Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in <Emphasis Type="Italic">Brassica rapa</Emphasis>

A glabrous, yellow-seeded doubled haploid (DH) line and a hairy, black-seeded DH line in Chinese cabbage (B. rapa) were used as parents to develop a DH line population that segregated for both hairiness and seed coat color traits. The data showed that both traits completely co-segregated each other, suggesting that one Mendelian locus controlled both hairiness and seed coat color in this population. A fine genetic map was constructed and a SNP marker that was located inside a Brassica ortholog of TRANSPARENT TESTA GLABRA 1 (TTG1) in Arabidopsis showed complete linkage to both the hairiness and seed coat color gene, suggesting that the Brassica TTG1 ortholog shared the same gene function as its Arabidopsis counterpart. Further sequence analysis of the alleles from hairless, yellow-seeded and hairy, black-seeded DH lines in B. rapa showed that a 94-base deletion was found in the hairless, yellow-seeded DH lines. A nonfunctional truncated protein in the hairless, yellow-seeded DH lines in B. rapa was suggested by the coding sequence of the TTG1 ortholog. Both of the TTG1 homologs from the black and yellow seeded B. rapa lines were used to transform an Arabidopsis ttg1 mutant and the results showed that the TTG1 homolog from the black seeded B. rapa recovered the Arabidopsis ttg1 mutant, while the yellow seeded homolog did not, suggesting that the deletion in the Brassica TTG1 homolog had led to the yellow seeded natural mutant. This was the first identified gene in Brassica species that simultaneously controlled both hairiness and seed coat color traits.     

Development of SRAP,SNP and Multiplexed SCAR molecular markers for the major seed coat color gene in <Emphasis Type="Italic">Brassica rapa L.</Emphasis>

Seed coat color inheritance in B. rapa was studied in F(1), F(2), F(3), and BC(1) progenies from a cross of a Canadian brown-seeded variety 'SPAN' and a Bangladeshi yellow sarson variety 'BARI-6'. A pollen effect was found when the yellow sarson line was used as the maternal parent. Seed coat color segregated into brown, yellow-brown and bright yellow classes. Segregation was under digenic control where the brown or yellow-brown color was dominant over bright yellow seed coat color. A sequence related amplified polymorphism (SRAP) marker linked closely to a major seed coat color gene (Br1/br1) was developed. This dominant SRAP molecular marker was successfully converted into single nucleotide polymorphism (SNP) markers and sequence characterized amplification region (SCAR) markers after the extended flanking sequence of the SRAP was obtained with chromosome walking. In total, 24 SNPs were identified with more than 2-kb sequence. A 12-bp deletion allowed the development of a SCAR marker linked closely to the Br1 gene. Using the five-fluorescence dye set supplied by ABI, four labeled M13 primers were integrated with different SCAR primers to increase the throughput of SCAR marker detection. Using multiplexed SCAR markers targeting insertions and deletions in a genome shows great potential for marker assisted selection in plant breeding.     

褐色种皮大豆与其黄色种皮衍生亲本的表型及基因型比较

大豆种皮色在从野生大豆到栽培大豆的选择过程中逐渐由黑色变成黄色,是重要的形态标记,因此,大豆种皮色相关基因的研究无论是对进化理论研究还是育种实践都具有非常重要的意义。利用褐色种皮J1265-2大豆及其衍生亲本黄色种皮大豆J1265-1为材料,通过SSR引物扩增片段,检验遗传背景的异同,同时对控制种皮的候选基因GmF3’H进行扩增和测序分析。结果表明,褐色种皮和黄色种皮材料不仅用161对SSR分子标记检测没有发现差异,其褐色种皮候选基因GmF3’H的编码区及起始密码子上游1465 bp序列也是一致的。因此,证明褐色种皮J1265-2大豆与其衍生亲本黄色种皮大豆J1265-1为近等基因系,其控制褐色种皮的基因型与已报道的基因型不同。     

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene*

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T₂ transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.     

Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus

To establish a cytoplasmic male-sterile/restored fertility (cms-Rf) system for F₁ seed production in Brassica napus, we transferred a gene from fertillity restored radish to B. napus by protoplast fusion. X-irradiated protoplasts, isolated from shoots of Raphanus sativus cv Kosena (Rf line), were fused with iodoacetamide-treated protoplasts of a B. napus cms cybrid. Among 300 regenerated plants, six were male-fertile. The fertile plants were characterized for petal color, chromosome number and the percentage of viable pollen grains. Three fertile plants had aneuploid chromosome numbers and white or cream petals, which is a dominant marker in radish. Of these three plants, one which had 2n = 47 chromosomes and white petals was used for further backcrosses. After two backcrosses, chromosome number and petal color became identical to that of B. napus. No female sterility was observed in the BC₃ generations.     

The inheritance of seed color in a resynthesizedBrassica napus line No. 2127-17 including a new epistatic locus

Yellow seed is an important trait inBrassica napus. To know the genet ic basis of yellow seed color inBrassica napus, we carried out genetic studies by using conventional genetics analyses. The conventional genetics was studied in generations (F1 F2 reciprocal F2, BC1, and F23) ofB. napus derived from crosses between a yellow-seeded (No. 2127-17) and nine different black-seeded parents. The results indicated that seed color was mainly controlled by the maternal genotype but influenced by the interact ion between the maternal and endosperm and/or embryonic genotypes. In the combinations which included black-seeded lines SW0780, 94560, 94545 and 1141B, the yellow seed is partially dominant over black with two or three dominance epistasis ratio. A dominant yellow-seeded gene Y which exhibits epistatic effects on the two independent dominant black-seeded genes B and C was ident ified in DH line No. 2127-17. These observations are in agreement with our previous reports. But in the rests, including the crosses with HS No.4, HS No. 3, XY No. 15, 94570 and ZS No. 10, the black seed color was dominant over yellow seed color. The inheritance of this trait in the segregating populations fits the model of a digenic dominance epistasis or triplicate dominance epistasis. A new locus was identified and designated as D: the dominant gene D for black seed color inhibits the dominant gene Y. Therefore, in combination with the Y, B and C, we found that the seed color was influenced by at least four genes. Identifying seed color genes and defining their inheritance should further our understanding of yellow seed color trait and facilitate development of new and better yellow-seeded cult ivars ofBrassics napus.     

Intersubgenomic heterosis in seed yield potential observed in a new type of Brassica napus introgressed with partial Brassica rapa genome

This paper reports the observation on the intersubgenomic heterosis for seed yield among hybrids between natural Brassica napus (AⁿAⁿCⁿCⁿ) and a new type of B. napus with introgressions of genomic components of Brassica rapa (A^rA^r). This B. napus was selected from the progeny of B. napus × B. rapa and (B. napus × B. rapa) × B. rapa based on extensive phenotypic and cytological observation. Among the 129 studied partial intersubgenomic hybrids, which were obtained by randomly crossing 13 lines of the new type of B. napus in F₃ or BC₁F₃ to 27 cultivars of B. napus from different regions as tester lines, about 90% of combinations exceeded the yield of their respective tester lines, whereas about 75% and 25% of combinations surpassed two elite Chinese cultivars, respectively. This strong heterosis was further confirmed by reevaluating 2 out of the 129 combinations in a successive year and by surveying hybrids between 20 lines of the new type of B. napus in BC₁F₅ and its parental B. napus in two locations. Some DNA segments from B. rapa were identified with significant effects on seed yield and yield components of the new type of B. napus in BC₁F₅ and intersubgenomic hybrids in positive or negative direction. It seems that the genomic components introgressed from B. rapa contributed to improvement of seed yield of rapeseed.     

Genetic Regulation on the Black Phenotype Mutants of Moth and Pupa of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae)

Genetic regulation of body color of mutant strain JBM of Helicoverpa armigera with black body color of pupae and adults was investigated. Reciprocal crosses between JBM and JBW (a wild strain with yellow brown body color of pupae and adults) were used to determine the inheritance characteristics of body color. Analysis of the ratio of phenotype segregation from the F₁ generation, F₂ generation, F₃ generation, BC₁ (F₁ × JBM) generation and F₁ generation of BC₁ indicated that the black body color was controlled by one recessive gene.__________From Genetika, Vol. 41, No. 5, 2005, pp. 702–704.Original English Text Copyright © 2005 by Wang Mo, Weihua Ma, Lizhen Chen, Fuxing Zhu, Jianhong Li.This article was submitted by the authors in English.     

Effects of Heat Stress during Seed Filling Stage on <i>Brassica napus</i> Seed Oil Accumulation and Chlorophyll Fluorescence Characteristics

As global temperature rise, the threat of heat stress to rapeseed production is becoming more obvious. Exploring the response characteristics of two important biological pathways, oil accumulation and photosynthesis, to heat stress during B. napus seed filling is helpful in the genetic improvement of heat-tolerant rapeseed. The effects of heat stress on seed oil accumulation and chlorophyll fluorescence characteristics of 29 B. napus germplasms with different oil content and environmental sensitivity, including 6 rapeseed varieties which exhibited environment-sensitive/insensitive and with high, medium or low oil content, were tested by whole plant heat stress or the in vitro silique culture system. Both assay exhibited similar trend on oil content of the rapeseed germplasms. The heat effect on the chlorophyll fluorescence kinetic parameters F_v/F_m, ETR and Y(II) were also consistent. Heat stress significantly decreased oil content, although there was abundant genetic variation on heat tolerance among the genotypes. Correlation analysis showed that the decrease rate of F_v/F_m of silique heat-stressed B. napus developing seed was positive correlative to the decrease rate of mature seed oil content of the whole plant heat-stressed rapeseed (R = 0.9214, P-value < 0.01). Overall, the results indicated that heat stress inhibited oil accumulation and photosynthesis in B. napus developing seed. The decrease rate of chlorophyll fluorescence parameter F_v/F_m of heat-stressed developing seed could be used as the index of heat tolerant rapeseed identification. Further, two heat insensitive rapeseed varieties with high oil content were identified.     

An ultradense genetic recombination map for <Emphasis Type="Italic">Brassica napus</Emphasis>, consisting of 13551 SRAP markers

Sequence related amplified polymorphism (SRAP) was used to construct an ultradense genetic recombination map for a doubled haploid (DH) population in B. napus. A total of 1,634 primer combinations including 12 fluorescently labeled primers and 442 unlabeled ones produced 13,551 mapped SRAP markers. All these SRAPs were assembled in 1,055 bins that were placed onto 19 linkage groups. Ten of the nineteen linkage groups were assigned to the A genome and the remaining nine to the C genome on the basis of the differential SRAP PCR amplification in two DH lines of B. rapa and B. oleracea. Furthermore, all 19 linkage groups were assigned to their corresponding N1–N19 groups of B. napus by comparison with 55 SSR markers used to construct previous maps in this species. In total, 1,663 crossovers were detected, resulting in a map length span of 1604.8 cM. The marker density is 8.45 SRAPs per cM, and there could be more than one marker in 100 kb physical distance. There are four linkage groups in the A genome with more than 800 SRAP markers each, and three linkage groups in the C genome with more 1,000 SRAP markers each. Our studies suggest that a single SRAP map might be applicable to the three Brassica species, B. napus, B. oleracea and B. rapa. The use of this ultra high-density genetic recombination map in marker development and map-based gene cloning is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular and cytological characterization of introgression lines in yellow seed derived from somatic hybrids between Brassica napus and Sinapis alba

Polymerase chain reaction and genomic in situ hybridization techniques were conducted to demonstrate the genomic introgressions in somatic hybrid progenies between Brassica napus and Sinapis alba. With minisatellite core sequence 33.6 as primer, an S. alba-specific band (288 bp) was amplified in yellow seed progenies. No hybridization signals were found using the genomic DNA of S. alba as probe. In addition, degenerate primers were used for the detection of related genes based on the genes related to flavonoid biosynthesis of the model plant Arabidopsis thaliana. Sequencing results show that flavonoid pathway genes are highly conserved in A. thaliana, S. alba and B. napus, and present subtle differences because of genetic evolution. A specific band (1,672 bp) consistent with S. alba was characterized in the yellow seed lines. This study demonstrates that the new yellow seed germplasms, derived from backcrossed and self-crossed progenies of B. napus–S. alba hybrids, are stable and homozygous introgression lines with yellow seed color, and differ from existing yellow seed materials.     

A knockout mutation in the lignin biosynthesis gene CCR1 explains a major QTL for acid detergent lignin content in Brassica napus seeds

Seed coat phenolic compounds represent important antinutritive fibre components that cause a considerable reduction in value of seed meals from oilseed rape (Brassica napus). The nutritionally most important fibre compound is acid detergent lignin (ADL), to which a significant contribution is made by phenylpropanoid-derived lignin precursors. In this study, we used bulked-segregant analysis in a population of recombinant inbred lines (RILs) from a cross of the Chinese oilseed rape lines GH06 (yellow seed, low ADL) and P174 (black seed, high ADL) to identify markers with tight linkage to a major quantitative trait locus (QTL) for seed ADL content. Fine mapping of the QTL was performed in a backcross population comprising 872 BC₁F₂ plants from a cross of an F₇ RIL from the above-mentioned population, which was heterozygous for this major QTL and P174. A 3:1 phenotypic segregation for seed ADL content indicated that a single, dominant, major locus causes a substantial reduction in ADL. This locus was successively narrowed to 0.75 cM using in silico markers derived from a homologous Brassica rapa sequence contig spanning the QTL. Subsequently, we located a B. rapa orthologue of the key lignin biosynthesis gene CINNAMOYL CO-A REDUCTASE 1 (CCR1) only 600 kbp (0.75 cM) upstream of the nearest linked marker. Sequencing of PCR amplicons, covering the full-length coding sequences of Bna.CCR1 homologues, revealed a locus in P174 whose sequence corresponds to the Brassica oleracea wild-type allele from chromosome C8. In GH06, however, this allele is replaced by a homologue derived from chromosome A9 that contains a loss-of-function frameshift mutation in exon 1. Genetic and physical map data infer that this loss-of-function allele has replaced a functional Bna.CCR1 locus on chromosome C8 in GH06 by homoeologous non-reciprocal translocation.     

Post-dispersal predation on Pinus sylvestris seeds by Fringilla spp: ground substrate affects selection for seed color

Background matching might lower the risk of seeds being eaten by seed predators that search visually. In aviary experiments, we analyzed the selection of diff erent-colored seeds by ground-feeding finches (Fringillacoelebs and F.montifringilla) against four naturally occurring forest soil substrates. The substrates were fresh burn (black), 6-year-old burn (brown), mineral soil (pale yellow) and Pleuroziumschreberi feather moss (green). We used color-sorted seeds of Pinussylvestris, a species with a large natural variation in seed color, ranging from pale yellow to black. Although seeds were scattered on the substrates at a density of only 91 seeds m⁻², birds removed seeds effectively. Both bird species found more pale than dark seeds on the fresh burn substrate. F. montifringilla also recovered more pale than dark seeds on the old burn, and more dark than pale seeds on mineral soil. In moss, the birds found very few seeds compared to the other substrates, and there was no color discrimination. P.sylvestris is frequently regenerating after fire, suggesting that dark seeds would be favored under selection from visually searching predators. Fire-adapted conifers with serotinous cones, e.g., Pinuscontorta ssp. latifolia, which spread their seeds primarily on freshly burnt surfaces, produce uniformly black or dark brown seeds. However, regeneration of the non-serotinous P.sylvestris is often extended for several years after a fire, during which substrate color and structure change. This may have helped to maintain variation in seed color. When regeneration of a plant species occurs on a substrate of uniform color, we believe that selection by visually searching seed predators will result in the evolution of cryptic seed color. Received: 16 August 1996 / Accepted: 30 November 1996     

Inheritance of seed color and molecular markers linked to the seed color gene in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Seed color inheritance in Brassica juncea was studied in F₁, F₂ and BC₁ populations. Seed color was found under the control of the maternal genotype, and the brown-seeded trait was dominant over the yellow-seeded trait. Segregation analysis revealed that one pair of major genes controlled the seed coat color. To develop markers linked to the seed color gene, AFLP (amplified fragments length polymorphism) combined with BSA (bulk segregant analysis) technology was used to screen the parents and bulks selected randomly from an F₂ population (Wuqi yellow mustard × Wugong mustard) consisting of 346 individuals. From a survey of 512 AFLP primer combinations, 15 AFLP markers located on either side of the gene were identified, and the average distance between markers was 2.59 cM. P11MG15 was a cosegregated marker, and the closest markers (P03MC08, P16MC02 and P11MG01) were at a distance of 0.3, 0.3 and 0.7 cM from the target gene, respectively. In order to utilize the markers for breeding of yellow-seeded varieties, four AFLP markers, P11MG01, P15MG15, P09MC12 and P16MC02 were successfully converted into SCAR (sequence characterized amplified region) markers. The seed color trait controlled by the single gene together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded varieties. The markers found in the present study could accelerate the step of map-based cloning of the target gene.     

The genetic basis of flowering time and photoperiod sensitivity in rapeseed <Emphasis Type="Italic">Brassica napus</Emphasis> L.

The objective of this study was to dissect the genetic control of days to flowering (DTF) and photoperiod sensitivity (PS) into the various components including the main-effect quantitative trait loci (QTLs), epistatic QTLs and QTL-by-environment interactions (QEs). Doubled haploid (DH) lines were produced from an F₁ between two spring Brassica napus cultivars Hyola 401 and Q2. DTF of the DH lines and parents were investigated in two locations, one location with a short and the other with a long photoperiod regime over two years. PS was calculated by the delay in DTF under long day as compared to that under short day. A genetic linkage map was constructed that comprised 248 marker loci including SSR, SRAP, and AFLP markers. Further QTL analysis resolved the genetic components of flowering time and PS into the main-effect QTLs, epistatic QTLs, and QEs. A total of 7 main-effect QTLs and 11 digenic interactions involving 21 loci located on 13 out of the 19 linkage groups were detected for the two traits. Three main-effect QTLs and four pairs of epistatic QTLs were involved in QEs conferring DTF. One QTL on linkage group (LG) 18 was revealed to simultaneously affect DTF and PS and explain for the highest percentage of the phenotypic variation. The implications of the results for B. napus breeding have been discussed. The text was submitted by the authors in English.     

Seed colour and post-fire bird predation in a Mediterranean pine forest

In a Pinus halepensis Mill. forest, a field experiment was designed to evaluate post-fire seed predation as affected by combinations of seed colour and soil substrates: light grey and black seeds combined with light grey ash, dark grey ash and pale brown sand. A survey of bird species inhabiting the area was also carried out and polyphenolic content of seed coat was assessed in seed lots of different colour. Light grey seeds were observed to be less predated on light grey ash, suggesting eucrypsis as a protective strategy against bird predation. On the contrary, no clear pattern was observed for the predation of black seeds on different substrates. In the study area both bird species breaking the seed coat and eating the endosperm and bird species swallowing the whole seed were monitored. We have estimated that more seeds were swallowed than broken, in all colour categories. Light grey seeds, which were found to have a higher content of polyphenols, were predated more than black seeds when exposed on the same substrate. Thus, no evidence was produced that the amount of polyphenols in seed coat could protect seeds from predation.     

Inheritance of seed condensed tannins and their relationship with seed-coat color and pattern genes in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Condensed tannins are major flavonoid end products that affect the nutritional quality of many legume seeds. They chelate minerals and interact with proteins, thus reducing their bioavailability. Tannins also contribute to seed coat color and pigment distribution or intensity. The objective of this study was to analyze the relationship between quantitative trait loci (QTL) for seed tannin concentration in common bean and Mendelian genes for seed coat color and pattern. Three populations of recombinant inbred lines, derived from crosses between the Andean and Mesoamerican genepools were used for QTL identification and for mapping STS markers associated with seed color loci. Seed coat condensed tannins were determined with a butanol–HCl method and a total of 12 QTL were identified on separate linkage groups (LGs) in each of the populations with individual QTL explaining from 10 to 64% of the phenotypic variation for this trait. Loci on linkage groups B3 and B10 were associated with the Mendelian genes Z and Bip for partly colored seed coat pattern, while a QTL on linkage group B7 was associated with the P gene which is the primary locus for the control of color expression in beans. In conclusion, this study found that the inheritance of tannin concentration fits an oligogenic model and identifies novel putative alleles at seed coat color and pattern genes that control tannin accumulation. The results will be important for the genetic improvement of nutritionally enhanced or biofortified beans that have health promoting effects from higher polyphenolics or better iron bioavailability.     

Germination response of black and yellow seed coated canola (<Emphasis Type="Italic">Brassica napus</Emphasis>) lines to chemical treatments under cold temperature conditions

Seed quality is a key critical component to produce well established and vigorous seedlings under cool soil (<10°C) conditions experienced in Western Canada. A simple, relatively quick germination assay is required to separate small differences in seed germination which can have a significant impact on seedling growth. It has long been established that phytohormones regulate seed germination: abscisic acid inhibits germination whereas gibberellins enhance germination. We investigated the effects of ABA, GA, ethylene and inhibitors of these phytohormones alone and in combination on the germination rate of a black and a yellow seed canola (Brassica napus) imbibed at 8°C. The effects of either saline solutions, osmotic solutions, fusicoccin or testa on the germination of canola seeds imbibed at 8°C were also investigated. This temperature is representative of the soil temperatures experienced in the early spring of Western Canada. The two canola seed lines, especially the yellow seed line, were very sensitive to increasing concentration of saline solutions at 8°C, but not at 23°C; however, iso-osmotic solutions that reduced water potential were more inhibitory. The seed coat (testa) including the endosperm was a major factor affecting the germination rate of the yellow seed line at 8°C, however, GA₄₊₇ overcame the inhibitory effect of the testa, whereas ABA exacerbated it. Fusicoccin was more stimulatory to germination than GA₄₊₇, however, unlike GA₄₊₇, it was unable to overcome the inhibitory effect of paclobutrazol, a GA biosynthesis inhibitor. Fluridone, an ABA biosynthesis inhibitor, was unable to overcome the inhibitory effects of a saline solution suggesting that the inhibitory effect was not due to elevated ABA levels. Ethylene, a stimulator of germination did not appear to be involved in the germination of these two lines. Controlled deterioration at 35°C, 85% RH could be either partially or completely overcome by exogenous GA₄₊₇. This study demonstrated the effect of hormones, salinity and testa on the germination of canola seeds under less than ideal environmental conditions.     

Perch color preference in juvenile green tree pythons,Chondropython viridis

Green tree pythons, Chondropython viridis, are polymorphic for color as juveniles, commonly being primarily yellow or brown until becoming mostly green at about 1 year of age. We tested the hypothesis that the different morphs arose as a result of selection for differential background matching, yellow morphs selecting light-colored backgrounds, and brown morphs selecting dark-colored backgrounds. Twelve yellow and eight brown morphs were placed repeatedly in individual testing enclosures and allowed to choose between black and white or yellow and brown halves of a t-perch. Trials showed that both color morphs preferred dark over light perches. We tentatively suggest that individuals chose dark-colored perches for purposes of concealment. © 1994 Wiley-Liss, Inc.