Determination of n+1 Gamete Transmission Rate of Trisomics and Location of Gene Controlling 2n Gamete Formation in Chinese Cabbage(Brassica rapa)

A set of trisomics of Chinese cabbage was used for determining the n-I-1 gamete transmission rate and locating the gene controlling 2n gamete formation on the corresponding chromosome. The results showed that the transmission rates of extra chromosomes in different trisomics varied from 0% to 15.38% by male gametes and from 0% to 17.39% by female gametes. Of the nine F2 populations derived from the hybridizations between each trisomic and Bp058 （2n gamete material）, only Tri- 4xBp058 showed that the segregation ratio of plants without 2n gamete formation to plants with 2n gamete formation was 10.38：1, which fitted the expected segregation ratio of the trisomics （AAa） based on the 7.37% of n＋l gamete transmission through female and 5.88% through male. In other populations the segregation ratios varied from 2.48：1 to 3.72：1, which fitted the expected 3：1 segregation ratio of the bisomics （Aa）. These results suggested that the gene controlling 2n gamete formation in Chinese cabbage Bp058 was located on chromosome 4. Further trisomic analysis based on the chromosome segregation and the incomplete stochastic chromatid segregation indicated that the gene locus was tightly linked to the centromere.

Overlapping functions of YDA and MAPKKK3/MAPKKK5 upstream of MPK3/MPK6 in plant immunity and growth/development

Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3(MAPK3 or MPK3)and MPK6 play important signaling roles in plant immunity and growth/development.MAPK KINASE4(MKK4)and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes.YODA(YDA),also known as MAPK KINASE KINASE4(MAPKKK4),is upstream of MKK4/MKK5 and forms a complete MAPK cascade(YDA–MKK4/MKK5–MPK3/MPK6)in regulating plant growth and development.In plant immunity,MAPKKK3 and MAPKKK5 function redundantly upstream of the same MKK4/MKK5–MPK3/MPK6 module.However,the residual activation of MPK3/MPK6 in the mapkkk3 mapkkk5 double mutant in response to flg22 pathogen-associated molecular pattern(PAMP)treatment suggests the presence of additional MAPKKK(s)in this MAPK cascade in signaling plant immunity.To investigate whether YDA is also involved in plant immunity,we attempted to generate mapkkk3 mapkkk5 yda triple mutants.However,it was not possible to recover one of the double mutant combinations(mapkkk5 yda)or the triple mutant(mapkkk3 mapkkk5 yda)due to a failure of embryogenesis.Using the clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPRassociated protein 9(Cas9)approach,we generated weak,N-terminal deletion alleles of YDA,yda-del,in a mapkkk3 mapkkk5 background.PAMP-triggered MPK3/MPK6 activation was further reduced in the mapkkk3 mapkkk5 yda-del mutant,and the triple mutant was more susceptible to pathogen infection,suggesting YDA also plays an important role in plant immune signaling.In addition,MAPKKK5 and,to a lesser extent,MAPKKK3 were found to contribute to gamete function and embryogenesis,together with YDA.While the double homozygous mapkkk3 yda mutant showed the same growth and development defects as the yda single mutant,mapkkk5 yda double mutant and mapkkk3 mapkkk5 yda triple mutants were embryo lethal,similar to the mpk3 mpk6 double mutants.These results demonstrate that YDA,MAPKKK3,and MAPKKK5 have overlapping functions upstream of the MKK4/MKK5–MPK3/MPK6 module in both plant immunity and growth/development.