Bifang Cheng

Linoleic acid (C18:2) is an important polyunsaturated fatty acid in the seed oil of many crops. Here, we report that mutations in the promoter, intron and CDS of the FAD2 genes SalFAD2.LIA1 and SalFAD2.LIA2 generate three alleles LIA^1a , LIA ^1b and lia ¹ and two alleles LIA² and lia², respectively, controlling the C18:2 variation (4.4–32.7%) in yellow mustard. The allelic effect on increasing C18:2 content is LIA ^1a > LIA ^1b > lia ¹ , LIA² > lia², and LIA ^1a > LIA². The five FAD2 alleles each contain two exons, one intron and a promoter adjacent to exon 1. LIA^1a has a 1152 bp CDS, a 1221 bp intron with promoter function and a 607 bp promoter. Compared with LIA ^1a , the intron of LIA ^1b has reduced promoter activity and that of LIA² and lia² has no promoter function due to extensive SNP and indel mutations. lia¹ differed from LIA^1b by having an insertion of 1223 bp retrotransposon in its intron. lia ² with mutations in the promoter has reduced promoter activity compared with LIA ² . This study revealed that complex quantitative variation of trait phenotype in plants could be modulated by multiple alleles of oligogenic loci resulting from mutations in the regulatory region and CDS.

Naturally occurring heritable variation provides a fundamental resource to reveal the genetic and molecular bases of traits in forward genetic studies. Here, we report the molecular basis of the differences in the four alleles E¹, E², E³, and e of the FATTY ACID ELONGATION1 (FAE1) gene controlling high, medium, low, and zero erucic content in yellow mustard (Sinapis alba). E¹ represents a fully functional allele with a coding DNA sequence (CDS) of 1521 bp and a promoter adjacent to the CDS. The null allele e resulted from an insertional disruption in the CDS by Sal-PIF, a 3100-bp PIF/Harbinger-like DNA transposon, whereas E² and E³ originated from the insertion of Sal-T1, a 4863-bp Copia-like retrotransposon, in the 5′ untranslated region. E³ was identical to E² but showed cytosine methylation in the promoter region and was thus an epiallele having a further reduction in expression. The coding regions of E² and E³ also contained five single-nucleotide polymorphisms (SNPs) not present in E¹, but expression studies in Saccharomyces cerevisiae indicated that these SNPs did not affect enzyme functionality. These results demonstrate a comprehensive molecular framework for the interplay of transposon insertion, SNP/indel mutation, and epigenetic modification influencing the broad range of natural genetic variation in plants.

Development of yellow mustard (Sinapis alba L.) with superior quality traits (low erucic and linolenic acid contents, and low glucosinolate content) can make this species as a potential oilseed crop. We have recently isolated three inbred lines Y1127, Y514 and Y1035 with low (3.8%), medium (12.3%) and high (20.8%) linolenic acid (C18∶3) content, respectively, in this species. Inheritance studies detected two fatty acid desaturase 3 (FAD3) gene loci controlling the variation of C18∶3 content. QTL mapping revealed that the two FAD3 gene loci responsible for 73.0% and 23.4% of the total variation and were located on the linkage groups Sal02 and Sal10, respectively. The FAD3 gene on Sal02 was referred to as SalFAD3.LA1 and that on Sal10 as SalFAD3.LA2. The dominant and recessive alleles were designated as LA¹ and la¹ for SalFAD3.LA1, and LA² and la² for SalFAD3.LA2. Cloning and alignment of the coding and genomic DNA sequences revealed that the SalFAD3.LA1 and SalFAD3.LA2 genes each contained 8 exons and 7 introns. LA¹ had a coding DNA sequence (CDS) of 1143 bp encoding a polypeptide of 380 amino acids, whereas la¹ was a loss-of-function allele due to an insertion of 584 bp in exon 3. Both LA² and la² had a CDS of 1152 bp encoding a polypeptide of 383 amino acids. Allele-specific markers for LA¹, la¹, LA² and la² co-segregated with the C18∶3 content in the F₂ populations and will be useful for improving fatty acid composition through marker assisted selection in yellow mustard breeding.

Yellow mustard (Sinapis alba) has a sporophytic self-incompatibility reproduction system. Genetically stable self-incompatible (SI) and self-compatible (SC) inbred lines have recently been developed in this crop. Understanding the S haplotype of different inbred lines and the inheritance of the self-(in)compatibility (SI/SC) trait is very important for breeding purposes. In this study, we used the S-locus gene-specific primers in Brassica rapa and Brassica oleracea to clone yellow mustard S-locus genes of SI lines Y514 and Y1130 and SC lines Y1499 and Y1501. The PCR amplification results and DNA sequences of the S-locus genes revealed that Y514 carried the class I S haplotype, while Y1130, Y1499, and Y1501 had the class II S haplotype. The results of our genetic studies indicated that self-incompatibility was dominant over self-compatibility and controlled by a one-gene locus in the two crosses of Y514 × Y1499 and Y1130 × Y1501. Of the five S-locus gene polymorphic primer pairs, Sal-SLGI and Sal-SRKI each generated one dominant marker for the SI phenotype of Y514; Sal-SLGII and Sal-SRKII produced dominant marker(s) for the SC phenotype of Y1501 and Y1499; Sal-SP11II generated one dominant marker for Y1130. These markers co-segregated with the SI/SC phenotype in the F₂ populations of the two crosses. In addition, co-dominant markers were developed by mixing the two polymorphic primer pairs specific for each parent in the multiplex PCR, which allowed zygosity to be determined in the F₂ populations. The SI/SC allele-specific markers have proven to be very useful for the selection of the desirable SC genotypes in our yellow mustard breeding program.

Yellow mustard (Sinapis alba L.) is an important condiment crop for the spice trade in the world. It has lagged behind oilseed Brassica species in molecular marker development and application. Intron length polymorphism (ILP) markers are highly polymorphic, co-dominant and cost-effective. The cross-species applicability of ILP markers from Brassica species and Arabidopsis makes them possible to be used for genetic linkage mapping and further QTL analysis of agronomic traits in yellow mustard.

Yellow mustard (Sinapis alba L.) has been grown as an important source of condiment for the spice trade in the world. It is an obligate outcrossing species due to its sporophytic self-incompatibility (SI). To utilize heterosis for yield potential, we have attempted to develop elite component inbred lines for producing high-yielding synthetic varieties for this crop. The open-pollinated variety Andante was used as the initial population. To circumvent the SI barrier, bud-pollination for selfing was performed on the selected initial (S0) plants. Various types of inbreeding depression were observed in the S1 generation. Elite inbred lines tolerant to inbreeding were produced by purging the deleterious alleles in each inbred generation. Self-compatible (SC) lines were developed for the first time in this species. There were three types of erucic variants (high: 49.9%, median: 23.9% and low: 1.4%), three types of linolenic variants (high: 18.5%, median: 13.8% and low: 3.8%) and two types of mucliage variants (high: 164.0 cS*mL/g and low: 12.0 cS*mL/g) among the developed inbred lines. These variants are being used to investigate the genetic and molecular mechanism underpinning the phenotypic variation of the seed oil profile and SI/SC traits in yellow mustard.