Cytological Observations and Bulked-Segregant Analysis Coupled Global Genome Sequencing Reveal Two Genes Associated with Pollen Fertility in Tetraploid Rice

Neo-tetraploid rice with high fertility is a useful germplasm for polyploid rice breeding, which was developed from the crossing of different autotetraploid rice lines. However, little information is available on the molecular mechanism underlying the fertility of neo-tetraploid rice. Here, two contrasting populations of tetraploid rice, including one with high fertility (hereafter referred to as JG) and another with low fertility (hereafter referred to as JD), were generated by crossing Huaduo 3 (H3), a high fertility neo-tetraploid rice that was developed by crossing Jackson-4x with 96025-4x, and Huajingxian74-4x (T452), a low fertility autotetraploid rice parent. Cytological, global genome sequencing-based bulked-segregant (BSA-seq) and CRISPR/Cas9 technology were employed to study the genes associated with pollen fertility in neo-tetraploid rice. The embryo sacs of JG and JD lines were normal; however, pollen fertility was low in JD, which led to scarce fertilization and low seed setting. Cytological observations displayed low pollen fertility (25.1%) and approximately 31.3 and 27.2% chromosome lagging at metaphase I and II, and 28.8 and 24.8% chromosome straggling at anaphase I and II in JD, respectively. BSA-seq of F_2–3 generations and RNA-seq of F₄ generation detected a common fragment, i.e., 18,915,234–19,500,000, at chromosome 7, which was comprised of 78 genes associated with fertility. Among 78 genes, 9 genes had been known to be involved in meiosis and pollen development. Two mutants ny1 (LOC_Os07g32406) and ny2 (LOC_Os07g32040) were generated by CRISPR/Cas9 knockout in neo-tetraploid rice, and which exhibited low pollen fertility and abnormal chromosome behavior. Our study revealed that two unknown genes, LOC_Os07g32406 (NY1) and LOC_Os07g32040 (NY2) play an important role in pollen development of neo-tetraploid rice and provides a new perspective about the genetic mechanisms of fertility in polyploid rice.     

Conjunctive Analyses of BSA-Seq and BSR-Seq Unveil the Msβ-GAL and MsJMT as Key Candidate Genes for Cytoplasmic Male Sterility in Alfalfa (Medicago sativa L.)

Knowing the molecular mechanism of male sterility in alfalfa is important to utilize the heterosis more effectively. However, the molecular mechanisms of male sterility in alfalfa are still unclear. In this study, the bulked segregant analysis (BSA) and bulked segregant RNA-seq (BSR) were performed with F2 separation progeny to study the molecular mechanism of male sterility in alfalfa. The BSA-seq analysis was located in a candidate region on chromosome 5 containing 626 candidate genes which were associated with male sterility in alfalfa, while the BSR-seq analysis filtered seven candidate DEGs related to male sterility, and these candidate genes including EF-Tu, β-GAL, CESA, PHGDH, and JMT. The conjunctive analyses of BSR and BSA methods revealed that the genes of Msβ-GAL and MsJMT are the common detected candidate genes involved in male sterility in alfalfa. Our research provides a theory basis for further study of the molecular mechanism of male sterility in alfalfa and significant information for the genetic breeding of Medicago sativa.     

Identifying QTLs for Grain Size in a Colossal Grain Rice (Oryza sativa L.) Line,and Analysis of Additive Effects of QTLs

Grain size is an important component of quality and harvest traits in the field of rice breeding. Although numerous quantitative trait loci (QTLs) of grain size in rice have been reported, the molecular mechanisms of these QTLs remain poorly understood, and further research on QTL observation and candidate gene identification is warranted. In our research, we developed a suite of F₂ intercross populations from a cross of 9311 and CG. These primary populations were used to map QTLs conferring grain size, evaluated across three environments, and then subjected to bulked-segregant analysis-seq (BSA-seq). In total, 4, 11, 12 and 14 QTLs for grain length (GL), grain width (GW), 1000-grain weight (TGW), and length/width ratio (LWR), respectively, were detected on the basis of a single-environment analysis. In particular, over 200 splicing-related sites were identified by whole-genome sequencing, including one splicing-site mutation with G>A at the beginning of intron 4 on Os03g0841800 (qGL3.3), producing a smaller open reading frame, without the third and fourth exons. A previous study revealed that the loss-of-function allele caused by this splicing site can negatively regulate rice grain length. Furthermore, qTGW2.1 and qGW2.3 were new QTLs for grain width. We used the near-isogenic lines (NILs) of these GW QTLs to study their genetic effects on individuals and pyramiding, and found that they have additive effects on GW. In summary, these discoveries provide a valuable genetic resource, which will facilitate further study of the genetic polymorphism of new rice varieties in rice breeding.