Comparative Mapping Combined With Map-Based Cloning of the Brassica juncea Genome Reveals a Candidate Gene for Multilocular Rapeseed

Abstract

Multilocular traits exist in a variety of plants and exert important effects on plant yield. Previous genetic studies have shown that multilocular trait of the Brassica juncea cultivar Duoshi is controlled by two recessive genes, Bjln1 and Bjln2. In previous studies, the Bjln1 gene is located on chromosome A07, and the Bjln1 candidate gene is BjuA07.CLV1. In this study, a BC4 mapping population for the Bjln2 gene was generated. This population was used to construct genetic linkage maps of the Bjln2 gene using amplified fragment length polymorphism (AFLP), intron length polymorphism (IP) and simple sequence repeat (SSR) methodology. The results showed that the Bjln2 gene was restricted to a 0.63 cM interval. BLAST alignment with B. juncea revealed the Bjln2 gene was located within a 11.81–16.65 Mb region on chromosome B07. Moreover, the candidate gene BjuB07.CLV1 (equivalent to Bjln2) was cloned by comparing mapping and map-based cloning, and BjuB07.CLV1 gene was shown to have the ability to restore the bilocular traits in a genetic complementation experiment. The sequencing revealed that a 4961 bp insertion interrupted the coding sequence of the BjuB07.CLV1 gene, resulting in an increase in locule number. Expression analysis revealed that BjuB07.CLV1 was expressed in all tissues and the expression level in bilocular plants was significantly higher than that in multilocular plants. In addition, markers closely linked to the Bjln2 gene were developed and used for molecular marker-assisted breeding of multilocular traits.

Increased seed number per silique in Brassica juncea by deleting cis‐regulatory region affecting BjCLV1 expression in carpel margin meristem

“…There was a significant difference in CMM size between the bilocular and trilocular plants but no significant difference in the sizes of SAM, IM and FM. However, the SAM, IM and FM of multilocular plants were larger than those of bilocular plants in B. rapa ml4 (Fan et al ., 2014) and B. juncea ‘ duoshi’ (Chen et al ., 2018; Xiao et al ., 2018). Thus, multilocular phenotype formation was concurrent with enlarged SAMs, which could lead to an enlarged FM and, further allowing the initiation of more floral organ primordia, with extra gynoecium inside the silique as the FM failed to terminate.…”

“…However, both Bjln1 and Bjln2 in B. juncea ‘duoshi’ are homologous genes of CLV1 . Bjln1 , BjA7.clv1 , is caused by a change in amino acids at positions 28 and 63 because of five SNPs in LRR domain of the coding region (Xiao et al ., 2018), and Bjln2 is caused by the insertion of a 4961‐bp fragment in the coding region that interferes with the normal BjB3.CLV1 transcription (Chen et al ., 2018). This could explain why J163‐4 and ‘duoshi’ have different phenotypes (Xiao et al ., 2013; Xu et al ., 2014), even though both have mutations in the BjCLV1 gene.…”

“…The cores of this complex network are WUS, a mobile homeodomain TF expressed in the organizing centre (OC) that can move to the central zone (CZ) to promote stem cell fate (Yadav et al ., 2011), especially by repressing differentiation (Mayer et al ., 1998) and CLV3 (Fletcher et al ., 1999), a small peptide ligand whose expression is induced by WUS in the CZ but can repress WUS expression when perceived by leucine‐rich repeat receptor‐like kinases (LRR‐RLKs), such as CLV1 (Clark et al ., 1997), and a leucine‐rich repeat receptor‐like protein (LRR‐RLP) CLV2 (Jeong et al ., 1999). Over the past 30 years, mutations in CLV1 in Arabidopsis and CLV1 orthologs in crops, such as the CLV1 orthologs in rice (Suzaki et al ., 2004), in maize (Bommert et al ., 2005), in tomato (Xu et al ., 2015) and in B. juncea (Chen et al ., 2018; Xiao et al ., 2018), often created great interest, which contains enlarged shoot meristems, flattened stems and increased floral and fruit organ number. However, despite mutations in CLV1 and CLV1 orthologs involved in shoot meristem activity that affect the formation of these gynoecial structures, little is known about their direct roles in CMM development.…”

Characterization of the BnA10.tfl1 Gene Controls Determinate Inflorescence Trait in Brassica napus L.

“…Previous studies have found that the determinate phenotype was caused by the development and differentiation of the shoot apical meristem (SAM) [3][4][5]14], To further understand the phenotypic differences of apical SAM, we observed the inflorescence SAM in homozygous indeterminate and determinate plants at different stage. Paraffin sectioning of the plant SAMs was performed as previously described [24]. The samples were treated with 50% FAA (Formalin-acetic acid-alcohol), then safranine-dyed, rinsed, dehydrated, cleared, infiltrated, embedded, sliced, sealed, and examined with a Nikon microscope (Nikon, Tokyo, Japan) [25].…”

“…The fragment of genomic DNA that included the upstream, full-length gene, and downstream sequences was amplified using Phusion Hot Start High Fidelity DNA polymerase (NEB, Ipswich, MA, USA) with recombinant primers (CP525-F/CP525-R, Table S1) from the indeterminate inflorescence line 2014. The complementation plasmid pBnA10.TFL1:BnA10.TFL1 was produced using a sequenced genomic fragment and the pCAMBIA2300 vector that had been digested with EcoRI and PstI using a One Step Cloning Kit (Vazyme, Nanjing, China), as previously described [24]. The confirmed recombinant plasmid was transformed into GV3101 (Agrobacterium tumefaciens) and stored at −80 • C after adding sterilized glycerin.…”