Sheng Chen

The genome sequence of allopolyploid Brassica juncea and analysis of differential homoeolog gene expression influencing selection.

The Brassica genus encompasses three diploid and three allopolyploid genomes, but a clear understanding of the evolution of agriculturally important traits via polyploidy is lacking. We assembled an allopolyploid Brassica juncea genome by shotgun and single-molecule reads integrated to genomic and genetic maps. We discovered that the A subgenomes of B. juncea and Brassica napus each had independent origins. Results suggested that A subgenomes of B. juncea were of monophyletic origin and evolved into vegetable-use and oil-use subvarieties. Homoeolog expression dominance occurs between subgenomes of allopolyploid B. juncea, in which differentially expressed genes display more selection potential than neutral genes. Homoeolog expression dominance in B. juncea has facilitated selection of glucosinolate and lipid metabolism genes in subvarieties used as vegetables and for oil production. These homoeolog expression dominance relationships among Brassicaceae genomes have contributed to selection response, predicting the directional effects of selection in a polyploid crop genome.

Quantitative trait loci for thermal time to flowering and photoperiod responsiveness discovered in summer annual-type Brassica napus L.

Time of flowering is a key adaptive trait in plants and is conditioned by the interaction of genes and environmental cues including length of photoperiod, ambient temperature and vernalisation. Here we investigated the photoperiod responsiveness of summer annual-types of Brassica napus (rapeseed, canola). A population of 131 doubled haploid lines derived from a cross between European and Australian parents was evaluated for days to flowering, thermal time to flowering (measured in degree-days) and the number of leaf nodes at flowering in a compact and efficient glasshouse-based experiment with replicated short and long day treatments. All three traits were under strong genetic control with heritability estimates ranging from 0.85–0.93. There was a very strong photoperiod effect with flowering in the population accelerated by 765 degree-days in the long day versus short day treatments. However, there was a strong genetic correlation of line effects (0.91) between the long and short day treatments and relatively low genotype x treatment interaction indicating that photoperiod had a similar effect across the population. Bivariate analysis of thermal time to flowering in short and long days revealed three main effect quantitative trait loci (QTLs) that accounted for 57.7% of the variation in the population and no significant interaction QTLs. These results provided insight into the contrasting adaptations of Australian and European varieties. Both parents responded to photoperiod and their alleles shifted the population to earlier flowering under long days. In addition, segregation of QTLs in the population caused wide transgressive segregation in thermal time to flowering. Potential candidate flowering time homologues located near QTLs were identified with the aid of the Brassica rapa reference genome sequence. We discuss how these results will help to guide the breeding of summer annual types of B. napus adapted to new and changing environments.

Functional markers developed from multiple loci in GS3 for fine marker-assisted selection of grain length in rice

The gene GS3 has major effect on grain size and plays an important role in rice breeding. The C to A mutation in the second exon of GS3 was reported to be functionally associated with enhanced grain length in rice. In the present study, besides the C-A mutation at locus SF28, three novel polymorphic loci, SR17, RGS1, and RGS2, were discovered in the second intron, the last intron and the final exon of GS3, respectively. A number of alleles at these four polymorphic loci were observed in a total of 287 accessions including Chinese rice varieties (Oryza sativa), African cultivated rice (O. glaberrima) and AA-genome wild relatives. The haplotype analysis revealed that the simple sequence repeats (AT)n at RGS1 and (TCC)n at RGS2 had differentiated in the wild rice whilst the C-A mutation occurred in the cultivated rice recently during domestication. It also indicated that A allele at SF28 was highly associated with long rice grain whilst various motifs of (AT)n at RGS1 and (TCC)n at RGS2 were mainly associated with medium to short grain in Chinese rice. The C-A mutation at SF28 explained 33.4% of the grain length variation in the whole rice population tested in this study, whereas (AT)n at RGS1 and (TCC)n at RGS2 explained 26.4 and 26.2% of the variation, respectively. These results would be helpful for better understanding domestication of GS3 and its manipulation for grain size in rice. The genic marker RGS1 based on the motifs (AT)n was further validated as a functional marker using two sets of backcross recombinant inbred lines. These results suggested that the functional markers developed from four different loci within GS3 could be used for fine marker-assisted selection of grain length in rice breeding.

Trigenomic bridges for Brassica improvement

We introduce and review Brassica crop improvement via trigenomic bridges. Six economically important Brassica species share three major genomes (A, B, and C), which are arranged in diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) species in the classical triangle of U. Trigenomic bridges are Brassica interspecific hybrid plants that contain the three genomes in various combinations, either triploid (ABC), unbalanced tetraploid (e.g., AABC), pentaploid (e.g., AABCC) or hexaploid (AABBCC). Through trigenomic bridges, Brassica breeders can access all the genetic resources in the triangle of U for genetic improvement of existing species and development of new agricultural species. Each of the three Brassica genomes occurs in several species, where they are distinguished as subgenomes with a tag to identify the species of origin. For example, the A subgenome in B. juncea (2n = AABB) is denoted as Aj and the A subgenome in B. napus (2n = AACC) as An. Trigenomic bridges have been used to increase genetic diversity in allopolyploid Brassica crop species, such as a new-type B. napus with subgenomes from B. rapa (Ar) and B. carinata (Cc). Recently, trigenomic bridges from several sources have been crossed together as the ‘founders’ of a potentially new allohexaploid Brassica species (AABBCC). During meiosis in a trigenomic bridge, crossovers are expected to form between homologous chromosomes of related subgenomes (for example Ar and An), but cross-overs may also occur between non-homologous chromosomes (for example between A and C genome chromosomes). Irregular meiosis is a common feature of new polyploids, and any new allotetraploid or allohexaploid Brassica genotypes derived from a trigenomic bridge must achieve meiotic stability through a process of diploidisation. New sequencing technologies, at the genomic and epigenomic level, may reveal the genetic and molecular basis of diploidization, and accelerate selection of stable allotetraploids or allohexaploids. Armed with new genetic resources from trigenomic bridges, Brassica breeders will be able to improve yield and broaden adaptation of Brassica crops to meet human demands for food and biofuel, particularly in the face of abiotic constraints caused by climate change.

Allelic diversity in a novel gene pool of canola-quality Brassica napus enriched with alleles from B. rapa and B. carinata

Brassica napus is an amphidiploid with genome AACC and is relatively deficient in genetic diversity. The abundant genetic diversity in other A- and C-genome Brassica species is a valuable resource to expand the narrow gene pool of B. napus. Recently the Ar genomic components from Chinese B. rapa (ArAr) and Cc genomic components from Ethiopian mustard B. carinata (BBCcCc) were introgressed into B. napus through interspecific hybridisation, and the Ar/Cc components were enriched through two generations of molecular marker-assisted selection. In this study, the simple sequence repeat (SSR) allelic diversity of 29 of these new-type B. napus lines, 12 from the first generation and 17 from the second generation, was compared with 66 international B. napus varieties from Australia, China and other countries. Hierarchical clustering and two-dimensional multidimensional scaling revealed that second generation lines and a few first generation lines, all selected for high Ar/Cc components, formed a unique population that was distantly separated from international B. napus. This novel gene pool had significantly higher richness of private SSR alleles and more alleles per SSR marker than the international B. napus varieties. The new-type B. napus lines showed variation in agronomic traits beyond the canola-quality B. napus parent. Many of the lines had low erucic acid and low glucosinolates in the seed (canola quality), indicating that they could be utilised immediately in canola breeding programs.

Doubled haploids of novel trigenomic Brassica derived from various interspecific crosses

To develop doubled haploid (DH) mapping populations of hexaploid Brassica, 10 F1 hybrids derived from crosses between allohexaploid Brassica parents were evaluated in this study. The allohexaploid Brassica parents were selfed progenies of unique interspecific crosses between Brassica rapa (genome AA)xa0×xa0B. carinata (BBCC), B. nigra (BB)xa0×xa0B. napus (AACC), and a complex cross between B. juncea (AABB), B. napus and B. carinata, with relatively stable chromosome number (2nxa0=xa054). Hexaploid status and chromosome behavior during meiosis I in four promising F1 hybrids were assessed using microscopy and flow cytometry, and progeny were obtained following microspore culture. Hybrids H11-2 and H16-1 demonstrated higher amenability for embryo generation, plantlet regeneration, and frequency of production of DH microspore-derived progeny of hexaploid DNA content (6x) compared to hybrids H08-1 and H24-1. A total of 370 6x DH progeny were selected out of 693 plantlets from H11-2, 241/436 from H16-1, 23/54 from H08-1, and 21/56 from H24-1. DH progenies of hybrids H11-2 and H16-1 were then designated as promising mapping populations of a new hexaploid Brassica species.

A genetic linkage map of Brassica carinata constructed with a doubled haploid population

Brassica carinata is an important oilseed crop with unique favourable traits that are desirable for other Brassica crops. However, given the limited research into genetic resources in B. carinata, knowledge of the genetic structure of this species is relatively poor. Nine homozygous, genetically distinct accessions of B. carinata were obtained via microspore culture, from which two divergent doubled haploid (DH) lines were used to develop a DH mapping population that consisted of 183 lines. The mapping population showed segregation of multiple traits of interest. A genetic map was constructed with PCR-based markers, and a total of 212 loci, which covered 1,703xa0cM, were assigned to eight linkage groups in the B genome and nine linkage groups in the C genome, which allowed comparison with genetic maps of other important Brassica species that contain the B/C genome(s). Loci for two Mendelian-inherited traits related to pigmentation (petal and anther tip colour) and one quantitative trait (seed coat colour) were identified using the linkage map. The significance of the mapping population in the context of genetic improvement of Brassica crops is discussed.

Evidence from Genome-wide Simple Sequence Repeat Markers for a Polyphyletic Origin and Secondary Centers of Genetic Diversity of Brassica juncea in China and India

The oilseed Brassica juncea is an important crop with a long history of cultivation in India and China. Previous studies have suggested a polyphyletic origin of B. juncea and more than one migration from the primary to secondary centers of diversity. We investigated molecular genetic diversity based on 99 simple sequence repeat markers in 119 oilseed B. juncea varieties from China, India, Europe, and Australia to test whether molecular differentiation follows Vavilovs proposal of secondary centers of diversity in India and China. Two distinct groups were identified by markers in the A genome, and the same two groups were confirmed by markers in the B genome. Group 1 included accessions from central and western India, in addition to those from eastern China. Group 2 included accessions from central and western China, as well as those from northern and eastern India. European and Australian accessions were found only in Group 2. Chinese accessions had higher allelic diversity per accession (Group 1) and more private alleles per accession (Groups 1 and 2) than those from India. The marker data and geographic distribution of Groups 1 and 2 were consistent with two independent migrations of B. juncea from its center of origin in the Middle East and neighboring regions along trade routes to western China and northern India, followed by regional adaptation. Group 1 migrated further south and west in India, and further east in China, than Group 2. Group 2 showed diverse agroecological adaptation, with yellow-seeded spring-sown types in central and western China and brown-seeded autumn-sown types in India.

Identification of quantitative trait loci associated with germination using chromosome segment substitution lines of rice (Oryza sativa L.)

Rapid and uniform seed germination under diverse environmental conditions is a desirable characteristic for most crop plants, such as rice, wheat, and maize. However, the genetic base of the variations in the rate of germination is not well understood. In this study, quantitative trait loci (QTL) for germination rate were mapped with a set of 143 chromosome segment substitution lines (CSSL) each contains a small genomic fragment from a japonica variety Nipponbare in the uniform genetic background of an indica variety Zhenshan97. Nine CSSL showed significantly lower germination rate than that in Zhenshan97. Four germination-related QTL were identified located on chromosomes 2, 5, 6 and 10, at which all japonica alleles decreased germination rate. By using the CSSL-derived F2 population, a major QTL (qGR2) on chromosome 2 was confirmed, and delimited to a 10.4xa0kb interval containing three putative candidate genes, of which OsMADS29 was only expressed preferentially in the seed. These results would facilitate cloning of the major gene that affects germination rate, and provide an insight into the genetic basis of germination.

Diversity and selective sweep in the OsAMT1;1 genomic region of rice

BackgroundAmmonium is one of the major forms in which nitrogen is available for plant growth. OsAMT1;1 is a high-affinity ammonium transporter in rice (Oryza sativa L.), responsible for ammonium uptake at low nitrogen concentration. The expression pattern of the gene has been reported. However, variations in its nucleotides and the evolutionary pathway of its descent from wild progenitors are yet to be elucidated. In this study, nucleotide diversity of the gene OsAMT1;1 and the diversity pattern of seven gene fragments spanning a genomic region approximately 150 kb long surrounding the gene were surveyed by sequencing a panel of 216 rice accessions including both cultivated rice and wild relatives.ResultsNucleotide polymorphism (Pi) of OsAMT1;1 was as low as 0.00004 in cultivated rice (Oryza sativa), only 2.3% of that in the common wild rice (O. rufipogon). A single dominant haplotype was fixed at the locus in O. sativa. The test values for neutrality were significantly negative in the entire region stretching 5 upstream and 3 downstream of the gene in all accessions. The value of linkage disequilibrium remained high across a 100 kb genomic region around OsAMT1;1 in O. sativa, but fell rapidly in O. rufipogon on either side of the promoter of OsAMT1;1, demonstrating a strong natural selection within or nearby the ammonium transporter.ConclusionsThe severe reduction in nucleotide variation at OsAMT1;1 in rice was caused by a selective sweep around OsAMT1;1, which may reflect the nitrogen uptake system under strong selection by the paddy soil during the domestication of rice. Purifying selection also occurred before the wild rice diverged into its two subspecies, namely indica and japonica. These findings would provide useful insights into the processes of evolution and domestication of nitrogen uptake genes in rice.