Many of our major crop species are polyploids, containing more than one genome or set of chromosomes. Polyploid crops present unique challenges, including difficulties in genome assembly, in discriminating between multiple gene and sequence copies, and in genetic mapping, hindering use of genomic data for genetics and breeding. Polyploid genomes may also be more prone to containing structural variation, such as loss of gene copies or sequences (presence-absence variation) and the presence of genes or sequences in multiple copies (copy-number variation). Although the two main types of genomic structural variation commonly identified are presence-absence variation and copy-number variation, we propose that homeologous exchanges constitute a third major form of genomic structural variation in polyploids. Homeologous exchanges involve the replacement of one genomic segment by a similar copy from another genome or ancestrally duplicated region, and are known to be extremely common in polyploids. Detecting all kinds of genomic structural variation is challenging, but recent advances such as optical mapping and long-read sequencing offer potential strategies to help identify structural variants even in complex polyploid genomes. All three major types of genomic structural variation (presence-absence, copy-number, and homeologous exchange) are now known to influence phenotypes in crop plants, with examples of flowering time, frost tolerance, and adaptive and agronomic traits. In this review, we summarize the challenges of genome analysis in polyploid crops, describe the various types of genomic structural variation and the genomics technologies and data that can be used to detect them, and collate information produced to date related to the impact of genomic structural variation on crop phenotypes. We highlight the importance of genomic structural variation for the future genetic improvement of polyploid crops.

Sequence-specific nucleases (SSN) that generate double-stranded DNA breaks (DSBs) in genes of interest are the key to site-specific genome editing in plants. Genome editing has developed into one method of reducing undesirable traits in crops by the induction of knockout mutations. Different SSN-mediated genome-editing systems, including LAGLIDADG homing endonucleases or meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats, are emerging as robust tools for introducing functional mutations in polyploid crops including citrus, wheat, cotton, soybean, rapeseed, potato, grapes, Camelina sativa, dandelion, and tobacco. The approach utilizes knowledge of biological mechanisms for targeted induction of DSBs and their error-prone repair, allowing highly specific changes at designated genome loci. In this review, we briefly describe genome-editing technologies and their application to genetic improvement of polyploid crops.

As an important wild relative of wheat, Agropyron cristatum has been successfully used for wheat improvement. Currently, a few useful agronomic traits of A. cristatum, such as high grain number per spike and resistance to diseases, have been transferred into common wheat. However, the effective detection of small A. cristatum segmental introgressions in common wheat is still difficult. The objective of this study was to identify A. cristatum-specific single nucleotide polymorphisms (SNPs) for the detection of small alien segments in wheat. The transcriptome sequences of A. cristatum were aligned against wheat coding DNA sequences (CDS) for SNP calling. As a result, we discovered a total of 167,613 putative SNPs specific to the P genome of A. cristatum compared with the common wheat genomes. Among 230 selected SNPs with functional annotations related to inflorescence development and stress resistance, 68 were validated as P genome-specific SNPs in multiple wheat backgrounds using Kompetitive Allele Specific PCR (KASP) assays. Among them, 55 SNPs were assigned to six homoeologous groups of the P genome using wheat-A. cristatum addition lines, and 6P-specific SNP markers were further physically mapped on different segments of chromosome 6P in 6P translocation lines. The P genome-specific SNPs were also validated by Sanger sequencing and used to detect the P chromatin in wheat-A. cristatum cryptic introgression lines. Two SNP markers (Unigene20217-182 and Unigene20307-1420) were detected in two wheat-A. cristatum introgression lines that showed enhanced grain number per spike and high resistance to powdery mildew. Together, the developed P genome-specific SNP markers will accelerate the detection of large numbers of wheat-A. cristatum derivatives and will be helpful for marker-assisted transfer of desirable traits from A. cristatum into adapted wheat cultivars in wheat breeding programs.

Thinopyrum intermedium and barley are two close relatives of wheat and carry many genes that are potentially valuable for the improvement of various wheat traits. In this study we created wheat double substitution lines by hybridizing different wheat-Th. intermedium and wheat-barley disomic alien substitution lines, with the aim of using genes in Th. intermedium and barley for wheat breeding and investigating the genetic behavior of alien chromosomes and their wheat homoeologs. As expected, we obtained two types of wheat double substitution lines, 2D2Ai#2(2B)2H(2A) and 2A2Ai#2(2B)2H(2D), in which different group 2 wheat chromosomes were replaced by barley chromosome 2H and Th. intermedium chromosome 2Ai#2. The new materials were characterized using molecular markers, genomic in situ hybridization (GISH), and fluorescent in situ hybridization (FISH). GISH and FISH experiments revealed that the double substitution lines harbor 42 chromosomes including 38 wheat chromosomes, a pair of barley chromosomes, and a pair of Th. intermedium chromosomes. Analysis using specific DNA markers showed that two pairs of wheat homoeologous group 2 chromosomes in the new lines were substituted by a pair of 2H and a pair of 2Ai#2 chromosomes. Chromosome 2H showed a higher transmission rate than 2Ai#2, and both chromosomes were preferentially transmitted between generations via female gametes. Evaluation of botanic and agronomic traits demonstrated that, compared with their parents, the new lines showed similar growth habits and plant type but differences in plant height, flowering date, and self-fertility. Cytological observations using different probes suggested that the double substitution lines showed nearly normal genetic behavior before and during meiosis. The novel substitution lines can potentially be used in wheat meiosis research and breeding programs.

Wheat crops in China are constantly challenged by stripe rust. Deployment of cultivars with diverse resistances is the best strategy to control the disease. A recombinant inbred line (RIL) population derived from a cross between the resistant cultivar Chakwal 86 and susceptible landrace Mingxian 169 was studied in multiple environments to examine the underlying genetics and to identify quantitative trait loci (QTL) for stripe rust resistance. One hundred and twenty-eight RILs were genotyped with wheat 35K SNP array and a genome-wide linkage map with 1480 polymorphic SNP markers, or bins, was constructed. Two major QTL on chromosomes 1BL and 3BS, and one minor QTL on 6BS had significant effects in reducing stripe rust severity. The QTL were validated using composite interval mapping (CIM) and inclusive composite interval mapping (ICIM). These methods explained 59.0%-74.1% of the phenotype variation in disease response. The QTL on chromosome 1BL was confirmed to be Yr29/Lr46 and the one on 3BS was the resistance allele identified in CIMMYT germplasm but was not Yr30/Sr2. The QTL on 6BS probably corresponded to previously known QTL. RILs with combined QTL were more resistant than those with single or no QTL. Kompetitive allele-specific PCR (KASP) assays for the QTL with largest effect QTL on chromosome 3BS were performed on a subset of RILs and 150 unrelated wheat lines. The QTL on 3BS with its linked KASP markers can be used in marker-assisted selection to improve stripe rust resistance in breeding programs.

Short coleoptiles associated with GA-insensitive Rht-1 alleles in wheat reduces yield due to poor seedling establishment under dry, or stubble-retained conditions. Hence there is a need for alternative dwarfing genes for wheat improvement programs. GA-sensitive dwarfing gene Rht14 confers semidwarf stature in wheat while retaining longer coleoptiles and early seedling vigor. Two RIL populations were used to identify the map position of Rht14 and to estimate its effect on plant height, coleoptile length, seedling shoot length, spike length and internode length. Rht14 on chromosome 6A was mapped in the genomic region 383-422?Mbp flanked by GA2oxA9 and wmc753 in a Bijaga Yellow/Castelporziano RIL population. Recombination events between Rht14 and GA2oxA9 in the RIL population indicated that Rht14 might not be allelic to GA2oxA9. The conserved DNA sequence of GA2oxA9 and its flanking region in Castelporziano also suggested that the point of mutation responsible for the Rht14 allele must be a few Mbp away from GA2oxA9. The dwarfing effects of Rht14 on plant height, internode length and seedling vigor were compared with those of Rht-B1b in an HI 8498/Castelporziano RIL population. Both genes significantly reduced plant height and internode length. Rht-B1b conferred a significant reduction in coleoptile length and seedling shoot length, whereas Rht14 reduced plant height, but not coleoptile and seedling shoot length. Therefore, Rht14 can be a used as an alternative to Rht-B1b for development of cultivars suitable for deeper sowing in dry environments and in conditions of conservation agriculture where crop residues are retained.

The cotton fiber is the most important raw material for the textile industry and an ideal model system for studying cell elongation. However, the genetic variation of fiber elongation in relation to miRNA is poorly understood. A high-throughput comparative RNA-seq of two lines differing in fiber length (FL) from a backcross inbred line (BIL) population of G. hirsutum?×?G. barbadense revealed differentially expressed (DE) miRNAs and their targets in rapidly elongating fibers. A real-time quantitative PCR analysis was further performed to validate the results. A total of 463 (including 47 DE) miRNAs were identified, and seven DE miRNAs were co-localized with seven FL quantitative trait loci (QTL) identified in the G. hirsutum?×?G. barbadense population. Of 82 (including 21 DE) targets identified, nine (including one DE) were also co-localized with the seven FL QTL. The relationship between the allopolyploid and its diploid ancestral species with respect to miRNAs and their targets was also characterized. These results will facilitate the understanding of the molecular genetic mechanism of fiber elongation with regards to miRNAs in cotton.

Plant architecture traits influence crop yield. An understanding of the genetic basis of cotton plant architecture traits is beneficial for identifying favorable alleles and functional genes and breeding elite cultivars. We collected 121 cotton accessions including 100 brown-fiber and 21 white-fiber accessions, genotyped them by whole-genome resequencing, and phenotyped them in multiple environments. This genome-wide association study (GWAS) identified 11 quantitative trait loci (QTL) for two plant architecture traits: plant height and fruit spur branch number. Negative-effect alleles were enriched in the elite cultivars. Based on these QTL, gene annotation information, and published QTL, candidate genes and natural genetic variations in four QTL were identified. Ghir_D02G017510 and Ghir_D02G017600 were identified as candidate genes for qD02-FSBN-1, and a premature start codon gain variation was found in Ghir_D02G017510. Ghir_A12G026570, the candidate gene of qA12-FSBN-2, belongs to the pectin lyase-like superfamily, and a significantly associated SNP, A12_105366045 (T/C), in this gene represents an amino acid change. The QTL, candidate genes, and associated natural variations in this study are expected to lay a foundation for studying functional genes and developing breeding programs for desirable architecture in brown-fiber cotton.

Optimizing the profile and quantity of fatty acids in rapeseed (Brassica napus L.) is critical for maximizing the value of edible oil and biodiesel. However, selection of these complex seed quality traits is difficult before haplotypes controlling their contents are identified. To efficiently identify genetic loci influencing these traits and underlying candidate genes and networks, we performed a genome-wide association study (GWAS) of eight seed quality traits (oil and protein content, palmitic, stearic, oleic, linoleic, eicosenoic and erucic acids content). The GWAS population comprised 370 diverse accessions, which were phenotyped in five environments and genotyped using 60K SNP arrays. The results indicated that oil and protein contents generally showed negative correlations, while fatty acid contents showed positive or negative correlations, with palmitic and erucic acid contents directly affecting oil content. Seven SNPs on five chromosomes were associated with both seed oil and protein content, and five genes orthologous to genes in Arabidopsis thaliana were predicted as candidates. From resequencing data, besides known haplotypes in BnaA.FAE1.a and BnaC.FAE1.a, three accessions harboring a new haplotype conferring moderate erucic acid content were identified. Interestingly, in a haplotype block, one haplotype was associated with high palmitic acid content and low oil content, while the others showed the reverse effects. This finding was consistent with a negative correlation between palmitic acid and oil contents, suggesting historical selection for high oil content. The identification by this study of genetic variation and complex correlations of eight seed quality traits may be beneficial for crop selection strategies.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) de Bary is one of the most devastating diseases of Brassica napus worldwide. Both SSR resistance and flowering time (FT) adaptation are major breeding goals in B. napus. However, early maturing rapeseed varieties, which are important for rice-rapeseed rotation in China, are often highly susceptible to SSR. Here, we found that SSR resistance was significantly negatively correlated with FT in a natural population containing 521 rapeseed inbred lines and a double haploid (DH) population with 150 individual lines, both of which had great variation in FT. Four chromosomal regions on A2, A6, C2, and C8 affecting both SSR resistance and FT were identified using quantitative trait loci (QTL) mapping after constructing a high-density genetic map based on single nucleotide polymorphism markers in the DH population. Furthermore, we aligned QTL for the two traits identified in the present and previous studies to the B. napus reference genome, and identified four colocalized QTL hotspots of SSR resistance and FT on A2 (0-7.7?Mb), A3 (0.8-7.5?Mb), C2 (0-15.2?Mb), and C6 (20.2-36.6?Mb). Our results revealed a genetic link between SSR resistance and FT in B. napus, which should facilitate the development of effective strategies in both early maturing and SSR resistance breeding and in map-based cloning of SSR resistance QTL.

The inheritance of pod- and seed-number traits (PSNT) in peanut (Arachis hypogaea L.) is poorly understood. In the present study, a recombinant inbred line (RIL) population of 188 lines was used to map quantitative trait loci (QTL) for number of seeds per pod (NSP), number of pods per plant (NPP), and numbers of one-, two-, and three-seeded pods per plant (N1PP, N2PP, and N3PP) in four environments. A total of 28 consensus QTL and 14 single QTL were identified, including 11 major and stable QTL. Four major and stable QTL including qN3PPA5.2, qN3PPA5.4, qN3PPA5.5, and qN3PPA5.7 each explained 12.3%-33.0% of phenotype variation. By use of another integrated linkage map for the A5 group (hereafter referred to as INT A5 group), QTL for PSNT were located in seven intervals of 0.73-9.68?Mb in length on chromosome A05, and candidate genes underlying N3PP were suggested. These findings shed light on the genetic basis of PSNT. Major QTL for N3PP could be used as candidates for further positional cloning.

To dissect the genetic mechanism of multi-seed pod in peanut, we explored the QTL/gene controlling multi-seed pod and analyzed the interaction effect of QTL and environment. Two hundred and forty eight recombinant inbred lines (RIL) from cross Silihong?×?Jinonghei 3 were used as experimental materials planted in 8 environments from 2012 to 2017. Three methods of analysis were performed. These included individual environment analysis, joint analysis in multiple environments, and epistatic interaction analysis for multi-seed pod QTL. Phenotypic data and best linear unbiased prediction (BLUP) value of the ratio of multi-seed pods per plant (RMSP) were used for QTL mapping. Seven QTL detected by the individual environmental mapping analysis and were distributed on linkage groups 1, 6, 9, 14, 19(2), and 21. Each QTL explained 4.42%-11.51% of the phenotypic variation in multi-seed pod, and synergistic alleles of 5 QTL were from the Silihong parent. One QTL, explaining 4.93% of the phenotypic variation was detected using BLUP data, and this QTL mapped in the same interval as qRMSP19.1 detected in the individual environment analysis. Seventeen additive QTL were identified by joint analysis across multiple environments. A total of 43 epistatic QTL were detected by ICIM-EPI mapping in the multiple environment trials (MET) module, and involved 57 loci. Two main-effect QTL related to multi-seed pod in peanut were filtered. We also found that RMSP had a highly significant positive correlation with pod yield per plant (PY), and epistatic effects were much more important than additive effects. These results provide theoretical guidance for the genetic improvement of germplasm resources and further fine mapping of related genes in peanut.

Tuber starch content and plant maturity are two important agronomic traits of potato. To investigate the complex genetic basis of these traits in the cultivated potato, as well as the relationship between them, we developed a linkage map in a tetraploid population of 192 clones derived from the cross Longshu 8?×?Zaodabai and mapped quantitative trait loci (QTL) for tuber starch content and plant maturity using data collected in three diverse environments over two years. We detected eleven QTL for tuber starch content distributed on seven chromosomes, of which four, on chromosomes I, II, and VIII, were expressed in at least three environments. For plant maturity, we identified six QTL on chromosomes II, IV, V, VII, and XI, one of which, on chromosome V, showed LOD peaks ranging from 45.2 to 62.5?cM and explained 21.6%-26.6% of phenotypic variation was expressed in five of the six environments. Because the reproducible QTL for plant maturity and tuber starch content mapped to different chromosomes and neither overlapping QTL, nor any genetic interaction between QTL were detected, we infer that tuber starch content and plant maturity are controlled by independent genetic loci. This inference supports the prospect of breeding potato for both early maturity and high starch content.