing Pm21 have been planted on more than 3.4 million hectares since 2002. Pm21 has been intractable to molecular genetic mapping because the 6VS does not pair and recombine with the 6AS. Moreover, all known accessions of H. villosa are immune to powdery mildew fungus. Pm21 is still defined by cytogenetics as a locus. In the present study, a putative serine and threonine protein kinase gene Stpk-V was cloned and characterized with an integrative strategy of molecular and cytogenetic techniques. Stpk-V is located on the Pm21 locus. The results of a single cell transient expression assay showed that Stpk-V could decrease the haustorium index dramatically. After the Stpk-V was transformed into a susceptible wheat variety Yangmai158, the characterized transgenic plants showed high and broad-spectrum powdery mildew resistance similar to T6VS·6AL. Silencing of the Stpk-V by virus-induced gene silencing in both T6VS·6AL and H. villosa resulted in their increased susceptibility. Stpk-V could be induced by Bgt and exogenous H(2)O(2), but it also mediated the increase of endogenous H(2)O(2), leading to cell death and plant resistance when the plant was attacked by Bgt.

Wheat Pm55 alleles exhibit distinct interactions with an inhibitor to cause different powdery mildew resistance.

Powdery mildew poses a significant threat to wheat crops worldwide, emphasizing the need for durable disease control strategies. The wheat-Dasypyrum villosum T5AL·5 V#4 S and T5DL·5 V#4 S translocation lines carrying powdery mildew resistant gene Pm55 shows developmental-stage and tissue-specific resistance, whereas T5DL·5 V#5 S line carrying Pm5V confers resistance at all stages. Here, we clone Pm55 and Pm5V, and reveal that they are allelic and renamed as Pm55a and Pm55b, respectively. The two Pm55 alleles encode coiled-coil, nucleotide-binding site-leucine-rich repeat (CNL) proteins, conferring broad-spectrum resistance to powdery mildew. However, they interact differently with a linked inhibitor gene, SuPm55 to cause different resistance to wheat powdery mildew. Notably, Pm55 and SuPm55 encode unrelated CNL proteins, and the inactivation of SuPm55 significantly reduces plant fitness. Combining SuPm55/Pm55a and Pm55b in wheat does not result in allele suppression or yield penalty. Our results provide not only insights into the suppression of resistance in wheat, but also a strategy for breeding durable resistance.

RLP1.1, a novel wheat receptor-like protein gene, is involved in the defence response against Puccinia striiformis f. sp. tritici.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most serious diseases of wheat; therefore, exploring effective resistance-related genes is critical for breeding and studying resistance mechanisms. However, only a few stripe rust resistance genes and defence-related genes have been cloned. Moreover, transgenic wheat with enhanced stripe rust resistance has rarely been reported. Receptor-like proteins (RLPs) are known to be involved in defence and developmental pathways. In this research, a novel RLP gene TaRLP1.1 was characterized as an important stripe rust defence gene. TaRLP1.1 was screened by GeneChip and was found to be induced by Pst specifically in the resistant variety. Knock down of TaRLP1.1 in the stripe rust-resistant plants resulted in increased susceptibility to Pst, and phenolic autofluorogen accumulation at the pathogen-host interaction sites, usually correlated with the hypersensitive response, was decreased dramatically. However, when the TaRLP1.1 gene was transformed into the susceptible wheat variety Yangmai158, the transgenic plants showed highly increased resistance to Pst, and the hypersensitive response was enhanced at the infection sites. Meanwhile, the expression of pathogenesis-related genes decreased in the TaRLP1.1-silenced plants and increased in the TaRLP1.1-overexpressing plants. Thus, it was proposed that TaRLP1.1 greatly contributed to the hypersensitive response during the pathogen-host interaction. Along with the functional analysis, an evolutionary study of the TaRLP1 family was performed. Characterization of TaRLP1.1 may facilitate breeding for stripe rust resistance and better understanding of the evolution of the RLP genes in wheat.

Induction of 4VS chromosome recombinants using the CS ph1b mutant and mapping of the wheat yellow mosaic virus resistance gene from Haynaldia villosa.

The wheat spindle streak mosaic virus (WSSMV) or wheat yellow mosaic virus (WYMV) resistance gene, Wss1, from Haynaldia villosa, was previously mapped to the chromosome arm 4VS by the development of 4V (4D) substitution and T4DL·4VS translocation lines. For better utilization and more accurate mapping of the Wss1, in this research, the CS ph1b mutant was used to induce new translocations with shortened 4VS chromosome fragments. Thirty-five homozygous translocations with different alien fragment sizes and breakpoints of 4VS were identified by GISH and molecular marker analysis. By field test, it was found that all the identified terminal translocations characterized as having smaller 4VS chromosome segments in the chromosome 4DS were highly resistant to WYMV, while all the interstitial translocations with 4VS inserted into the 4DS were WYMV susceptible. Marker analysis using 32 4VS-specific markers showed that both the terminal and interstitial translocations had different alien fragment sizes. Five specific markers could be detected in the WYMV-resistant terminal translocation line NAU421 with the shortest introduced 4VS fragment, indicating they can be used for marker-assisted selection in wheat breeding. Based on the resistance evaluation, GISH and molecular marker analysis of the available translocations, the gene(s) conferring the WYMV resistance on 4VS could be further cytologically mapped to the distal region of 4VS, immersed in the bin of FL 0.78-1.00. The newly developed small fragment translocations with WYMV resistance and 4VS specific markers have laid solid groundwork for the utilization in wheat breeding for WYMV resistance as well as further cloning of Wss1.

Leymus EST linkage maps identify 4NsL-5NsL reciprocal translocation, wheat-Leymus chromosome introgressions, and functionally important gene loci.

Allotetraploid (2n = 4x = 28) Leymus triticoides and Leymus cinereus are divergent perennial grasses, which form fertile hybrids. Genetic maps with n = 14 linkage groups (LG) comprised with 1,583 AFLP and 67 heterologous anchor markers were previously used for mapping quantitative trait loci (QTLs) in these hybrids, and chromosomes of other Leymus wildryes have been transferred to wheat. However, identifications of the x = 7 homoeologous groups were tenuous and genetic research has been encumbered by a lack of functional, conserved gene marker sequences. Herein, we mapped 350 simple sequence repeats and 26 putative lignin biosynthesis genes from a new Leymus EST library and constructed one integrated consensus map with 799 markers, including 375 AFLPs and 48 heterologous markers, spanning 2,381 centiMorgans. LG1b and LG6b were reassigned as LG6b* and LG1b*, respectively, and LG4Ns and LG4Xm were inverted so that all 14 linkage groups are aligned to the x = 7 Triticeae chromosomes based on EST alignments to barley and other reference genomes. Amplification of 146 mapped Leymus ESTs representing six of the seven homoeologous groups was shown for 17 wheat-Leymus chromosome introgression lines. Reciprocal translocations between 4L and 5L in both Leymus and Triticum monococcum were aligned to the same regions of Brachypodium chromosome 1. A caffeic acid O-methyltransferase locus aligned to fiber QTL peaks on Leymus LG7a and brown midrib mutations of maize and sorghum. Glaucousness genes on Leymus and wheat chromosome 2 were aligned to the same region of Brachypodium chromosome 5. Markers linked to the S self-incompatibility gene on Leymus LG1a cosegregated with markers on LG2b, possibly cross-linked by gametophytic selection. Homoeologous chromosomes 1 and 2 harbor the S and Z gametophytic self-incompatibility genes of Phalaris, Secale, and Lolium, but the Leymus chromosome-2 self-incompatibility gene aligns to a different region on Brachypodium chromosome 5. Nevertheless, cosegregation of self-incompatibility genes on Leymus presents a powerful system for mapping these loci.

Mapping and validation of quantitative trait loci associated with wheat yellow mosaic bymovirus resistance in bread wheat.

Wheat yellow mosaic (WYM) caused by wheat yellow mosaic bymovirus (WYMV) has been growing as one of the most serious diseases affecting wheat production in China. In this study, the association of quantitative trait loci (QTLs) governing WYMV resistance with molecular markers was established using 164 recombinant inbred lines (RILs) derived from 'Xifeng Wheat' (highly resistant) × 'Zhen 9523' (highly susceptible). Phenotypic data of WYMV resistance of the RILs were collected from 4-year, two-location replicated field trials. A molecular marker-based linkage map, which was comprised of 273 non-redundant loci and represented all the 21 wheat chromosomes, was constructed with the JoinMap 4.0 software. Using the Windows QTL Cartographer V2.5 software, three QTLs associated with WYMV resistance, QYm.njau-3B.1, QYm.njau-5A.1 and QYm.njau-7B.1, were detected on chromosomes 3BS, 5AL, and 7BS, respectively. The favorable allele effects were all contributed by 'Xifeng Wheat'. Among the three QTLs, QYm.njau-3B.1 and QYm.njau-5A.1 were detected in all the four trials and the overall mean, and could explain 3.3-10.2% and 25.9-53.7% of the phenotypic variation, respectively, while QYm.njau-7B.1 was detected in one trial and the overall mean and explained 4.9 and 3.3% of the phenotypic variation, respectively. A large portion of the variability for WYMV response was explained by a major QTL, QYm.njau-5A.1. The relationship of the molecular markers linked with QYm.njau-5A.1 and the WYMV resistance was further validated using a secondary F(2) population. The results showed that three markers, i.e., Xwmc415.1, CINAU152, and CINAU153, were closely linked to QYm.njau-5A.1 with the genetic distances of 0.0, 0.0, and 0.1 cM, respectively, indicating they should be useful in marker-assisted selection (MAS) wheat breeding for WYMV resistance. A panel of germplasm collection consisting of 46 wheat varieties with known WYMV response phenotypes was further used to validate the presence and effects of QYm.njau-5A.1 and the above three markers. It was found that QYm.njau-5A.1 was present in 12 of the 34 WYMV-resistant varieties.

Molecular and cytogenetic characterization of a small alien-segment translocation line carrying the softness genes of Haynaldia villosa.

The wheat-alien small segment translocation (SAST) lines carrying the beneficial genes from wild species are useful genetic stocks for wheat improvement. In this study, to introduce the grain hardness-related genes of Haynaldia villosa (L.) Schur. into common wheat (Triticum aesitivum L.), the mature female gametes of whole-arm wheat--H. villosa translocation line T5VS·5DL was irradiated by 60CO-γ ray to develop SAST lines involving 5VS. Among the BC2F2 population, six homozygous SAST lines with different fragment sizes of 5VS were identified by GISH, and the exact fragment sizes were further defined using four 5VS-specific markers and four Ha gene-based markers. The results showed that five lines (NAU5VS-1 to NAU5VS-5) carried the softness gene Dina/Dinb of H. villosa, and that NAU5VS-5 had the smallest alien translocation segment, identified to be a 5VS-6AS·6AL terminal translocation. The translocation chromosome 5VS-6AS·6AL was proved to be stably inherited to the successive generations. In the BC3F2 generation, the individuals having the homozygous 5VS-6AS·6AL translocation chromosomes all showed soft grain texture, with an approximately 50% reduction in the SKCS hardness index compared with that of their backcrossing parent. Both the 5VS-6AS·6AL translocation line and the molecular markers developed in this study will be valuable in wheat breeding for soft grain quality improvement.

A fast-neutron induced chromosome fragment deletion of 3BS in wheat landrace Wangshuibai increased its susceptibility to Fusarium head blight.

Fusarium head blight (FHB), also called wheat scab, is an important disease in warm and humid regions worldwide, which not only reduces crop yield and grain quality, but also is a major safety concern in food and feed production due to mycotoxin contamination. Growing wheat cultivars with FHB resistance is one of the most economical and effective means to control the disease. Chinese wheat landrace Wangshuibai is an important resistant source from southern China. Several resistance QTLs in Wangshuibai were identified and mapped on chromosomes or chromosomal arms including 3BS, 4B, 6BS, 7AL, etc. In the present research, a mutant with increased FHB susceptibility, designated as NAUH117, was identified from the M(1) progenies of Wangshuibai irradiated by fast neutron. Genetic analysis of the F (1), F (2), and F (2:3) families from the reciprocal cross of Wangshuibai and NAUH117 indicated that NAUH117 was a recessive mutant. Genome-wide molecular marker analysis identified a deletion in the short arm of chromosome 3B of NAUH117, spanning the region of FL0.57 to FL1.00 that covers the locus of Fhb1 previously mapped on chromosome 3BS. Further molecular cytogenetics characterization by bi-color fluorescence in situ hybridization using three repetitive sequences, pSc119.2, pAs1 and GAA-satellite indicated that a multiple chromosome rearrangements occurred in chromosomes 3B, 6B, 3D, 4D, and 3A of the mutant. During these processes, a distal fragment of chromosome arm 3BS was eliminated, which is confirmed by molecular marker analysis. Four markers covered the deletion fragment were used for analysis of the F (2) population. The result showed that the 3BS deletion was only present in the susceptible plants, indicating that the deletion of 3BS fragment in NAUH117 increased susceptibility to FHB. The susceptible mutant will be valuable for the validation of the contribution of the resistant QTL located on 3BS, and for the characterization of the molecular mechanisms of FHB resistance in Wangshuibai.

Collinearity-based marker mining for the fine mapping of Pm6, a powdery mildew resistance gene in wheat.

The genome sequences of rice (Oryza sativa L.) and Brachypodium distachyon and the comprehensive Triticeae EST (Expressed Sequence Tag) resources provide invaluable information for comparative genomics analysis. The powdery mildew resistance gene, Pm6, which was introgressed into common wheat from Triticum timopheevii, was previously mapped to the wheat chromosome bin of 2BL [fraction length (FL) 0.50-1.00] with limited DNA markers. In this study, we saturated the Pm6 locus in wheat using the collinearity-based markers by extensively exploiting these genomic resources. All wheat ESTs located in the bin 2BL FL 0.50-1.00 and their corresponding orthologous genes on rice chromosome 4 were firstly used to develop STS (Sequence Tagged Site) markers. Those identified markers that flanked the Pm6 locus were then used to identify the collinear regions in the genomes of rice and Brachypodium. Triticeae ESTs with orthologous genes in these collinear regions were further used to develop new conserved markers for the fine mapping of Pm6. Using two F(2) populations derived from crosses of IGVI-465 × Prins and IGVI-466 × Prins, we mapped a total of 29 markers to the Pm6 locus. Among them, 14 markers were co-segregated with Pm6 in the IGVI-466/Prins population. Comparative genome analysis showed that the collinear region of the 29 linked markers covers a ~5.6-Mb region in chromosome 5L of Brachypodium and a ~6.0-Mb region in chromosome 4L of rice. The marker order is conserved between rice and Brachypodium, but re-arrangements are present in wheat. Comparative mapping in the two populations showed that two conserved markers (CINAU123 and CINAU127) flanked the Pm6 locus, and an LRR-receptor-like protein kinase cluster was identified in the collinear regions of Brachypodium and rice. This putative resistance gene cluster provides a potential target site for further fine mapping and cloning of Pm6. Moreover, the newly developed conserved markers closely linked to Pm6 can be used for the marker-assisted selection (MAS) of Pm6 in wheat breeding programs.